TW201617090A - Precautionary, preventive, or therapeutic substance for corneal disease or corneal damage, cell sheet, cell culture supplement, and method of culturing cells - Google Patents

Abstract

A precautionary, preventive, or therapeutic substance for corneal disease or corneal damage of the present invention is characterized by including a polypeptide as an active ingredient, which includes a region consisting of any one of a polypeptide selected from the group consisting of the following (a) to (c): (a) a polypeptide composed of at least 70 consecutive amino acids in an amino acid sequence represented by sequence identification number 4 and including a region of 166aa to 235aa in the sequence identification number 4, (b) a polypeptide composed of at least 70 consecutive amino acids in an amino acid sequence represented by sequence identification number 2 and including a region of 166aa to 235aa in the sequence identification number 2, and (c) a polypeptide having at least 80 percent of sequence homology with the polypeptide (a) or (b).

Description

Prophylactic, inhibitory or therapeutic agent for corneal diseases or corneal damage, cell sheet, cell culture supplement Agent and cell culture method Field of invention

The present invention relates to a preventive, inhibitory or therapeutic agent for corneal diseases or corneal damage containing semaphorin 3A as an active ingredient. Further, the present invention relates to a cell sheet cultured in the presence of brain signaling protein 3A, a cell culture supplement containing brain signal protein 3A as an active ingredient, and a cell culture method cultured under brain signal protein 3A.

This case is based on the priority of Japan's special offer 2014-041017, which was filed in Japan on March 3, 2014, and its content is used here.

Background of the invention

The cornea is an avascular transparent tissue located on the outermost surface of the eye. In order to obtain a healthy visual function, it is a necessary condition for the cornea to be invisible and transparent without scar tissue or blood vessels. It plays an important role in maintaining the transparency of the cornea, which is the corneal endothelial cells located in the innermost layer of the cornea. Corneal endothelial cells control corneal parenchyma by shielding function and pump function (Parenchyma) A cell of water, and is thought to have little ability to divide in humans.

According to the report, the density is usually about 3,000 per square millimeter at birth, but it gradually decreases with 0.5%/year with increasing age. At this rate, corneal endothelial cells will have a life span of 200 years. However, due to corneal hereditary diseases such as contact lenses, various ophthalmic surgeries, corneal infections, Fuchs' corneal endothelial degeneration, glaucoma, and uveitis, the rate of corneal endothelium reduction will change. high. Once the corneal endothelial cells are reduced to about 400 per square millimeter, the control of the cornea that depends on the corneal parenchyma of the corneal endothelial cells cannot be achieved. Therefore, the cornea as a whole becomes edema and exhibits a turbidity like ground glass. Such a condition is called bullous keratopathy. The opacity of bullous keratopathy is irreversible, and once it becomes such a state, the patient will exhibit a very strong visual dysfunction accompanied by pain and become a social blindness state.

There is no drug treatment with corneal endothelium as the standard. When the formation of bullous keratopathy, the only method is corneal transplantation such as whole corneal transplantation or corneal endothelial transplantation. Large-foam keratopathy now accounts for the first cause of keratoplasty. However, compared with other diseases, keratoplasty for bullous keratopathy has indicated that the graft has a short life span and a poor prognosis. In addition, in the country (Japan), the supply of donated cornea is not sufficient. At present, even the only treatment of corneal transplantation cannot be fully implemented. Many patients are suffering from a decrease in visual function due to corneal opacity caused by corneal endothelial dysfunction. From this background, the development of the function of the corneal endothelium, Or the treatment for the purpose of preventing bullous keratopathy is highly anticipated, however, the status quo is no way.

It is said that human corneal endothelial cells (HCEC) do not divide in vivo, and many attempts are being made to overcome this phenomenon.

In recent years, it has been reported that cells having stem cell characteristics are isolated from mouse corneal parenchyma (Non-Patent Document 4). By using the induced differentiation medium, the stem cells derived from the neural ridges may even have the ability to differentiate into nerve cells or fat cells and the like. It has been reported that the tissue stem cells/precursor cells can be induced to differentiate into corneal endothelial cells by performing subsequent culture in a medium to which TGFb is added (Patent Document 4), but whether or not it is used as a corneal endothelial cell The function (for example, the pump function) is nothing.

So far, it has been reported that agglomerates (Spheres) obtained by suspension culture of corneal endothelial cells have properties of corneal endothelial progenitor cells (Patent Document 5 and Non-Patent Document 3). However, the aggregate obtained by this method does not express the marker p75 of neural stem cells derived from corneal endothelium, and its undifferentiation is still unclear.

Although corneal endothelial cells are said to not divide in vivo, they may proliferate in a certain degree in vitro. Attempts have been made to proliferate corneal endothelial cells in vitro and to treat them by using them (Non-Patent Document 1 and Patent Documents 1 to 3). However, in the culture method using the previous serum, once the corneal endothelial cells are cultured for a long period of time, the cells will change and lose the function of the corneal endothelium, and eventually the proliferation will be completely stopped. In general, it can only be propagated to the subcultures 5~ 7 (Non-Patent Document 2).

Others have developed cells using cultured corneal endothelial cells. Technology for culturing corneal endothelial cells, such as treatment, gene delivery technology, or eye drop treatment of Rho kinase inhibitors, cannot be performed due to poor cell conditions, low production, high technical requirements, and high cost. The transplantation of corneal endothelial cells is difficult to apply to the clinical situation.

The representative brain signaling protein molecule Sema3A, which has been clarified in the function of the immune system, has been actively analyzed in the world as a function of a guidance factor (Non-Patent Document 5).

Regarding the association between Sema3A and the cornea, there have been several reports in recent years. Non-Patent Document 6 shows that Sema3A is expressed in the corneal epithelium, the corneal parenchyma, and the corneal endothelium, respectively. It has also been reported that the performance of Sema3A is enhanced by corneal wounds or inflammation (Non-Patent Document 7 and Non-Patent Document 8).

However, until now, no report has clearly stated the role and function of Sema3A in the cornea.

Advanced technical literature Patent literature

Patent Document 1: International Publication No. 2006/092894

Patent Document 2: International Publication No. 2010/084970

Patent Document 3: International Publication No. 2013/012087

Patent Document 4: International Publication No. 2013/051722

Patent Document 5: International Publication No. 2011/096593

Non-patent literature

Non-Patent Document 1: MICHIHARU KIKUCHI ET AL.: 'Suihosei Kakumakusho ni Okeru Kakumaku Naihi Saibo no Zoshokuno' JOURNAL OF JAPANESE OPHTHALMOLOGICAL SOCIETY vol. 106, 2002, page 103, 47

Non-Patent Document 2: TADASHI SENOO ET AL.: 'Suihosei Kakumakusho no Kakumaku Naihi Saibo Zoshokuno' JOURNAL OF JAPANESE OPHTHALMOLOGICAL SOCIETY vol. 105, 2001, page 196, P65

Non-Patent Document 3: YAMAMOTO, N. ET AL.: 'Basic study of retinal stem/progenitor cell separation from mouse iris tissue' MED MOL MORPHOL vol. 43, no. 3, 2010, pages 139 - 144

Non-Patent Document 4: TOMA, J.G. ET AL.: 'Isolation of multipotent adult stem cells from the dermis of mammalian skin' NAT. CELL BIOL. vol. 3, no. 9, September 2001, pages 778-84.

Non-Patent Document 5: Kolodkin AL, Matthes DJ, Goodman CS. "The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules". Cell 75 (7): 1389-99 (1994)

Non-Patent Document 6: Morishige N et al. Exp Eye Res. Vol. 86 p835-843.

Non-Patent Document 7: Morishige N et al. Biochem Biophys Res Commun. 2010 Vol.395 p451-457.

Non-Patent Document 8: Ko JA et al. Biochem Biophys Res Commun. 2008 Vol. 377 p104-108.

Summary of invention

The present invention has been made in view of the above circumstances, and provides a prophylactic, inhibitory or therapeutic agent for preventing, inhibiting or treating corneal diseases or corneal damage, and providing corneal endothelial cells. Well cultured cell culture supplement, cell sheet containing corneal endothelial cells, and cell culture method of corneal endothelial cells.

The present inventors have found that the brain signal protein 3A (Sema3A), which is known as a guiding factor in the nervous system, has an excellent proliferation promoting effect on corneal endothelial cells, and the present invention has been completed. That is, the present invention is as follows.

(1) A prophylactic, inhibitory or therapeutic agent for corneal diseases or corneal damage, which comprises a polypeptide as an active ingredient, and the polypeptide comprises a group selected from the group consisting of (a) to (c) below. a region consisting of any of the polypeptides: (a) consisting of a continuous 70 or more amino acids in the amino acid sequence of SEQ ID NO: 4, and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 4; a contiguous 70 or more amino acids in the amino acid sequence of SEQ ID NO: 2, and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 2; (c) 80% of the polypeptide having (a) or (b) More sequence identity Peptide.

(2) The prophylactic, inhibitory or therapeutic agent for corneal diseases or corneal damage according to (1), wherein the corneal disease is a disorder accompanied by a decrease in the number of corneal endothelial cells or a dysfunction.

(3) A cell sheet characterized by culturing a corneal endothelial cell in the presence of a polypeptide comprising a region consisting of any one selected from the group consisting of (a) to (c) below; (a) a polypeptide consisting of 70 or more amino acids in the amino acid sequence shown in SEQ ID NO: 4, and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 4; (b) an amino group represented by SEQ ID NO: a polypeptide comprising 70 or more amino acids in the acid sequence and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 2; (c) a polypeptide having 80% or more sequence identity to the polypeptide of (a) or (b).

(4) A cell culture supplement for corneal endothelial cells, which comprises a polypeptide as an active ingredient, and the polypeptide comprises any polypeptide selected from the group consisting of (a) to (c) below. The region: (a) consists of a continuous 70 or more amino acids in the amino acid sequence shown in SEQ ID NO: 4, and contains the polypeptide of the region 166aa to 235aa in SEQ ID NO: 4; (b) is shown by SEQ ID NO: a continuous amino acid comprising 70 or more amino acids in the amino acid sequence and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 2; (c) a polypeptide having 80% or more sequence identity to the polypeptide of (a) or (b).

(5) A cell culture method comprising the step of culturing a corneal endothelial cell in the presence of a polypeptide comprising a region consisting of any one selected from the group consisting of (a) to (c) below; (a) a polypeptide consisting of 70 or more amino acids in the amino acid sequence shown in SEQ ID NO: 4, and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 4; (b) an amino group represented by SEQ ID NO: a polypeptide comprising 70 or more amino acids in the acid sequence and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 2; (c) a polypeptide having 80% or more sequence identity to the polypeptide of (a) or (b).

When the prophylactic, inhibitory or therapeutic agent for corneal diseases or corneal damage of the present invention is used, it is possible to prevent, inhibit or treat corneal diseases or corneal damage in vivo which cannot be performed in the past. Further, when the cell culture supplement of the present invention is used, corneal endothelial cells can be well cultured. Furthermore, according to the present invention, it is possible to provide a high-quality cell sheet containing corneal endothelial cells and a cell culture method suitable for culturing corneal endothelial cells.

Fig. 1A is a graph showing the results of primary culture of human corneal endothelium in culture experiment 1.

Figure 1B is a diagram showing the primary culture of human corneal endothelium in culture experiment 1. A picture of the results.

Fig. 2 is a graph showing the results of proliferation of human corneal endothelial cells in the culture experiment 2-1.

Fig. 3 is a graph showing the results of proliferation of human corneal endothelial cells in the culture experiment 2-2.

Fig. 4 is a photograph showing the corneal endothelial cell appearance after 12 hours of culture in the cell culture experiment 2-1.

Fig. 5 is a photograph showing the results of the Wound healing experiment in the culture experiment 3.

Fig. 6 is a photograph showing the results of the Wound healing experiment in the culture experiment 4.

Fig. 7 is a graph showing the Wound healing rate in the culture experiment 4.

Fig. 8 is a photograph showing the anterior ocular portion of a mouse which is a result of prevention and/or treatment of corneal oxidative stress.

Fig. 9 is a photograph showing the observation of corneal endothelial cells as a result of prevention and/or treatment of corneal oxidative stress.

Fig. 10 is a photograph showing the corneal tissue after BrdU immunostaining for the prevention and/or treatment of corneal oxidative stress.

Form for implementing the invention

Preferred embodiments of the invention are described below, but the invention is not limited by the examples. Additions, omissions, substitutions, and other changes to the structure are possible without departing from the scope of the present invention. .

≪Prevention, inhibition or treatment ≫

The prophylactic, inhibitory or therapeutic agent for corneal diseases or corneal damage of the present invention contains brain signal protein 3A as an active ingredient.

The brain signal protein 3A of the active ingredient may be a natural type or a variant. Specific examples of the natural brain signaling protein 3A or the brain signaling protein 3A variant include any region consisting of the following polypeptides (a) to (c), and have prevention of corneal diseases or corneal damage. , inhibiting or therapeutically active polypeptide: (a) consisting of more than 70 consecutive amino acids in the amino acid sequence of SEQ ID NO: 4, and containing 166aa~235aa in SEQ ID NO: 4 (meaning "166th amine (a) consisting of a continuous amino acid of 70 amino acids in the amino acid sequence of SEQ ID NO: 2, and comprising a region of 166aa to 235aa in SEQ ID NO: 2 a polypeptide; (c) a polypeptide having 80% or more sequence identity to the polypeptide of (a) or (b).

Here, the term "prophylaxis, inhibition or therapeutic activity of corneal diseases or corneal damage" means an activity for improving the symptoms of corneal lesions of corneal diseases or corneal damage, or an activity for preventing the deterioration of the symptoms. Whether a polypeptide has a prophylactic, inhibitory or therapeutic activity against corneal disease or corneal damage can be investigated by an in vitro experiment, for example, by administering a polypeptide to a corneal endothelial cell and promoting proliferation or migration of the cell. Proliferation or migration of corneal endothelial cells can be achieved by a Wound healing experiment as shown in <Cultural Experiment 3> and <Cultivation Experiment 4> in [Examples] to be described later. Alternatively, whether the polypeptide has this activity can also be investigated by in vivo experiments, for example by diagonal Membrane diseases or diseases in which corneal damage is occurring are investigated by administering the polypeptide via eye drops, subconjunctival administration, etc., and evaluating whether the symptoms of the lesion are improved. For the corneal disease or the corneal damage animal, for example, a model animal such as the ultraviolet keratitis-infected mouse described in the following examples can be used. This mouse causes the onset of ultraviolet keratitis by irradiating ultraviolet rays to the eyes. The evaluation of the prevention, inhibition or therapeutic activity of the corneal disease or corneal damage using the mouse can be specifically described by the following examples, for example, by administering eye drops and subconjunctival injection to the corneal lesion of the mouse. The polypeptide was administered for a fixed period of time and evaluated for improvement in the symptoms of the lesion. The improvement of the symptoms of the conjunctival lesions can be evaluated, for example, as scars, corneal transparency, cell morphology, and the like as described in the following examples. For example, when the average value of the keratitis symptom score of the keratitis-infected individual group administered with the polypeptide at a fixed period is significantly reduced as compared with the mean value of the keratitis smear score in the untreated ultraviolet keratitis group, or for example, When the average score of the polypeptide administration group is about 2/3 or less of the average value of the components of the untreated individual, and preferably is 1 or less, it is judged that the polypeptide has corneal disease or prevention of corneal damage, Inhibition or therapeutic activity.

Furthermore, in the present invention, the term "polypeptide" is called a plurality (more than 2). The amino acid is a molecule formed by a peptide bond, and is not only a high molecular weight molecule constituting a large amount of an amino acid, but also a low molecular weight molecule (oligopeptide) having a small amount of an amino acid, or a protein.

Further, in the present invention, the phrase "having an amino acid sequence" means an amine. The base acid residues are arranged in this order. Therefore, for example, "a polypeptide having an amino acid sequence represented by SEQ ID NO: 2" means having Met Gly Trp Leu... (middle)...Proamino acid sequence of Pro Arg Ser Val and a polypeptide of size 771 amino acid residue.

Brain signal protein with SEQ ID NO: 2 and 4 The sequences of the 3A proteins (human and chicken, respectively) were registered in GenBank under accession numbers NM006080 and U02528. The brain signaling protein is a protein that is identified as a molecule that directs elongation of axons. It is sometimes referred to as Collapsin because it was originally found to cause the growth cone at the anterior end of the neurite to collapse. Brain signaling proteins constitute a secretory or transmembrane family of proteins, and more than 30 members have been identified from vertebrates such as nematodes to human vertebrates. Brain signal protein 3A (Sema3A) is a member of the secreted protein of brain signaling proteins and is known as a protein molecule that acts as a repulsive axon guiding molecule. The secretory brain signaling protein of Sema3A or the like has three domains: a sema domain of a ~500 aa amino terminal, a C-2 immunoglobulin (Ig) domain, and a base terminal domain. It is known that the physiological activity-requiring region such as the inhibition of nerve elongation of Sema3A is in the sema domain, and particularly in the region of 70 amino acids located in No. 166-235 (Koppel et al., Neuron, Vol. .19, 531-537, 1997). The region of the 70 amino acids (hereinafter referred to as "70aa region") is a region of 166aa to 235aa in either of sequence number 2 and sequence number 4. Sema3A has been identified from various animals such as humans, chickens, mice, and rats. If the protein is compared as a whole, the similarity of these Sema3A is as high as about 85%; if only the above 70 amino acid regions are compared, it is as high as about 95%. Furthermore, in the chicken Sema3A having the sequence number 4 amino acid sequence, the sema domain is 61aa~567aa, Ig The domain is 592aa~655aa, and the base end is 726aa~772aa (see Feiner et al., Neuron, Vol. 19, 539-545, 1997). In the human Sema3A having the SEQ ID NO: 2 amino acid sequence, the sema domain is 61aa~567aa, the Ig domain is 591aa~654aa, and the base end domain is 726aa~771aa.

The above (a) polypeptide is composed of an amine having the sequence number 4 The polypeptide consisting of a region of at least a 70 aa region of the chicken Sema3A protein of the basal acid sequence is specifically a polypeptide consisting of the full length of SEQ ID NO: 4 or a fragment comprising a 70 aa region. One example of a polypeptide which is an active ingredient contained in the prophylactic, inhibitory or therapeutic agent of the present invention is a polypeptide comprising a region consisting of (a) a polypeptide and having a prophylactic, inhibitory or therapeutic activity against corneal disease or corneal damage. (The following is referred to as "active ingredient polypeptide a" for convenience). The active ingredient polypeptide a may be composed only of (a) a polypeptide as long as it has a prophylactic, inhibitory or therapeutic activity of corneal disease or corneal damage, or may be an amino acid added to one or both ends of the (a) polypeptide. Or a polypeptide. The length of the amino acid sequence constituting the active ingredient polypeptide a is not limited, and may be, for example, 70 to 10000 aa, preferably 70 to 3000 aa, and preferably 70 to 1500 aa.

Specific examples of the active ingredient polypeptide a composed of only the (a) polypeptide The aspect is not particularly limited, and examples thereof include a polypeptide consisting of the entire length of the amino acid sequence shown in SEQ ID NO: 4 (that is, a chicken Sema3A protein). The case where the chicken Sema3A protein has a prophylactic, inhibitory or therapeutic activity against corneal diseases or corneal damage is specifically described in the following examples. Further, even a fragment lacking a part of the N-terminus or the C-terminus of the chicken Sema3A protein is an active ingredient polypeptide a as long as it has a prophylactic, inhibitory or therapeutic activity against corneal disease or corneal damage and is included in the scope of the present invention.

Further, the (a) polypeptide is added with an active ingredient polypeptide a such as a polypeptide. Specific examples are not particularly limited, and examples thereof include a chimera protein in which a 70 aa region of SEQ ID NO: 4 is replaced with a secretory brain signal protein corresponding region other than Sema 3A, and (a) a polypeptide is attached. There are polypeptides such as various markers such as a His tag, a FLAG tag, and a myc tag. The secretory brain signaling proteins other than the above Sema3A may be, for example, brain-depleting protein 2, brain-depleting protein 3, brain-depleting protein 5, etc., but are not limited thereto. Further, amino acid sequences of brain-depleting protein 2, brain-depleting protein 3 and brain-depleting protein 5 are known as Feiner et al., Neuron, vol. 19, 539-545 (1997). As disclosed in the above paper by Koppel et al., the chimeric protein constructed by replacing the region containing at least the 70 aa region of Sema3A with the corresponding region of brain-damage protein 2 still retains the DRG withdrawal activity of Sema3A and exerts the same function as Sema3A. Physiological activity. In view of this, such a chimeric protein also has a prophylactic, inhibitory or therapeutic activity against corneal disease or corneal damage, and thus is also considered to be the active ingredient polypeptide a and is included in the scope of the present invention. Even Sema3A, which has deleted the Ig domain, also has DRG withdrawal activity (Koppel et al., 1997). However, this polypeptide can be used as a 70aa region in the Sema3A that is closer to the N-terminal side than the Ig domain. A polypeptide to which the base domain of Sema3A is added is also included in the active ingredient polypeptide a and is included in the scope of the present invention. Further, the specific examples described above are merely exemplified, and regardless of the amino acid or polypeptide to which the (a) polypeptide is added, as long as it has a prophylactic, inhibitory or therapeutic activity against corneal diseases or corneal damage, it is the active ingredient polypeptide a and is included in The scope of the invention.

The above (b) polypeptide is composed of an amine having the sequence number 2 a region of at least 70 aa of the human Sema3A protein of the acid sequence The polypeptide to be composed specifically includes a polypeptide consisting of the full length of SEQ ID NO: 2 or a fragment comprising a 70 aa region. Another example of a polypeptide which is an active ingredient contained in the prophylactic, inhibitory or therapeutic agent of the present invention is a region comprising (b) a polypeptide and having a prophylactic, inhibitory or therapeutic activity against corneal disease or corneal damage. The polypeptide (hereinafter referred to as "active ingredient polypeptide b" for convenience). The Sema3A (sequence number 4) of chicken is identical to human Sema3A (SEQ ID NO: 2), which is 86% in terms of the full length of the protein. If only the 70 aa region important for the physiological activity of Sema3A is compared, it is as high as 95%. As described in the following examples, chicken Sema3A can prevent, inhibit or treat corneal diseases or corneal damage in chickens other than chickens, so it is also possible to use chicken Sema3A to prevent, inhibit or treat corneas other than chickens and mice. Disease or corneal damage. Therefore, the active ingredient polypeptide b such as the human Sema3A protein, which is the same as the above-mentioned active ingredient polypeptide a such as the chicken Sema3A protein, can also prevent, inhibit or treat corneal diseases or corneal damage such as macrovesicular keratopathy and ultraviolet keratitis. The agent is useful. The active ingredient polypeptide b is also the same as the above-mentioned active ingredient polypeptide a, and may have only the (b) polypeptide, or may be one of the (b) polypeptides, as long as it has the prevention, inhibition or therapeutic activity of corneal disease or corneal damage. Or an amino acid or polypeptide is added to both ends. The length of the amino acid sequence constituting the active ingredient polypeptide b is infinitely limited, and 70 to 10000 aa is an example, and 70 to 3000 aa is preferred, and 70 to 1500 aa is preferred. Specific examples of the polypeptide are the same as the above-mentioned active ingredient polypeptide a. For example, the polypeptide consisting of the full length of SEQ ID NO: 2 (that is, the human Sema3A protein) is exemplified, and the corneal disease or corneal damage is prevented or inhibited. Or a therapeutically active fragment thereof; according to the above aspect, the invasive brain signal protein of Sema3A is embedded A protein; a polypeptide having various labels attached to the (b) polypeptide. These specific examples are also merely exemplified, and regardless of the aspect of the amino acid or polypeptide to which the (b) polypeptide is attached, as long as it has the prophylactic, inhibitory or therapeutic activity of corneal disease or corneal damage, the active ingredient polypeptide b is It is included in the scope of the invention.

The above (c) polypeptide is a few of the above (a) or (b) polypeptides (suitable a polypeptide having 1 to several amino acid residues substituted, deleted, and/or inserted, and having 80% or more, preferably 90% or more, preferably 95% or more, and more preferably 98% or more of the original sequence. The identity of the polypeptide. In general, it is well known to those skilled in the art that in proteins, even when a small number of amino acid residues in the amino acid sequence of the protein are substituted, deleted or inserted, sometimes The original protein has almost the same function. Therefore, since the region composed of the polypeptide (c) described above may have the physiological activity of the same Sema3A protein as the above 70aa region, it is the same as the above-mentioned effective component polypeptides a and b, and is composed of the above (c) polypeptide. A polypeptide having a prophylactic, inhibitory or therapeutic activity against corneal disease or corneal damage (hereinafter referred to as "active ingredient polypeptide c" for convenience) is also useful for preparing a prophylactic, inhibitory or therapeutic agent of the present invention.

Here, the "identity" of the amino acid sequence means that the response should be Comparing the amino acid residues of the two amino acid sequences as many as possible to juxtapose the two amino acid sequence, and dividing the number of identical amino acid residues by the number of total amino acid residues is expressed as a percentage. . At the time of the above juxtaposition, a space is appropriately inserted in one or both of the two sequences to be compared as needed. Such sequence parallelization can be performed using well-known programs such as BLAST, FASTA, and CLUSTAL W. In the case where a space is inserted, the above-mentioned peramino acid residue The cardinality is the number of residues in which one space is counted as one amino acid residue. The number of total amino acid residues calculated according to this is different between the two sequences being compared, and the identity (%) is the number of consistent amino acid residues divided by the number of total amino acid residues of the elderly sequence. To calculate. Furthermore, it is known that the 20 amino acids constituting the natural protein can be grouped with similar properties, such as a neutral amino acid having a low polarity side chain (Gly, Ile, Val, Leu, Ala, Met, Pro). Hydrophilic side chain neutral amino acid (Asn, Gln, Thr, Ser, Tyr Cys), acidic amino acid (Asp, Glu), basic amino acid (Arg, Lys, His), aromatic Amino acid (Phe, Tyr, Trp), if the substitution between these, the nature of the polypeptide does not change. Therefore, when the amino acid residue in the above (a) or (b) polypeptide is substituted, the polypeptide used as an active ingredient is used as a component of the cornea to maintain conjunctival disease or corneal damage by substituting between the groups. The possibility of preventing, inhibiting or treating activity will become higher.

The above-mentioned (c) polypeptide, specifically, has a sequence A polypeptide consisting of a polypeptide consisting of the full length of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 (i.e., Sema3A protein) having 80% or more sequence identity, or having 80% or more sequence identity with a Sema3A protein fragment having a 70 aa region. The polypeptide. Therefore, the active ingredient polypeptide c is a polypeptide composed only of the polypeptides, or an amino acid or a polypeptide is added to one or both ends of the (c) polypeptide, and has prevention and inhibition of corneal diseases or corneal damage. Or therapeutically active polypeptide. The specific examples are not particularly limited as long as the active ingredient polypeptides a and b, and may, for example, have a sequence identity of 80% or more with a polypeptide consisting of the full length of SEQ ID NO: 2 or 4 (that is, Sema3A protein). Polypeptide; with prevention, inhibition or treatment of corneal diseases or corneal damage More than 80% sequence identity of the active Sema3A protein fragment; chimeric protein fused to a region having more than 80% sequence identity to the region containing the 70aa region, and other regions of the secreted brain signaling protein other than Sema3A In the (c) polypeptide, various labeled polypeptides and the like are attached. These specific examples are also merely exemplified, and regardless of the aspect of the amino acid or polypeptide to which the (c) polypeptide is attached, as long as it has the prophylactic, inhibitory or therapeutic activity of corneal disease or corneal damage, the active ingredient polypeptide c is It is included in the scope of the invention.

The active ingredient polypeptide c is preferably a polypeptide having a higher identity in the 70 a region than the entire region of the (c) polypeptide. For example, when the active ingredient polypeptide c is a polypeptide having 80% identity with a polypeptide having the amino acid sequence of SEQ ID NO: 2, the identity in the 70 a region is preferably more than 80%, and particularly the identity in the 70 a region is More than 90%, and more than 95% are preferred, more preferably 98% or more, and most preferably the sequence in the 70 a region is the same polypeptide. Further, for example, when the active ingredient polypeptide c is a chimeric protein which is fused to a region other than the sema domain of a secretory brain signaling protein other than Sema3A in a region having 80% sequence identity with the sema domain of the human Sema3A protein, The 70aa region in the sema domain is preferably more than 80% identical, especially in the 70aa region, the homology is more than 90%, and more than 95% is better, more than 98% is better chimeric protein, and the best is The sequence in the 70aa region is the same chimeric protein.

In the above-mentioned active ingredient polypeptides a to c, it is particularly preferred to have a polypeptide having 80% or more identity with a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and preferably 90% or more, 95. More preferably, more preferably, a polypeptide having 98% or more identity, among which, as described above, It is preferred that the identity in the 70 a region is higher than that of the polypeptide as a whole. Most preferably, the polypeptide which is an active ingredient contained in the prophylactic, inhibitory or therapeutic agent of the present invention is a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

Generally, in a medicine consisting of a polypeptide, in order to improve the stability of a polypeptide in vivo, a polypeptide is added with a sugar chain or a polyethylene glycol (PEG) chain, and a D-amino acid is used as a constituent polypeptide. Techniques such as at least a portion of an amino acid, an additional Fc domain, and the like are well known and used. By adding a sugar chain or a PEG chain, using D-amino acid as at least a part of the amino acid constituting the polypeptide, etc., it will become difficult to be decomposed by the peptide in vivo, and the half-life of the polypeptide in vivo will be lengthen. The polypeptide to be used in the present invention may be modified for the purpose of stabilization in vivo, as long as it has a prophylactic, inhibitory or therapeutic activity for corneal diseases or corneal damage, and is within the scope of the present specification and the patent application. The term "polypeptide" is used in addition to the fact that it is not obvious in the context of the context. It is also used to refer to the inclusion of modifications for the stabilization of the body.

It is known to attach a sugar chain to a polypeptide, for example, Sato M, Furuike T, Sadamoto R, Fujitani N, Nakahara T, Niikura K, Monde K, Kondo H, Nishimura S., "Glycoinsulins: dendritic sialyloligosaccharide-displaying insulins showing a prolonged Blood-sugar-lowering activity.", J Am Chem Soc. 2004 Nov 3;126(43):14013-22 or Sato M,Sadamoto R,Niikura K,Monde K,Kondo H,Nishimura S,"Site-specific introduction Of sialic acid into insulin.", Angew Chem Int Ed Engl.2004 Mar 12;43(12): 1516-20, etc. The sugar chain may be bonded to the N-terminus, the C-terminus or the amino acid between the two, but in order not to inhibit the activity of the polypeptide, it is preferably bonded at the N-terminus or the C-terminus. Further, the number of added sugar chains is preferably one or two, and one is preferably one. The sugar chain is preferably from a monosaccharide to a 4 sugar, and preferably a 2 or 3 sugar. The sugar chain may be bonded to the free amine or carboxyl group of the polypeptide directly or, for example, through a spacer structure of a methylene chain having a carbon number of about 1 to 10.

It is also known to attach a PEG chain to a polypeptide, such as, for example, Ulbricht K, Bucha E, Poschel KA, Stein G, Wolf G, Nowak G., "The use of PEG-Hirudin in chronic hemodialysis monitored by the Ecarin Clotting Time: influence on Clotting of the extracorporeal system and hemostatic parameters.", Clin Nephrol. 2006 Mar; 65(3): 180-90. or Dharap SS, Wang Y, Chandna P, Khandare JJ, Qiu B, Gunaseelan S, Sinko PJ, Stein S , Farmanfarmaian A, Minko T., "Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.", Proc Natl Acad Sci USA. 2005 Sep 6; 102(36): 12962-7." The chain may be bonded to the N-terminus, the C-terminus or the amino acid between the two, usually one or two PEG chains may be bonded to the free amine group or carboxyl group on the polypeptide. The molecular weight of the PEG chain is not particularly limited. Usually 3,000 ~ 7000 can be used, and about 5000 is appropriate.

A method of forming at least a portion of the amino acid constituting the polypeptide into a D body is also known, for example, as Brenneman DE, Spong CY, Hauser JM, Abebe D, Pinhasov A, Golian T, Gozes I., "Protective " peptides that are orally active and mechanistically nonchiral.", J Pharmacol Exp Ther. 2004 Jun; 309(3): 1190-7 or Wilkemeyer MF, Chen SY, Menkari CE, Sulik KK, Charness ME., "Ethanol antagonist peptides: structural Specificity without stereospecificity.", J Pharmacol Exp Ther. 2004 Jun; 309(3): 1183-9. etc. Although a part of the amino acid constituting the polypeptide can be made into a D body, in order not to inhibit the polypeptide as much as possible. For the activity, it is preferred that all of the amino acids constituting the polypeptide be made into a D-amino acid.

A method of adding an Fc domain to a polypeptide is also known, and can be produced, for example, by using an Fc fusion protein expression vector such as commercially available pFUSEN-Fc (InvivoGen).

The brain signaling protein 3A used as an active ingredient in the present invention can be easily prepared by a usual method, for example, using a commercially available peptide synthesizer. It can also be easily modulated using well-known genetic engineering techniques. For example, RNA can be extracted from a tissue having a Sema3A gene, and the cDNA of the Sema3A gene can be modulated from the RNA by RT-PCR, and then the full length or a desired portion of the cDNA can be transferred to a expression vector and introduced into a host cell to obtain a target. The polypeptide. The extraction of RNA, RT-PCR, cDNA transfer to a vector, and introduction into a host cell can be carried out by a known method. Further, the vectors or host cells used are also well known and are sold in a variety of ways. The method for producing a chimeric protein is also known in the art, and chimeric proteins can be produced, for example, by the method described in the above-mentioned Koppel et al. Further, the above-described stabilization modification can be easily carried out by a known method described in each of the above documents.

Brain signal protein 3A, which is an active ingredient in the prophylactic, inhibitory or therapeutic agent of the present invention, is effective for the prevention, inhibition or treatment of corneal diseases or corneal damage.

The cause of the corneal disease is not particularly limited, and may be a corneal disease locally generated in the cornea or a corneal disease accompanied by a disease other than the cornea.

The corneal disease is preferably a corneal endothelium disorder with a decrease in corneal endothelial cells or dysfunction. Once the corneal endothelial cells are reduced or dysfunctional, the corneal endothelial cells rely on the control of the corneal parenchyma to become unsettled, not only the corneal endothelium, but also the cornea as a whole. Moreover, by improving the function of the corneal endothelial cells, it is possible to effectively prevent, inhibit or treat the disorder of the cornea as a whole.

Corneal diseases associated with decreased or insufficiency of corneal endothelial cells include bullous keratopathy, desquamation syndrome (PE), desquamation keratitis, pseudo desquamous corneal endothelium, congenital hereditary corneal endotrophic disorder with corneal herpes For the representative of congenital corneal endothelium degeneration, smear cornea, F. sinus corneal endothelium, F. sinus corneal endotrophic disorders, posterior pleomorphic corneal dystrophy, iris endothelium syndrome, etc., corneal endothelial degeneration, corneal endothelial cells Malnutrition, keratocontinitis due to viral infection (cytomegalovirus corneal endotheliitis, herpes simplex virus corneal endotheliitis), bacterial corneal infection, corneal fungal disease, amoebic keratitis, rejection after corneal transplantation, etc.

External factors that may be the cause of corneal endothelium disorders include corneal leukoplakia and corneal parenchyma. In addition, physical damage that may be the cause of corneal endothelium damage may be due to trauma, surgical injury, and blue Light eye hair is represented by a sharp increase in intraocular pressure, damage due to long-term wear of contact lenses, degeneration due to age, oxidative stress due to ultraviolet rays, and the like. Trauma also includes signs of trauma, infection, or chemical trauma due to flying into alkaline or acidic fluids during childbirth.

The surgical procedure for causing damage to the corneal endothelium may be intraocular surgery, laser treatment, etc., and is not limited thereto. Specific surgical examples can be exemplified by corneal transplantation, retinal ‧ vitreous surgery, cataract surgery, postoperative treatment for PE patients, glaucoma surgery (filtration surgery, peripheral erythratic incision, laser treatment), laser treatment for secondary cataract (YAG), laser iridescent incision (LI), laser angle formation (LGP), angle of adhesion separation (GSL), laser myopia surgery, tissue evapotranspiration using excimer lasers.

Due to the above-mentioned exemplified causes of corneal endothelium, the final disease reached is a macrolens keratopathy.

The prophylactic, inhibitory or therapeutic agent of the present invention is administered to a mammal, and may be, for example, a human, a dog, a cat, a rabbit, a hamster or the like. Furthermore, the use of brain signaling protein 3A of the same biological species as the patient for prevention, inhibition or treatment is considered to be high in prevention, inhibition or therapeutic effect, and therefore it is preferable from the viewpoint of safety of clinical application. . According to this, for example, when the prophylactic, inhibitory or therapeutic agent of the present invention is administered to a human, it is particularly preferable to use a prophylactic, inhibitory or therapeutic agent containing the above-mentioned active ingredient polypeptide b as an active ingredient.

The prophylactic, inhibitory or therapeutic agent of the present invention may be composed only of brain signal protein 3A, or may be a pharmacologically acceptable excipient, a stabilizer, a preservative, a buffer, and a solution suitable for each administration form. Subsist, emulsifier, Additives such as a diluent and a tonicity agent are suitably mixed to prepare a preparation. Formulation methods and additives which can be used are well known in the field of pharmaceutical preparations, and any of the methods and additives can be used.

The prophylactic, inhibitory or therapeutic agent of the present invention is administered to the corneal portion to be prevented or to the corneal lesion to be treated. The administration method may be a topical administration (instillation on the cornea, administration under the subconjunctival injection, etc.). The dosage form may be a contact lens type, an eye ointment, an injection, an eye drop, or the like.

In the above contact lens type, the transparent film having the contact lens-like spherical surface contains the brain signal protein 3A, and the brain signal protein 3A released from the membrane can be administered to the cornea.

The dosage can be appropriately selected depending on the symptoms, age, body weight, and administration method, and is not particularly limited. However, in terms of the amount of eye drops, the amount of the active ingredient in the target animal is usually about 6000 to 60000 U per day, and It is appropriate to be around 4000~40000U (refer to International Publication No. 2013/005603).

In the case of subconjunctival injection, the amount of the active ingredient in the target animal is usually about 500 to 5000 U on the 1st, about 500 to 5000 U, and can be divided into 1 to several administrations. It can be set, for example, that the subconjunctival injection amount is about 0.001 to 10 ng, about 0.05 to 5 ng, and is administered to each eye at a concentration of, for example, 10 ng/ml, 100 ng/ml, 300 ng/ml, and 1000 ng/ml. μ l, and should be administered regularly, depending on the degree of symptom improvement, between 1 day and several months, once or several times a day, or once or several times a few days apart.

In the case of eye drop administration or subconjunctival injection, it can be set, for example, that the amount of eye drop is about 1 to 3000 ng, about 20 to 900 ng, about 50 to 900 ng, about 50 to 500 ng, and about 80 to 500 ng. And, for example, at a concentration of 10 ng/ml, 100 ng/ml, 300 ng/ml, 1000 ng/ml, 2 μl is administered to each eye, and it is preferably between several days and several months, depending on the degree of symptom improvement. Regular injections are made once or several times a day, or once or several times a few days apart.

The prophylactic, inhibitory or therapeutic agent of the present invention may be used alone or in combination with other prophylactic, inhibitory or therapeutic agents. Other preventive, inhibiting or therapeutic agents include anti-biomass, anti-inflammatory agents, antiviral agents, cell proliferation promoters, wound therapeutic agents, extracellular matrix components, vitamins and the like.

For example, the above-mentioned ophthalmic ointment may be exemplified as Sema3A/hyaluronic acid ointment, Sema3A/Neo-Medrol EE ointment, Sema3A/eye ‧ earring Rinderon-A ointment, Sema3A/flavin adenine Flavitan eye ointment 0.1%. Sema3A/hyaluronic acid ointment can be used as a hyaluronic ophthalmic surgical preparation. The above Neo-Medrol EE ointment is a blending agent of anti-biomass and synthetic para-adrenocortical agent, and can be expected to have a high anti-inflammatory effect by being used in combination with Sema3A. This is the same for the above Lin Delong A ointment. In addition, the components of the Neo-Medrol EE ointment may be used to reduce the components of the adrenal cortexin.

The prophylactic, inhibitory or therapeutic agent of the present invention may be used in combination with ascorbic acid, ascorbic acid derivative, or a salt or hydrate thereof, a Rock inhibitor or a substance thereof. The ascorbic acid derivative is preferably a phosphate derivative of ascorbic acid such as ascorbic acid-2-phosphate.

It is known that the amount of brain signaling protein 3A present in the body is stabilized by feedback. Therefore, brain signaling protein 3A will have a stable effect in vivo. Moreover, since the administration of brain signaling protein 3A does not cause significant morphological changes in corneal endothelial cells, it can activate the dividing ability, so The side effects of the prophylactic, inhibitory or therapeutic agents of the invention are low.

In one embodiment, the present invention provides a method for preventing, inhibiting or treating corneal diseases or corneal damage, comprising the step of administering cerebral signal protein 3A to a mammal.

In one embodiment, the invention provides a brain signaling protein 3A for use in preventing, inhibiting or treating corneal disease or corneal damage.

In one embodiment, the present invention provides a use of brain signaling protein 3A, which is a prophylactic, inhibitory or therapeutic agent for the manufacture of corneal diseases or corneal damage.

≪ cell culture supplement ≫

The cell culture supplement for corneal endothelial cells of the present invention contains brain signal protein 3A as an active ingredient. The brain signal protein 3A can be used in the same manner as shown in the above-mentioned sputum prevention, inhibition or therapeutic agent ≫. In addition to the primary corneal endothelial cells and corneal endothelial stem cells that have been isolated from the cornea, corneal endothelial cells also mean the use of subcultured corneal endothelial cells that have been subcultured, have been induced from corneal endothelial progenitor cells or any other cells. Differentiated corneal endothelial cells, corneal endothelial stem cells, corneal endothelial-like cells exhibiting the same properties as corneal endothelial progenitor cells or corneal endothelial cells. Whether or not the cell is the above-mentioned corneal endothelial cell can be discriminated by investigating whether or not there is at least one of various prominent phenotypic characteristics in the above-mentioned corneal endothelial cells.

Further, the cell culture supplement of the present invention may further contain nutrients, growth factors, cell proliferation factors, differentiation inducing factors, antibacterial agents, antifungal agents, etc., in addition to the above-mentioned brain signal protein 3A. As a component of the cell culture supplement, it may further contain the adrenal gland An anti-inflammatory component such as a corticosteroid, an ascorbic acid, an ascorbic acid derivative or a salt or a hydrate thereof, a Rock inhibitor or a related substance thereof. The ascorbic acid derivative is preferably a phosphate derivative of ascorbic acid such as ascorbic acid-2-phosphate.

The cell culture supplement of the present invention may be added to a medium in which a proper amount of corneal endothelial cells are cultured, or the cell culture supplement may contain a medium component and the cell culture supplement itself may be used as a medium. The concentration of the brain signal protein 3A contained in the cell culture supplement or the medium may be appropriately selected depending on the type, state, amount, and the like of the corneal endothelial cells to be used, and each medium should be set to 1 to 3000 ng/mL, and It is preferably 20 to 900 ng/mL, preferably 50 to 900 ng/mL, more preferably 50 to 500 ng/mL, and particularly preferably 80 to 500 ng/mL.

The culture medium is not particularly limited as long as it is a culture medium capable of culturing corneal endothelial cells, and examples thereof include DMEM medium, BME medium, IMDM medium, and mixed mediums thereof.

The medium component may be a component contained in a normal animal cell culture medium, a nutrient component such as glucose, sodium chloride, vitamins/minerals, or an amino acid; a growth factor, a cell proliferation factor, a differentiation inducing factor, an antibacterial agent, and an antibiotic. Fungal agents, etc.

According to the cell culture supplement of the present invention, corneal endothelial cells close to the state of the original corneal endothelial cells can be cultured with high productivity.

Cell culture method, cell pellet

The cell culture method of the present invention has a step of culturing corneal endothelial cells in the presence of brain signaling protein 3A. Brain signaling protein 3A can be used and The above-mentioned sputum prevention, inhibition or therapeutic agent is the same as shown. The corneal endothelial cells can be used in the same manner as shown in the above-mentioned sputum cell culture supplement ≫. In the presence of cerebral signal protein 3A, a medium containing brain signal protein 3A may be mentioned, and the concentration of brain signal protein 3A contained in the medium may be appropriately selected according to the type, state, amount, etc. of the corneal endothelial cells to be cultured, and each medium should be appropriately selected. It is set to 1 to 3000 ng/mL, preferably 20 to 900 ng/mL, preferably 50 to 900 ng/mL, more preferably 50 to 500 ng/mL, and particularly preferably 80 to 500 ng/mL.

In addition to the brain signal protein 3A, the nutrient component, the growth factor, the cell proliferation factor, the differentiation inducing factor, the antibacterial agent, the antifungal agent, and the like are conventionally known for use in culturing corneal endothelial cells. The ingredients. Further, it may be used in combination with an anti-inflammatory component such as an adrenal corticosteroid, ascorbic acid, an ascorbic acid derivative, or a salt or a hydrate thereof, a Rock inhibitor or a related substance thereof. The ascorbic acid derivative is preferably a phosphate derivative of ascorbic acid such as ascorbic acid-2-phosphate.

According to the cell culture method of the present invention, corneal endothelial cells close to the state of the original corneal endothelial cells can be cultured with high productivity.

In one embodiment, the present invention provides a corneal endothelial cell obtained by culturing in the presence of brain signaling protein 3A.

The cell sheet of the present invention is obtained by culturing corneal endothelial cells in the presence of brain signaling protein 3A, and preferably contains corneal endothelial cells. The cell sheet of the present invention can be obtained by the cell culture method of the present invention. Therefore, in one embodiment, the present invention provides a method of manufacturing a cell sheet, Contains a step of culturing corneal endothelial cells in the presence of brain signaling protein 3A. The cell sheet of the present invention may be composed only of cells, and may be further in a state of containing a scaffold such as a collagen gel or a graft carrier. The cell sheet containing corneal endothelial cells means that the ratio of corneal endothelial cells in the cells constituting the cell sheet is preferably 10% or more, 30% or more, 50% or more, and preferably 80% or more, and 90% by volume. The above is better.

The cell sheet of the present invention is a high-quality cell which is suitable for use as a corneal tissue for transplantation, and is a highly useful cell sheet which has never been used.

The culture of the corneal endothelial cells contained in the cell sheet can be carried out, for example, at 37 ° C under 5% CO ₂ . In the culture of the corneal endothelial cells on the culture substrate, the concentration of the corneal endothelial cells in the culture solution can be set, for example, in the range of 1 × 10 ³ to 6 × 10 ³ /ml. The corneal endothelial cells cultured on the culture substrate are formed into cell sheets by mutual adhesion. The cell sheet is peeled off from the aforementioned culture medium and can be used for transplantation surgery. Alternatively, a living tissue-transplanted cell culture substrate may be used as a graft carrier in the culture medium, and corneal endothelial cells may be cultured thereon and used for transplantation surgery. The culture period can be appropriately determined according to the degree of cell proliferation, and can be set to about 1 to 3 weeks. The transplant can be performed by the same method as the conventional corneal transplantation from donation.

The culture medium is not particularly limited as long as it is used for cell culture, and examples thereof include a polymer material derived from natural materials such as fibronectin, collagen, gelatin, hyaluronic acid, alginic acid, and laminin; Synthetic polymer materials such as polyester, polyethylene, and polypropylene; biodegradable polymer materials such as polylactic acid and polyglycolic acid; inorganic materials such as glass, quartz, and hydroxyapatite; and amniotic membrane.

The density of the corneal endothelial cells in the cell sheet is not particularly limited as long as it can form a cell sheet, and the density of the corneal endothelial cells is preferably in the range of 1.0 × 10 ⁴ to 5.0 × 10 ⁶ cells/cm ² and is 1.0 × 10 ^{A range of 5} to 1.0 × 10 ⁶ cells/cm ² is preferred. The corneal endothelial cells are preferably in a density that works well in the eye of the transplant.

The cell sheet can be suitably used for corneal transplantation. The thickness of the cell sheet is not particularly limited, but it is preferably a thickness that can be used as a graft. Therefore, the thickness of the cell sheet is preferably about the same as the thickness of the cornea, and is preferably the same as the thickness of the layer of the corneal endothelium. More specifically, the thickness of the cell sheet is preferably in the range of 10 to 200 μm, preferably in the range of 10 to 100 μm, more preferably in the range of 15 to 50 μm.

[Examples]

The invention will be further illustrated in the following examples, but the invention is not limited by the following examples.

≪in vitro≫

Sema3A used in the following in vitro experiments was Recombinant Human Semaphorin 3A/Fc Chimera (manufactured by R & D Systems).

<Cultivation Experiment 1>

Culture of human corneal endothelial cells in the presence of Sema3A was performed.

(Sema3A group)

On the bottom of the 6 cm hole, 150 (7.5/cm ² ) corneal endothelial primary cells were seeded into the bottom of the wells of SMEM 3A-containing DMEM Low Glucose medium, and cultured in a 37 ° C incubator for 25 days. The medium was prepared to contain Sema3A at a concentration of 100 ng/mL. The primary cells of the corneal endothelium were obtained from Sight Life or Rocky Mountain Lions Eye Bank.

(control group)

Human corneal endothelial cells were cultured for the same 25 days as the Sema3A group except that the medium containing no Sema3A was used.

After 25 days of culture, the number of cells was counted using a blood cell calculation disk. The results are shown in Figure 1. The number of cells in the Sema3A group after 25 days of culture was more significantly increased than the number of cells in the control group. From this, it can be seen that Sema3A has an effect of promoting proliferation of corneal endothelial cells. The rate of cell proliferation is also in the range of normal cell proliferation rates, thus showing a high degree of safety.

<Cultivation Experiment 2-1>

The effects of corneal endothelial cell culture supplements and Sema3A on human corneal endothelial cells were compared.

(Sema3A-10 group)

The corneal endothelial cells were placed in the bottom of a 6-well plate containing 1 ng of DMEM Low Glucose medium containing 1 ng of Sema3A, and the corneal endothelial cells were subcultured 3 to 4 times at a cell density of 4000/cm ² and cultured in a 37 ° C incubator.

(Sema3A-100 group)

The culture was carried out in the same manner as described above (Sema 3A-10 group) except that the medium containing 100 ng of Sema3A per 1 mL was used.

(Sema3A-1000 group)

The culture was carried out in the same manner as described above (Sema 3A-10 group) except that the medium containing 1000 ng of Sema3A per 1 mL was used.

(ROCK inhibitor group)

In addition to using 10 μM ROCK inhibitor (Y-27632) in each medium The cells were cultured in the same manner as the above (Sema 3A-10 group) except that the cell culture adjuvant was used instead of Sema3A.

(ASC group)

The culture was carried out in the same manner as described above (Sema 3A-10 group) except that 0.3 mM of ascorbic acid-2-phosphate was used as a cell culture supplement in place of Sema3A in each medium.

(control group)

The culture was carried out in the same manner as described above (Sema 3A-10 group) except that the medium containing no Sema3A and other cell culture supplements was used.

The cell proliferation promoting effect of human corneal endothelial cells was compared with each of the culture adjuvants and Sema3A. For the control group, the ASC group, the Sema3A-10 group, the Sema3A-100 group, the Sema3A-1000 group, and the Rock inhibitor group, the cells from the start of the culture 3 days after the start of culture and 6 days after the start of the culture were measured using a blood cell calculation disk. number.

The results are shown in Figure 2. In the Rock inhibitor group and the Sema3A-10 group, the proliferation rate of the corneal endothelial cells was equal to that of the control group at any time after 3 days from the start of the culture and 6 days after the start of the culture. In contrast, in the ascorbate-2-phosphate group, the Sema3A-100 group, and the Sema3A-1000 group, the proliferation rate of corneal endothelial cells was significantly higher than that of the control group.

<Cultivation Experiment 2-2>

The cell proliferation promoting effect of human corneal endothelial cells was compared with the corneal endothelial cell culture supplement used in the past and Sema3A.

(Sema3A-100 group)

The corneal endothelial cells were placed in the bottom of a 12-well dish containing 1 ng of DMEM Low Glucose medium containing 100 ng of Sema3A, and the corneal endothelial cells were subcultured 3 to 4 times at a cell density of 4000/cm ² and cultured in a 37 ° C incubator.

(Sema3A-300 group)

The culture was carried out in the same manner as described above (Sema3A 100 group) except that the medium containing 300 ng of Sema3A per 1 mL was used.

(Sema3A-1000 group)

The culture was carried out in the same manner as described above (Sema 3A-100 group) except that the medium containing 1000 ng of Sema3A per 1 mL was used.

(ASC group)

The culture was carried out in the same manner as described above (Sema 3A-100 group) except that 0.3 mM ascorbic acid-2-phosphate was used as a cell culture supplement in place of Sema3A in each medium.

(Sema3A-100+ASC group)

The culture was carried out in the same manner as described above (Sema 3A-100 group) except that a medium containing 100 ng of Sema3A and 0.3 mM ascorbic acid-2-phosphate per 1 mL was used.

(Sema3A-1000+ASC group)

The culture was carried out in the same manner as described above (Sema 3A-100 group) except that the medium containing 1000 ng of Sema3A and 0.3 mM ascorbic acid-2-phosphate per 1 mL was used.

(control group)

The culture was carried out in the same manner as described above (Sema 3A-100 group) except that the medium containing no Sema3A and other cell culture supplements was used.

For the Sema3A-100 group, the Sema3A-300 group, the Sema3A-1000 group, the ASC group, the Sema3A-100+ASC group, the Sema3A-1000+ASC group, and the control group, the blood cell calculation disk was used to measure from the start of the culture for 3 days (12well disk). The number of cells at the time of the).

The results are shown in Figure 3. In the case of using Sema3A, ascorbic acid-2-phosphate, and Sema3A and ascorbic acid-2-phosphate, the number of cells was significantly increased compared with the control group.

<cell status confirmation>

Next, the appearance of corneal endothelial cells after 12 hours of culture in the control group of the <Cell Culture Experiment 2-1>, the ASC group, the Sema3A-100 group, and the Rock inhibitor is shown in Fig. 4 .

In the Rock inhibitor group and the ASC group, the morphology and size of corneal endothelial cells were irregular, and many cells forming pseudopods were also observed. Moreover, the formation state of the tight junction and the state of the cell subsequent state are slightly poor.

On the other hand, in the Sema3A-100 group, the morphology and size of the corneal endothelial cells were stone-like and regular, and the cells forming the pseudopod were few and the cell morphology was also good. Moreover, the state in which the cells are in contact with each other is also good.

Compared with the group of corneal endothelial cell culture supplements used in the past, the morphology of the corneal endothelial cells of the Sema3A-100 group showed the morphology closest to the state of the original corneal endothelial cells. Further, since the formation of the tight junction is good, it is presumed to be excellent in the shielding function and the pump function of the important functions of the corneal endothelial cells.

It is clear from the above that by administering ascorbic acid-2-phosphate The proliferation rate of human corneal endothelial cells is increased, and the cell state produced is superior in the case where Sema3A has been administered. By using Sema3A, human corneal endothelial cells can be cultured to a state close to the original corneal endothelial cells. It has also been shown that high-quality human corneal endothelial cell cell sheets can be obtained with high productivity by culturing human corneal endothelial cells in the presence of Sema3A.

<Cultivation Experiment 3>

A Wound healing experiment of human corneal endothelial cells in the presence of Sema3A was performed.

(Sema3A group)

The corneal endothelial cells after 3 to 4 passages were seeded at a cell density of 4000/cm ^{2 to} the bottom of the wells of DMEM Low Glucose medium containing Sema3A, and cultured in a 37 ° C incubator for 5 days. The medium was prepared to contain Sema3A at a concentration of 100 ng/ml per 1 mL. The cells are removed from the bottom surface of the hole to expose the bottom surface (Fig. 5 (Wound healing day 0, Ctrl)). After that, the culture was continued, and the state of the cells three days after the bottom surface was exposed was observed.

(control group)

The culture was carried out in the same manner as described above (Sema 3A group) except that the medium containing no Sema3A and other cell culture supplements was used.

The appearance of human corneal endothelial cells in the control group is shown in Figure 5 (Wound healing day 3, Ctrl). As shown in Fig. 5 (Wound healing day 3, Ctrl), in a region where the bottom surface is exposed in a circular shape, the cells are sparsely dot-shaped, and particularly in the central portion of the region, the bottom surface is maintained in an exposed state.

The appearance of human corneal endothelial cells in the Sema3A group is shown in the figure. 5 (Wound healing day 3, Sema3A). As shown in Fig. 5 (Wound healing day 3, Sema 3A), the entire area in which the bottom surface is exposed to a circle is covered by human corneal endothelial cells as a whole.

It is thus apparent that the proliferation and migration of human corneal endothelial cells can be promoted by using Sema3A.

<Cultivation Experiment 4>

Further, a Wound healing comparison experiment using a corneal endothelial cell culture supplement and a human corneal endothelial cell of Sema3A was carried out.

(Sema3A group)

The corneal endothelial cells after 3 to 4 passages were seeded at a cell density of 4000/cm ^{2 to} the bottom of a well placed in a DMEM Low Glucose medium containing Sema3A, and cultured in a 37 ° C incubator for 24 hours. The medium was prepared to contain Sema3A at a concentration of 100 ng/ml per 1 mL. After the culture, the cells were removed from the bottom of the well to remove the cells, and the bottom surface was exposed (Wound healing). Thereafter, the culture was continued, and the state of the cells 24 hours after the bottom surface was exposed was observed.

(ROCK inhibitor group)

The culture was carried out in the same manner as described above (Sema 3A group) except that 10 μM ROCK inhibitor (Y-27632) was used as a cell culture supplement in place of Sema3A in each medium.

(ASC group)

The culture was carried out in the same manner as described above (Sema 3A group) except that 0.3 mM of ascorbic acid-2-phosphate was used as a cell culture supplement in place of Sema3A in each medium.

(Sema3A+ASC group)

The culture was carried out in the same manner as described above (Sema 3A group) except that the medium containing 100 ng of Sema3A and 0.3 mM ascorbic acid-2-phosphate per 1 mL was used. (control group)

The culture was carried out in the same manner as described above (Sema 3A group) except that a medium containing no Sema3A and other cell culture adjuvants was used.

The appearance of human corneal endothelial cells in the control group is shown in Figure 6 (Ctrl). The cell density is low in the region where the bottom surface is exposed to the strip shape, and particularly in the central portion of the region, the bottom surface remains exposed.

The appearance of human corneal endothelial cells in the ROCK inhibitor group is shown in Figure 6 (ROCK inhibitor). The cell density is low in the region where the bottom surface is exposed to the strip shape, and particularly in the central portion of the region, the bottom surface remains exposed.

The appearance of human corneal endothelial cells in the ASC group is shown in Figure 6 (ASC). The entire area where the bottom surface is exposed in a strip shape is covered by human corneal endothelial cells. However, irregular cell morphology and size can be observed.

The appearance of human corneal endothelial cells in the Sema3A group is shown in Figure 6 (Sema3A). The entire area where the bottom surface is exposed in a strip shape is covered by human corneal endothelial cells. And the shape and size of the cells are uniform.

The appearance of human corneal endothelial cells in the Sema3A+ASC group is shown in Figure 6 (Sema3A+ASC). The entire area where the bottom surface is exposed in a strip shape is covered by human corneal endothelial cells. And the shape and size of the cells are uniform.

The Wound healing speed of each group is shown in Fig. 7. The vertical axis of the graph of Fig. 7 is the exposed area of the bottom surface in the region where the strip is exposed. The initial exposed area is shown as 100%, and the entire area is covered by cells. It is 0%.

As is clear from the graph of Fig. 7, the proliferation and migration of human corneal endothelial cells can be promoted by using Sema3A, ascorbic acid-2-phosphate, and Sema3A and ascorbyl-2-phosphate. Sema3A has the best Wound healing rate when compared with the previous cultured cell supplement and Sema3A.

≪in vivo≫

Sema3A used in the following in vivo experiments was a Recombinant Mouse Semaphorin 3A/Fc Chimera (manufactured by R & D Systems).

<Protection and / or treatment of ultraviolet keratitis> (Sema3A vote group)

Experiments were carried out using C57BL6 mice (white, 4 week old male, each group N=8) as the target animals. In order to induce ultraviolet corneal inflammation, the eyes of the mice were irradiated with UV-A 3.9 J, once a day / 2 days (5 times, 0, 2, 4, 6, and 8 days) after the anesthesia. After irradiation of each UV-A, 2 μl of PBS containing Sema3A (100 ng/ml or 300 ng/ml) was injected subconjunctivally. On the 10th day, the front eye photograph was taken and the eyeball was taken out.

(control group)

Except for the subconjunctival injection of PBS containing no Sema3A, the same experiment as the aforementioned Sema3A administration group was carried out.

<Confirm the state of the cornea>

A photograph of the anterior eye of the mouse on the 10th day after the start of the experiment is shown in Fig. 8. The formation of neovascularization was confirmed in the control group and the scar area was >100 ng/ml. In contrast, in the Sema3A administration group, the formation of neovascularization and scarring were not confirmed, and the corneal transparency was also good.

The corneal tissue was stained using the Whole mount method.

First, alizarin red staining was performed to observe corneal endothelial cells. The photo is shown in Figure 9. In the control group, the formation of new blood vessels and cell shedding were confirmed. In the Sema3A administration group, the state of cell alignment was good. Little neovascularization and cell shedding were observed.

Next, cell division in tissues was examined by immunostaining with bromodeoxyuracil (BrdU). The staining results are shown in Figure 10. When the portion which is caused to cause inflammation by UV irradiation (the inner portion of the broken line in Fig. 10) was compared, a clear fluorescent signal was observed in the corneal tissue of the Sema3A-administered group as compared with the control group, and cell proliferation was confirmed.

Similarly, using DAPI nuclear staining, a clear fluorescent signal was observed in the corneal tissue of the Sema3A-administered group compared with the control group, confirming the repair of corneal endothelium.

From the above results, it can be inferred that Sema3A promotes cell proliferation in the cornea and repairs corneal damage due to ultraviolet rays. It has also been shown that the prevention and/or treatment of ultraviolet keratitis can be carried out by administering Sema3A in vivo.

[Industrial availability]

The prophylactic, inhibitory or therapeutic agent cell sheet, the cell culture supplement, and the cell culture method of the present invention are widely used in the fields of medicine, pharmacy, biology, and bioengineering, including regenerative medicine.

<sequence number 1>

SEQ ID NO: 1 shows the DNA sequence of the human brain signaling protein 3A gene.

<sequence number 2>

SEQ ID NO: 2 shows the amino acid sequence of human brain signaling protein 3A.

<sequence number 3>

SEQ ID NO: 3 shows the DNA sequence of chicken brain signaling protein 3A gene.

<sequence number 4>

SEQ ID NO: 4 shows the amino acid sequence of chicken brain signaling protein 3A.

<110> University of Tokyo

<120> Prophylactic, inhibitory or therapeutic agents for corneal diseases or corneal damage, cell sheets, cell culture supplements, and cell culture methods

<130> OSP57768

<150> JP 2014-041017

<151> 2014-03-03

<160> 4

<170> PatentIn version 3.1

<210> 1

<211> 5672

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (316)..(2631)

<223>

<400> 1

<210> 2

<211> 771

<212> PRT

<213> Homo sapiens

<400> 2

<210> 3

<211> 3263

<212> DNA

<213> Gallus gallus

<220>

<221> CDS

<222> (168)..(2486)

<223>

<400> 3

<210> 4

<211> 772

<212> PRT

<213> Gallus gallus

<400> 4

Claims (5)

A prophylactic, inhibitory or therapeutic agent for corneal diseases or corneal damage, characterized in that a polypeptide is contained as an active ingredient, and the polypeptide contains any one selected from the group consisting of (a) to (c) below. A region consisting of a peptide: (a) consisting of a continuous 70 or more amino acids in the amino acid sequence shown in SEQ ID NO: 4, and containing a polypeptide of 166aa to 235aa in SEQ ID NO: 4; (b) by SEQ ID NO: 2 a continuous amino acid of 70 or more in the amino acid sequence shown, and comprising a polypeptide of the region 166aa to 235aa in SEQ ID NO: 2; (c) having the same sequence as 80% or more of the polypeptide of (a) or (b) Sexual polypeptide. The prophylactic, inhibitory or therapeutic agent for corneal diseases or corneal damage according to claim 1, wherein the corneal disease is a disorder accompanied by a decrease in the number of corneal endothelial cells or a dysfunction. A cell sheet characterized by culturing a corneal endothelial cell in the presence of a polypeptide comprising a region consisting of any one selected from the group consisting of (a) to (c) below: (a a polypeptide consisting of more than 70 consecutive amino acids in the amino acid sequence of SEQ ID NO: 4, and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 4; (b) from the amino acid sequence shown in SEQ ID NO: It is composed of more than 70 amino acids in succession and contains the region of 166aa~235aa in SEQ ID NO:2 a polypeptide; (c) a polypeptide having greater than 80% sequence identity to the polypeptide of (a) or (b). A cell culture supplement for corneal endothelial cells, which comprises a polypeptide as an active ingredient, and the polypeptide comprises a region consisting of any one selected from the group consisting of (a) to (c) below: (a) a polypeptide consisting of 70 or more amino acids in the amino acid sequence of SEQ ID NO: 4, and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 4; (b) an amino acid represented by SEQ ID NO: a polypeptide consisting of 70 or more amino acids in the sequence and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 2; (c) a polypeptide having 80% or more sequence identity to the polypeptide of (a) or (b). A cell culture method comprising the step of culturing a corneal endothelial cell in the presence of a polypeptide comprising a region consisting of any one selected from the group consisting of (a) to (c): (a a polypeptide consisting of more than 70 consecutive amino acids in the amino acid sequence of SEQ ID NO: 4, and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 4; (b) from the amino acid sequence shown in SEQ ID NO: a polypeptide consisting of 70 or more consecutive amino acids and comprising a polypeptide of 166aa to 235aa in SEQ ID NO: 2; (c) having more than 80% sequence identity with the polypeptide of (a) or (b) Peptide.