JPH06145065A - Immobilized hepatocyte proliferation agent - Google Patents

Abstract

(57) [Summary] [Structure] Hepatocyte, dextran sulphate, heparan sulphate, chondroitin sulphate, etc. Glycan or glucan or a derivative thereof is bound to hepatocyte growth factor to immobilize hepatocyte growth factor To get [Effects] According to the present invention, hepatocyte growth factor can be immobilized by using an appropriate glycosaminoglycan, glucan or a derivative thereof while maintaining the activity. As a result, this solid phase can be immobilized. In growth factor
Now available in vivo or in vitro. By devising a solid phase carrier for hepatocyte growth factor based on the present invention, it can be expected to be widely used in the medical field, and further, a sustained release effect / accumulation effect can be expected upon administration to a living body.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hepatocyte growth factor, and more specifically, by binding hepatocyte growth factor to glycosaminoglycan, glucan or a derivative thereof, the growth factor is immobilized on a solid phase. The present invention relates to a modified hepatocyte proliferation agent.

[0002]

2. Description of the Related Art In recent years,
A variety of cell growth factors have been cloned, among which a group of growth factors with a strong affinity for heparin has been found. These are collectively called heparin-binding growth factors, and among them are fibroblast growth factors (hereinafter FG).
Abbreviated as F), keratinocyte growth factor (KGF), pleiotrophin, granulocyte / macrophage colony formation stimulating factor (GM-CSF), interleukins 3 and 7, vascular endothelial cell growth factor (VEGF), etc. , Various known or unidentified growth factors (Experimental Medicine, 9 (1
4), 1772-176 (1991)). These factors are known to bind to glycosaminoglycans represented by heparin and heparan sulfate, and among them, F
It was confirmed that GF is stored on the cell surface or extracellular matrix by binding to heparan sulfate proteoglycan, and that the biological activity of FGF can be regulated by heparin (Cell, 64, 841-848 ( 1
991)).

Okuhara et al., A human-derived protein factor capable of taking out hepatocytes from a living body and promoting their growth in vitro, that is, human hepatocyte growth factor (hHG).
F) was found in plasma of patients with fulminant hepatitis (JP-A-63-2).
2526), and Kitamura et al., The amino acid sequence of hHGF protein and the gene encoding it (cDN
A) Sequence (JP-A-3-72883), a method for producing hHGF protein using this cDNA, and a transformant have been invented (JP-A-3-285693). The hHGF protein produced by such an invention is recognized to have a function of promoting the proliferation of hepatocytes in vitro. Further, it has been found that not only hHGF but also HGF protein is one of the above-mentioned heparin-binding growth factors having a strong affinity for heparin.

On the other hand, it is generally considered that not only a humoral factor but also an extracellular matrix (ECM) adjacent to a cell plays an important role in regulating cell growth and motility. E
The main constituents of CM are fibrous proteins such as collagen and elastin, adhesive proteins such as fibronectin and laminin, and complex carbohydrates such as proteoglycans having glycosaminoglycan sugar chains. Of these, it is not easy to prepare proteoglycans from biomaterials, and analysis of the physiological significance of glycosaminoglycans in ECM and the interaction with growth factors has been delayed.

[0005] Proteoglycan is a general term for covalent compounds of glycosaminoglycans and proteins. In recent years, various glycosaminoglycans have been covalently bonded to other proteins or phospholipids instead of natural core proteins. A model compound has been prepared (neoproteoglycan; J. Biol. Chem., 264 (14), 8).
012-8018 (1989), JP-A-4-80201
And No. 4-80202). This neoproteoglycan enabled for the first time a method of efficiently immobilizing glycosaminoglycan sugar chains on a culture dish.

[0006] In the prior art, it was possible to add various growth factors including heparin-binding growth factors such as hHGF to a solution to see their effects, but the growth factors retained their activity. Since it could not be solid-phased in the state, the action of the solid-phased growth factor has not been analyzed. Further, when the growth factor is administered in vivo, it is technically difficult to keep the growth factor at the local site of action.

[0007]

Means for Solving the Problems The present inventors have attempted to immobilize the growth factor by utilizing the binding property of HGF to glycosaminoglycan and to examine its action. As a result, interestingly, it was revealed that HGF was immobilized via glycosaminoglycan and could stimulate proliferation of hepatocytes, thus completing the present invention.

That is, the gist of the present invention resides in a solid-phased hepatocyte proliferating agent comprising a glycosaminoglycan, glucan or a derivative thereof bound to a hepatocyte growth factor. Hereinafter, the present invention will be described in detail. Hepatocyte growth factor (HGF) used in the present invention
Any of those obtained by purifying biomaterials and those obtained by expressing its cDNA by a recombinant method can be used. Specific examples of human HGF (hHGF) include protein chemical separation and purification from plasma of patients with fulminant hepatitis according to the method described in JP-A-63-22526, or JP-A-3-285693. CDNA encoding hHGF according to the method described in the publication
Examples include those obtained by constructing an expression vector containing the above and expressing it in a host such as CHO cells. Such hHGF starts to show the proliferative activity of hepatocytes at a concentration of about 0.5 to 2 ng / ml, and about 5 to 10 ng / ml.
The highest growth-inducing activity is reached at the concentration of.

In the present invention, glycosaminoglycan, glucan or a derivative thereof is used as a carrier for immobilizing the above HGF. Glycosaminoglycan is a general term for polysaccharides containing hexosamine as a constituent sugar, and specifically, hyaluronic acid, chondroitin, teichronic acid,
Colominic acid, chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, chondroitin sulfate D and E (chondroitin polysulfate), heparin, keratosulfate (keratan sulfate), heparitin sulfate (heparan sulfate), polyacetylgalactosamine phosphorus Examples thereof include acidic glycosaminoglycans such as acids, chitin, and galactosaminoglycans. On the other hand, glucan is a general term for polysaccharides containing glucose as a constituent sugar,
Specific examples thereof include dextran, dextran sulfate, laminaran, lihenan, cellulose, amylose, amylopectin and the like, and dextran or dextran sulfate is preferable in the present invention. These can be appropriately prepared using a known method.

The derivative of such glycosaminoglycan or glucan is not particularly limited, but in the present invention, it is preferably a protein capable of covalently bonding with glycosaminoglycan or glucan, or a derivative with phospholipid. . Examples of proteins include in vivo proteins such as albumin, and examples of phospholipids include glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol, and sphingophospholipids such as sphingomyelin. Can be mentioned. These protein derivatives are prepared by a known method (for example, J. Biol. Chem., 264 (14), 80) as a method for producing so-called neoproteoglycan.
12-8018 (1989)), and the phospholipid derivative is disclosed in JP-A-4-80.
It can be prepared according to the method described in No. 201, No. 4-80202, etc.

In the present invention, HGF is bound to glycosaminoglycan, glucan or a derivative thereof. At this time, since HGF is originally heparin-binding, it can be easily bound by mixing both, but phosphate buffer-physiological saline and the like can coexist as long as the properties are not impaired. Further, it is preferable that HGF and glycosaminoglycan, glucan or a derivative thereof are bound at a molar ratio of 1: 1 to 1: 1000. Furthermore, in the present invention, glycosaminoglycan,
If glucan or a derivative thereof is previously bound to a cell adhesion protein such as collagen, elastin, fibronectin, laminin, vitronectin, or cadherin (hereinafter abbreviated as "culture substrate"), it can be more stably immobilized. It is preferable because it is possible. At that time, glycosaminoglycan, glucan or a derivative thereof may be directly or indirectly coated on a culture substrate, and HGF may be added thereto in a solution state to solidify the culture substrate. Instead of, a sheet-shaped polymer base material may be used. The polymer base material is not particularly limited as long as it is a material compatible with a living body. When a culture substrate is used, it may be further bound to a polymer substrate for use. The thus immobilized HGF can locally induce the original physiological activity of the growth factor by exposing it to cells or tissues.

[0012]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the invention is not limited to the following examples as long as the gist thereof is not exceeded. Example 1: hHGF immobilized with neoproteoglycan
Hepatocyte proliferation inducing ability of carrier: Preparation of a bovine-derived type I collagen solution (10 μg / ml, co-containing solution) in a multi-well plastic dish (Corning) having a well with a diameter of 2.3 cm.
As a culture substrate, and then heparin-phosphatidylethanolamine (Hep-P) as a synthetic glycosaminoglycan lipid derivative (GAG-PE).
E) was coated at a concentration of 10 μg / ml to prepare a solid-phase carrier (Hep-PE was prepared according to the method described in Example 1 of JP-A-4-80201). As a control experiment, wells not coated with the synthetic glycosaminoglycan lipid derivative were also prepared.

Immobilization of hHGF: Recombinant hHG
F (prepared according to the method described in Japanese Patent Application Laid-Open No. 3-285693) containing 10 mM phosphate buffer containing 1% bovine serum albumin (BSA) to a final concentration of 10 ng / ml.
Prepared with physiological saline, pH 7.4 (PBS (-)),
It was added to the above carrier. After reacting at 4 ° C. for 6 hours, the solution was removed, and the carrier was washed 5 times with PBS (−). As a comparative experiment, a well in which the carrier was further washed twice with 1 M NaCl was also prepared.

Preparation of cells: Method of Seglen (Methods in Cell Biolog)
y, vol13, p29, Academic Pres
S., New York (1976)), and hepatocytes were isolated from male Wistar rats (body weight 200 g) using 0.05% collagenase (type I, Sigma). The hepatic parenchymal cells were 2.5 × 10 ⁴ cells / 0.25 ml / cm ² in a multi-well plastic dish (Corning Co.) coated with the above carrier and hHGF.
At a concentration of 5% carbon dioxide gas in the air, 37
Monolayer culture was carried out at ℃ (Tanaka et al., J.
Biochem. 84, 937-946 (197)
8)). As the culture medium, 5% fetal calf serum (FCS, Filtron), 10 ^-8 M insulin, 10 ^-8 M dexamethasone, and Williams E (WE) medium supplemented with 60 µg / ml gentamicin (Flo Laboratories, Inc.) Hereinafter, abbreviated as "basic medium") was used.

Measurement of cell proliferation-inducing activity: The basal medium was replaced with a new basal medium 2 hours after the start of culture, and 18 hours later, WE medium (10 ⁻⁷ M insulin and 6
(Containing 0 μg / ml gentamicin). 28 hours after the start of culture, 1.25 μCi of [methyl
³ H] thymidine (Amersham) was added, and 10 μg / ml of aphidicolin (Sigma) was added as needed. After 24 hours, cells were fixed with 1 ml of cold 10% trichloroacetic acid for 1-2 hours, washed 3 times with 95% ethanol and then lysed with 1M NaOH. A scintillator was added to the neutralized cell lysate, mixed well, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter. The replicated DNA synthesis value was calculated by subtracting the count in the presence of aphidicolin from each radioactivity count.
As a control group, the radioactivity in the case where GAG-PE was not added to each sample was determined, and the result was shown as a ratio to that value (FIG. 1). In addition, as a comparative experiment, hHGF
A similar experiment was conducted using epidermal growth factor (EGF), which has no affinity for glycosaminoglycan, in place of
The results are shown in FIG. The vertical axis represents the DNA synthesis induction activity of each sample as a ratio (%) to the DNA synthesis induction activity of the control group in which GAG-PE was not used. It can be seen that when Hep-PE is used as the culture substrate, the hHGF added before the cell seeding is effectively solid-phased and can stimulate the DNA synthesis of hepatocytes even after the medium exchange. That is, 66% enhancement was recognized as compared with the control (a). Further, even when hHGF was post-added as a solution to hepatocytes on Hep-PE as in the conventional case, about 20% of DNA synthesis enhancement was observed as compared with the control (b). When the immobilized hHGF was washed with 1 M NaCl, the DNA synthesis enhancement was reduced from 66% to 33% (c). This is considered to be due to the removal of part of the immobilized hHGF by washing with 1 M NaCl.
In EGF having no heparin-binding ability, no difference was observed depending on the presence or absence of GAG-PE (d and e). This suggests that hHGF binds to GAG-PE by the binding property with glycosaminoglycan as expected and is immobilized on the culture substrate.

From the above results, by combining hepatic parenchymal cell growth factor with glycosaminoglycan, glucan or a derivative thereof, the growth factor is effectively immobilized and the immobilized growth factor has a physiological action. You can see that it can be demonstrated.

[0017]

EFFECTS OF THE INVENTION In the past, when utilizing hepatocyte growth factor, no method for immobilizing such a protein in the state of retaining the activity of the protein or utilization of the immobilization factor has been known. As shown by the present invention, hepatocyte growth factor is
By using an appropriate glycosaminoglycan, glucan or a derivative thereof, it is possible to immobilize while retaining the activity, and as a result, this immobilized growth factor is
It is now available in vivo or in vitro. By devising a solid phase carrier for hepatocyte growth factor based on the present invention, it can be expected to be widely used in the medical field, and further, a sustained release effect / accumulation effect can be expected upon administration to a living body.

[Brief description of drawings]

FIG. 1 is a synthetic glycosaminoglycan lipid derivative (Hep
-PE) is a drawing showing solid-phase immobilization of hHGF.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C08B 37/08 Z 7329-4C 37/10 7329-4C

Claims (7)

[Claims] 1. A solid-phase hepatocyte proliferation agent comprising a glycosaminoglycan, a glucan or a derivative thereof bound to a hepatocyte growth factor. 2. The hepatocyte proliferation agent according to claim 1, wherein the glycosaminoglycan or glucan is selected from heparin, dextran sulfate, heparan sulfate and chondroitin sulfate. 3. The hepatocyte proliferation agent according to claim 1, wherein a glycosaminoglycan or a glucan derivative is used. 4. The hepatocyte proliferation agent according to claim 3, wherein the derivative is a glycosaminoglycan or glucan bound to a protein or phospholipid. 5. The hepatocyte proliferation agent according to claim 4, wherein the derivative is a glycosaminoglycan or glucan and a phospholipid bound to each other. 6. The hepatocyte proliferation agent according to claim 1, wherein glycosaminoglycan, glucan or a derivative thereof is bound to a polymer substrate or a cell adhesive protein. 7. The hepatocyte proliferation agent according to claim 6, wherein the cell adhesive protein is selected from collagen, elastin, fibronectin, laminin, vitronectin and cadherin.