CN1803193A - Use of stroma stem cell derived from bone marrow in preparing formulation for treating hypoxic ischemic cerebral palsy - Google Patents

Abstract

The invention belongs to the field of stem cells and tissue engineering, and relates to the use of bone marrow-derived mesenchymal stem cells for preparing preparations for treating hypoxic-ischemic cerebral palsy. The preparation is formulated from bone marrow-derived mesenchymal stem cells and normal saline, and the effective dose for the neonatal rat model of hypoxic-ischemic encephalopathy is 5×10 ⁵ cells/time, and it is used for neonatal cerebral palsy patients The effective dosage for patients is 1.0-5.0×10 ⁶ cells/time. The invention opens up a new way of cell therapy for the clinical treatment of hypoxic-ischemic cerebral palsy.

Description

Use of bone marrow-derived mesenchymal stem cells for preparing preparations for treating hypoxic-ischemic cerebral palsy

technical field

The invention belongs to the field of stem cells and tissue engineering, and relates to new uses of mesenchymal stem cells, in particular to the use of bone marrow-derived mesenchymal stem cells for preparing preparations for treating hypoxic-ischemic cerebral palsy.

Background technique

Hypoxic-ischemic brain injury (HIE) caused by perinatal asphyxia is the main cause of neonatal cerebral palsy. In the past 20 years, despite the rapid development of obstetrics and newborn medical care, the incidence of cerebral palsy in newborns has not been significantly reduced. According to a sample survey conducted by Beijing Medical University and other units in 1998, it was found that children aged 0-6 in my country had cerebral palsy. There are 310,000 children, and the rate is increasing by 46,000 every year. A prospective survey by the American Perinatal Cooperation Project found that the incidence of cerebral palsy in children is as high as 4‰. The main manifestations of children are non-progressive central motor dysfunction, or accompanied by mental retardation. The current treatment methods mainly include: (1) comprehensive rehabilitation medicine based on sports rehabilitation; (2) drug therapy based on nourishing nerves, relaxing muscles, and promoting blood circulation; (3) selective posterior rhizotomy therapy; (4) Chinese medicine therapy mainly including massage and acupuncture. However, since cerebral palsy is caused by fixed brain lesions, the above treatment methods cannot remove or correct the cause, and the curative effect is uncertain. However, for patients with cerebral palsy, especially severe ones, the children not only suffer great physical pain, but also suffer from suffocation caused by cerebral palsy, which leads to brain hypoxia and progressive decline in intelligence. Therefore, if a new and effective treatment method for cerebral palsy can be developed, it will bring happiness to countless families.

In the past few decades, the use of stem cell transplantation to treat patients with cerebral palsy has not been reported, but some progress has been made in the treatment of other diseases of the central nervous system.

The main sources of stem cells are: 1. Embryonic neural stem cells: According to research reports, using 6-9W aborted fetal brain cells to treat Parkinson's disease can significantly improve the function of some patients, and even synthesize dopamine in some patients. However, due to many factors such as cell source and ethics, the clinical application of embryonic neural stem cells in the treatment of nervous system diseases is limited. 2. Bone marrow-derived mesenchymal stem cells: In the 1970s, Friedenstein et al. isolated and cultured a kind of mesenchymal stem cells from bone marrow. In-depth research found that mesenchymal stem cells can not only survive and migrate in brain tissue, but also express neurons and glia. Cells and other specific markers of nerve cells, significantly improving the function of stroke and other model rats. At present, mesenchymal stem cells are mainly derived from adult bone marrow, but the source of adult bone marrow cells is limited, and pathogen contamination is inevitable. There is no report on the use of embryonic bone marrow as a source of stem cells.

Obviously, under the existing conditions, there are various defects and deficiencies in the relevant technologies and methods for the treatment of hypoxic-ischemic cerebral palsy.

Contents of the invention

The purpose of the present invention is to provide a use of bone marrow-derived mesenchymal stem cells for preparing preparations for treating hypoxic-ischemic cerebral palsy.

The preparation is formulated from bone marrow-derived mesenchymal stem cells and physiological saline, and the effective dosage for the neonatal rat model of hypoxic-ischemic encephalopathy is 5×10 ⁵ cells/time. The effective dose is determined by obtaining therapeutic effects through animal model experiments.

The bone marrow-derived mesenchymal stem cells are prepared according to the following method: use aseptic technique to isolate the bone marrow nucleated cells of rat second-trimester embryos, inoculate them in a culture bottle containing low-sugar DMEM medium containing fetal bovine serum, and adhere the cells to the wall. When the proliferation is close to fusion, digestion and passage are carried out, and this method is repeatedly passaged to achieve the purpose of purifying and expanding mesenchymal stem cells.

The preparation prepared from bone marrow-derived mesenchymal stem cells is used for intracerebral injection of neonatal rats at a dose of 5.0×10 ⁵ cells/time, within 24 hours after the establishment of the hypoxic-ischemic encephalopathy model transplant.

The preparation is prepared from bone marrow-derived mesenchymal stem cells and physiological saline, and the effective dose for neonatal cerebral palsy patients is 1.0-5.0×10 ⁶ cells/time. The effective dose is determined by obtaining therapeutic effects through animal model experiments and common knowledge of human medicine.

The bone marrow-derived mesenchymal stem cells are prepared according to the following method: use aseptic technique to separate embryonic bone marrow nucleated cells from human embryo abortion materials, inoculate them in a culture bottle containing low-sugar DMEM medium containing fetal bovine serum, and the cells When adherent proliferation is close to confluence, digestion and passage are carried out, and this method is repeatedly passaged to achieve the purpose of purifying and expanding mesenchymal stem cells. The cells are collected, frozen and recovered for later use.

The preparation prepared from bone marrow-derived mesenchymal stem cells is used for localized injection in the brain of neonatal patients with cerebral palsy, with a dose of 1.0-5.0×10 ⁶ /time, 24-48 hours after the occurrence of hypoxic-ischemic encephalopathy Intragraft.

In the present invention, mesenchymal stem cells derived from embryonic bone marrow are transplanted to children with hypoxia-deficiency cerebral palsy through brain positioning and infusion, so as to promote the repair of nerve cells in their brain tissue, improve their balance and coordination, motor function, and improve their learning and memory abilities. The purpose of improving the quality of life of children and reducing family and social burdens is to open up a new way of cell therapy for the clinical treatment of cerebral palsy.

Evaluation of the effect of the preparation prepared from the bone marrow-derived mesenchymal stem cells for the treatment of hypoxic-ischemic cerebral palsy:

1. Viability: including

a) Survival rate of model rats within 1 week;

b) Body weight gain of model rats within 1 month;

2. Learning and memory functions on the 42nd day after transplantation;

3. Anatomy and pathological features of brain tissue at the 10th week;

4. Immunohistochemistry and indirect immunofluorescence were used to analyze the survival, migration, and expression of nerve cell-specific antigens of exogenous cells in brain tissue.

The advantages of the present invention are:

(1) Transplantation of embryonic bone marrow-derived mesenchymal stem cells into the brain of neonatal hypoxic-ischemic model rats can significantly improve the viability of the model rats; improve their motor, learning, and memory abilities; environment, promoting nerve cell regeneration and/or restoration of function.

(2) Open up a new way of cell therapy for the clinical treatment of hypoxic-ischemic encephalopathy. The invention utilizes embryonic bone marrow-derived mesenchymal stem cells to be original, has high proliferation potential, is easy to prepare, has multi-directional differentiation potential after continuous culture and cryopreservation and recovery, has high cell homogeneity, and has normal karyotype and telomerase active.

(3) In the present invention, bone marrow-derived mesenchymal stem cells are injected into the brain to treat hypoxic-ischemic cerebral palsy model. It is proved that bone marrow-derived mesenchymal stem cells can block and reverse the progression of hypoxic-ischemic cerebral palsy. Development, opening up a new way of cell therapy for the clinical treatment of hypoxic-ischemic cerebral palsy.

Description of drawings

Figure 1 is the results of pathological examination of SD rat brain tissue sections (H&E staining, ×200). Among them, Figure A shows the distribution of cortical cells in the normal control group; Figure B shows the degeneration and necrosis of neurons in the HIE model (↑); Figure C shows the pyknosis of the cerebellar Purkinje cells in the HIE model (↑); Figure D shows HIE/hFBMMSCs In the transplantation group, cortical nerve cells increased extensively.

Figure 2 is the results of immunohistochemical staining of brain tissue in the hFBMMSCs transplantation group (DAB, ×100). Panel A shows positive anti-human NSE (↑); panel B shows positive anti-human GFAP (↑).

Fig. 3 is the results (×100) of indirect immunofluorescence detection of HIE/hFBMMSCs. Among them, 1A and 1B show: the same part of the same slice, the one with green fluorescence in A is positive for anti-human RNP; the one with orange-red fluorescence in B is positive for anti-GFAP; 2A and 2B show: the same part of the same slice, the one with green fluorescence in A Those with anti-human RNP are positive; those with orange-red fluorescence in B are anti-NSE positive.

Detailed ways

Example 1: Preparation of bone marrow-derived mesenchymal stem cells (MSCs):

The bone marrow nucleated cells of the second-trimester embryo were isolated by aseptic technique, inoculated in the culture flask of low-sugar DMEM medium containing 8% fetal bovine serum, and the medium was changed once every other day depending on the growth situation, and the cells adhered to the wall and proliferated close to the fusion of the cells. When 90% of the fusion state is reached, digestion and passage are carried out, and repeated passages are carried out in this way to achieve the purpose of purifying and expanding mesenchymal stem cells.

Described low-sugar DMEM growth base selects the CAT NO.SH30002.01 product of HYCLONE Company, or the similar product of GIBCO Company.

Example 2: Preparation of bone marrow-derived mesenchymal stem cells (MSCs):

Isolate the nucleated cells from the bone marrow of the second-trimester embryo by aseptic technique, inoculate them in a culture bottle containing 10% fetal bovine serum low-sugar DMEM medium, change the medium once every 3 days depending on the growth situation, and the cells adhere to the wall and proliferate close to the fusion of the cells. When the fusion state is 80%, carry out digestion and passaging, and repeat passaging according to this method to achieve the purpose of purifying and expanding mesenchymal stem cells.

Described low-sugar DMEM culture medium selects the CAT NO.SH30002.01 product of HYCLONE Company, or the similar product of GIBCO Company.

Example 3: Preparation of bone marrow-derived mesenchymal stem cells (MSCs):

The bone marrow nucleated cells of the second-trimester embryo were isolated by aseptic technique, inoculated in the culture flask of low-sugar DMEM medium containing 15% fetal bovine serum, and the medium was changed once every 4 days depending on the growth situation, and the cells adhered to the wall and proliferated close to the fusion of the cells. 70% confluent state, digested, passaged, repeated passages according to this method, to achieve the purpose of purification and expansion of mesenchymal stem cells, collected cells cryopreserved, resuscitated for later use.

Described low-sugar DMEM culture medium selects the CAT NO.SH30002.01 product of HYCLONE Company, or the similar product of GIBCO Company.

Example 4: Modeling of newborn rats with hypoxic-ischemic cerebral palsy:

One-day-old newborn F344 rats (body weight 5.85±0.39g) were fixed in the supine position after ether inhalation anesthesia, the skin was incised in the middle of the neck, the left common carotid artery was freed, the incision was ligated with No. In a constant-temperature airtight container, a mixed gas of 8% O2 and 92% N2 was input at a rate of 1 L/min for 3 hours, and after taking it out, it was placed in the original feeding environment to accept feeding of female mice.

Example 5: Infusion experiment of embryonic bone marrow-derived mesenchymal stem cells:

1. Timing of treatment: within 24 hours after model making.

2. Dose: 5.0×105/g.

3. Approach: brain localized injection.

4. Experimental control group: group design was adopted, and littermate rats were divided into 4 groups: model/stem cell transplantation group, model/normal saline group, model/untreated group, and normal control group.

Evaluation:

1. Viability

(1) Survival of rats within one week: the survival rate of the stem cell transplantation group had no significant difference compared with the normal control group; it was higher than that of the normal saline group and the untreated group. See Table 1 for details

(2) Body weight gain of newborn rats within 1 month: From the 5th day after transplantation, the weight gain of the rats in the transplanted group accelerated, and there was no significant difference from the normal control group on the 30th day, which was heavier than that of the normal saline group and the untreated group. Group. See Table 2 for details

2. Functional changes: On the 42nd day after cell transplantation, the Morris water maze test was used to test the learning and memory abilities of the rats in the transplanted group. The learning and memory abilities of the rats in the transplanted group were similar to those of the normal group, and stronger than those of the normal saline injection and untreated groups. See Table 3 for details

3. Anatomical results: In the 10th week, the brain tissue structure of the experimental rats was analyzed. The rate of brain atrophy and edema in the stem cell transplantation group was significantly lower than that in the normal saline injection group and the untreated group, and there was no significant difference with the normal control group.

4. Pathological examination results of brain tissue: H&E staining, under the light microscope, the stem cell transplantation group can see the proliferation of brain nerve cells, especially in the cerebral cortex, hippocampus and other parts; part of the cerebral blood vessel wall, a large number of cells can be seen migrating around; stem cell transplantation A large number of cells can be seen in agglomerates or scattered distribution at the needle site, migrating to the surrounding in a directional manner. In the untreated group and the normal saline group, neurons in the cerebellum, cortex, and hippocampus were significantly reduced, neuronal nucleus shrinkage, necrosis, glial cell phagocytosis, and glial cell reactive hyperplasia to form glial cell nodules and other lesions . See Figure 1 for details. Figure 1 is the results of pathological examination of SD rat brain tissue sections (H&E staining, ×200). Among them, Figure A shows the distribution of cortical cells in the normal control group; Figure B shows the degeneration and necrosis of neurons in the HIE model (↑); Figure C shows the pyknosis of the cerebellar Purkinje cells in the HIE model (↑); Figure D shows HIE/hFBMMSCs In the transplantation group, cortical nerve cells increased extensively.

5. Immunohistochemical analysis results: Immunohistochemical analysis showed that there were anti-human NSE and GFAP reactive cells in the brain tissue of the rats in the transplantation group, especially in the cortex and hippocampus; no NSE was found in the other three groups of experimental animals or GFAP positive cells. See Figure 2 for details. Figure 2 is the results of immunohistochemical staining of brain tissue in the hFBMMSCs transplantation group (DAB, ×100). Figure A shows positive anti-human NSE (↑), and picture B shows positive anti-human GFAP (↑).

6. Indirect immunofluorescence test results: anti-human RNP/CD45, anti-human RNP/NSE or anti-human RNP/GFAP double-positive intact cells were detected in the cerebral cortex and hippocampus on both sides of the transplantation group, especially at the needle insertion site. A large number of double-positive cells existed in agglomerates or scattered, and migrated to the surroundings with a gradient decrease in density, while no anti-human RNP, CD45, NSE, and GFAP positive cells appeared in the brain tissue sections of animals in the other three groups. See Figure 3 for details. Fig. 3 is the results (×100) of indirect immunofluorescence detection of HIE/hFBMMSCs. Among them, 1A and 1B show: the same part of the same slice, the one with green fluorescence in A is positive for anti-human RNP; the one with orange-red fluorescence in B is positive for anti-GFAP; 2A and 2B show: the same part of the same slice, the one with green fluorescence in A Those with anti-human RNP are positive; those with orange-red fluorescence in B are anti-NSE positive.

Table 1 Survival rate of newborn rats within 1 week Nest Total number of newborn mice (N) HIE/hFBMMSC survival rate (%) HIE/NS survival rate (%) HIE/untreated survival rate (%) Normal control survival rate (%) 1 10 100.0% (3/3) 66.7% (2/3) 50.0% (1/2) 100.0% (2/2) 2 14 ^* 75.0% (3/4) 33.3% (1/3) 66.7% (2/3) 100.0% (3/3) 3 13 ^** 66.7% (2/3) 0.0% (0/2) 50.0% (1/2) 100.0% (3/3) 4 8 ^* 100.0% (2/2) 50.0% (1/2) 50.0% (0/1) 100.0% (2/2) 5 16# 66.7% (2/3) 33.3% (1/3) 66.7% (2/3) 33.3% (1/3) 6 9## 100.0% (2/2) 0.0% (0/2) 100.0% (1/1) 100.0% (2/2) 7 11## 33.3% (1/3) 50.0% (1/2) 0.0% (0/2) 50.0% (1/2) Total (N) 81 75.0% (15/20) 35.3% (6/17) 50.0% (7/14) 82.4% (14/17)

Table 2 Body weight growth within 1M of 4 groups of neonatal rats (x±SD) days (d) Normal control (g) HIE/hFBMMSCs (g) HIE/NS(g) HIE/untreated (g) 5 8.89±0.82 5.48±0.91 4.28±0.39 4.82±0.61 10 15.87±1.34 13.99±0.93 12.44±0.93 12.59±0.93 15 22.80±1.84 20.96±1.85 17.93±1.68 17.98±1.38 20 33.94±1.62 33.61±2.63 27.39±1.84 27.82±1.01 25 52.54±1.67 51.80±3.12 45.79±1.53 46.79±1.14 30 73.92±6.27 71.01±2.66 63.53±5.35 64.93±4.25 Total (N) 30 9 10 4 4

Table 3 Morris water maze training and test results of 4 groups of experimental rats (X±SD)sec time (d) Normal control (n=4) HIE/hFBMMSCs (n=4) HIE/NS (n=4) HIE/untreated (n=4) 1 105.0±1.5 110.0±7.1 111.0±1.4 ^* 118.0±1.5# 2 50.0±2.7 90.0±2.5 96.0±22.6 ^* 100.0±2.5 3 30.0±9.8 47.5±12.5 71.7±24.7 80.0±15.3 4 13.0±3.2 14.0±4.3 33.0±20.9 29.0±11.0 5 7.0±2.1 7.5±2.1 23.7±14.9 24.0±9.0

^* : Experimental results of 2 mice; #: Experimental results of 3 mice

Claims (7)

1. Use of bone marrow-derived mesenchymal stem cells for preparing preparations for treating hypoxic-ischemic cerebral palsy. 2. The use according to claim 1, characterized in that the preparation is formulated from bone marrow-derived mesenchymal stem cells and normal saline, and is used for the effective dose of the neonatal rat model of hypoxic-ischemic encephalopathy 5×10 ⁵ cells/time. 3. The use according to claim 2, characterized in that the bone marrow-derived mesenchymal stem cells are prepared according to the following method: use aseptic technique to isolate nucleated cells from the bone marrow of rat second-trimester embryos, and inoculate them in embryo-containing cells. In the culture flask of low-sugar DMEM medium with bovine serum, when the cells adhere to the wall and proliferate close to confluence, they are digested and passaged. Repeated passages in this way achieve the purpose of purifying and expanding mesenchymal stem cells. The cells are collected for freezing and recovery for later use. 4. The use according to claim 2, characterized in that: the preparation prepared from bone marrow-derived mesenchymal stem cells is used for localized injection in the brain of neonatal rats, the dose is 5×10 ⁵ /time, Transplant within 24 hours after the establishment of the hypoxic-ischemic encephalopathy model. 5. The use according to claim 1, characterized in that the preparation is prepared from bone marrow-derived mesenchymal stem cells and normal saline, and the effective dose for neonatal cerebral palsy patients is 1.0-5.0×10 ⁶ cells /Second-rate. 6. The use according to claim 5, characterized in that, the bone marrow-derived mesenchymal stem cells are prepared by the following method: use aseptic technique to separate nucleated cells of embryo bone marrow from human embryo abortion materials, and inoculate them in In the culture flask of low-sugar DMEM medium containing fetal bovine serum, when the cells adhere to the wall and proliferate close to confluence, they are digested and passaged, and this method is repeatedly passaged to achieve the purpose of purifying and expanding mesenchymal stem cells, and the collected cells are frozen and recovered. spare. 7. The use according to claim 5, characterized in that: the preparation prepared from bone marrow-derived mesenchymal stem cells is used for intracerebral localized injection of neonatal cerebral palsy patients, and the dose is 1.0-5.0×10 ⁶ / Second, within 24-48 hours after the onset of hypoxic-ischemic encephalopathy.