Culture method, application and detection method of mesenchymal stem cells for treating nervous system diseases

Abstract

The invention provides a culture method, application and detection method of mesenchymal stem cells for treating nervous system diseases, and belongs to the technical field of biological medicines. The invention provides a culture medium of mesenchymal stem cells, a method for culturing and subculturing the mesenchymal stem cells by using the culture medium, and a method for detecting the biological efficacy of the mesenchymal stem cells for treating nervous system diseases. The mesenchymal stem cell preparation disclosed by the invention has a relatively good treatment effect on nervous system diseases, and can be used for improving the new object recognition capability and the cerebral infarction range, inhibiting the release of inflammatory factors, reducing the oxidative stress level and narrowing the spinal cord lesion range so as to improve nervous system injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a culture method, application and detection method of mesenchymal stem cells for treating nervous system diseases. Background Art

[0002] Mesenchymal stem cells (MSCs) are an important type of stem cell derived from mesoderm cells. They are found in various connective tissues in the body and possess the ability to self-renew and undergo multidirectional differentiation. MSCs can be divided into various types based on their source, such as bone marrow mesenchymal stem cells (BMSCs), adipose-derived mesenchymal stem cells (ADSCs), dental pulp stem cells (DPSCs), and umbilical cord mesenchymal stem cells (UB-MSCs). MSCs possess multidirectional differentiation potential, strong proliferation capacity, few ethical issues, low immunogenicity, and are easily accessible, making them of great scientific value and promising clinical application prospects.

[0003] The central nervous system, comprising the brain and spinal cord, is the core regulatory center for human neural activity. Stroke, Alzheimer's disease (AD), and spinal cord injury (SCI) are common CNS diseases. Stroke is a common cerebrovascular disease primarily caused by stenosis, occlusion, or rupture of cerebral arteries, leading to acute cerebral blood circulation impairment. Alzheimer's disease is a degenerative condition of the central nervous system characterized by progressive cognitive impairment and behavioral impairment. Spinal cord injury is damage to the spinal cord caused by trauma, inflammation, or tumors, resulting in functional impairment below the level of spinal cord injury, including loss of sensory and motor function and autonomic dysfunction. CNS diseases have a profound negative impact on patients' quality of life and social functioning. These diseases often lead to severe functional impairments, such as limb paralysis, cognitive decline, and language disorders. Current treatments and effectiveness are very limited, making it difficult to reverse neural damage.

[0004] The production process and quality inspection of MSCs still restrict their drug development. Because they need to be separated from tissues such as the umbilical cord, they may encounter problems such as slow growth, easy aging or differentiation during the isolation and culture process. The obtained stem cell products have no significant therapeutic effect on central nervous system diseases and lack specific molecular markers. Therefore, their culture conditions and preparation process require process optimization and quality inspection. Summary of the Invention

[0005] The present invention provides a culture method, use and detection method of mesenchymal stem cells for treating nervous system diseases. The preparation prepared using the mesenchymal stem cells has a good therapeutic effect on nervous system diseases.

[0006] The invention provides a culture medium for culturing mesenchymal stem cells, which uses DMEM / F12 culture medium as a basic culture medium and further comprises 8-20% v / v FBS, 4-15 μg / L bFGF and 5-10 μg / L glutamine.

[0007] In a preferred embodiment of the present invention, the culture medium further comprises hydrogen, and the concentration of the hydrogen is 1 to 1.8 μg / mL.

[0008] In a preferred embodiment of the present invention, in the culture medium, the concentration of FBS is 10-15% v / v, the concentration of bFGF is 5-10 μg / L, and the concentration of glutamine is 5-8 μg / L.

[0009] The present invention also provides a method for culturing mesenchymal stem cells, comprising the following steps: incubating a tissue containing mesenchymal stem cells in a cell-free culture medium for a period of time, placing the tissue in the above-mentioned culture medium for culturing, and digesting the tissue after the cell confluence reaches 70-90% to obtain P0 mesenchymal stem cells;

[0010] The P0 mesenchymal stem cells were collected after centrifugation and subcultured using the above culture medium.

[0011] In a preferred embodiment of the present invention, the incubation comprises breaking the tissue into tissue blocks, directly inoculating the blocks into a culture device, and sequentially performing a first incubation for 1 hour and a second incubation for 5 hours;

[0012] The first incubation temperature is 38-39°C, the CO2 concentration is 4-6%, the O2 concentration is 2-20%, and the time is 1-2 hours;

[0013] The second incubation temperature is 36-37° C., the CO 2 concentration is 4-6%, the O 2 concentration is 18-20%, and the time is 5-7 hours.

[0014] In a preferred embodiment of the present invention, the types of mesenchymal stem cells include any one of the following: adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells.

[0015] In a preferred embodiment of the present invention, the tissue comprises at least one of the following: fat, umbilical cord, placenta, umbilical cord blood, bone marrow and dental pulp.

[0016] In a preferred embodiment of the present invention, the culture temperature is 36-37°C, the CO2 concentration is 4-6%, the O2 concentration is 2-20%, and the culture time is 48-72 hours;

[0017] The temperature of the subculture is 37° C., the CO 2 concentration is 4-6%, the O 2 concentration is 18-20%, and the subculture time of each generation is 48-72 hours.

[0018] The present invention also provides an injection of mesenchymal stem cells, comprising mesenchymal stem cells cultured using the above method and a cell preservation solution, wherein the content of mesenchymal stem cells in the injection is (1-10)×10 6 / mL;

[0019] The cell preservation solution contains the following components at the following concentrations: a compound electrolyte injection solution of 10-20 mg / mL human serum albumin and 60-80 IU / mL heparin.

[0020] In a preferred embodiment of the present invention, the compound electrolyte injection comprises the following components in the following concentrations: 5.26 g / L sodium chloride, 5.02 g / L sodium gluconate, 3.68 g sodium acetate; 0.37 g / L potassium chloride and 0.30 g / L magnesium chloride.

[0021] The present invention also provides a method for preparing the above-mentioned injection, comprising the following steps: culturing the mesenchymal stem cells obtained by the above-mentioned method to 80% cell confluence, then digesting the cells, collecting the cells by centrifugation, washing them, and resuspending them in a cell preservation solution to prepare the injection of the mesenchymal stem cells.

[0022] The present invention also provides the use of mesenchymal stem cells cultured by the above method in preparing medicines for treating nervous system diseases.

[0023] In a preferred embodiment of the present invention, the neurological disease includes at least one of the following: Alzheimer's disease, ischemic stroke, Parkinson's disease, multiple sclerosis, spinal cord injury, epilepsy, dementia, depression, neuropathic pain, nervous system infection, demyelinating disease of the nervous system, movement disorders and mental disorders.

[0024] The present invention also provides a medicine for treating nervous system diseases, comprising mesenchymal stem cells cultured by the above method and pharmaceutically acceptable excipients.

[0025] In a preferred embodiment of the present invention, the dosage form of the drug includes any one of the following: drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, powders, emulsions, solutions, pellets, pills, buccal tablets, freeze-dried powder injections, gels, suppositories and aerosols.

[0026] The present invention also provides a method for detecting the biological efficacy of mesenchymal stem cells in treating nervous system diseases, comprising detecting the ability of mesenchymal stem cells to secrete sTNFR1, HGF, BDNF and / or GDNF;

[0027] The mesenchymal stem cells include any one of the following sources: mesenchymal stem cells cultured by the above method, the above injection solution and the above medicine.

[0028] In a preferred embodiment of the present invention, the judgment criteria for the biological efficacy detection of mesenchymal stem cells in treating nervous system diseases include at least one of the following:

[0029] Every 1×10 6 The amount of sTNFR1 secreted by mesenchymal stem cells within 48 hours was 200-1000 pg;

[0030] Every 1×10 6 The amount of HGF secreted by each mesenchymal stem cell within 48 hours is 100-300 ng;

[0031] Every 1×10 6 The amount of BDNF secreted by mesenchymal stem cells within 48 hours is 300-1200 pg;

[0032] Every 1×10 6 The amount of GDNF secreted by mesenchymal stem cells within 48 hours is 300-800 pg.

[0033] Beneficial Effects: The present invention provides a culture medium for culturing mesenchymal stem cells, comprising DMEM / F12 medium as a basal medium and further comprising 8-20% v / v FBS, 4-15 μg / L bFGF, and 5-10 μg / L glutamine. The culture medium can be used to culture and passage mesenchymal stem cells. The cells obtained after culture and passage exhibit adipogenic and osteogenic differentiation capabilities and highly express sTNFR1, HGF, BDNF, and GDNF.

[0034] The present invention can prepare mesenchymal stem cells after culture and passage into drugs, and it is proven that the drugs have a good therapeutic effect on nervous system diseases. For example, for Alzheimer's disease, mesenchymal stem cells can improve the ability to recognize new objects, reduce the escape latency, increase the number of platform crossings, reduce the concentration of inflammatory factors, and reduce the expression of Aβ and Tau proteins; for ischemic stroke, mesenchymal stem cells can improve the scope of cerebral infarction, reduce the level of oxidative stress, and thus improve nervous system damage; mesenchymal stem cells can improve nervous system damage caused by spinal cord injury, reduce the scope of spinal cord lesions, and inhibit the release of inflammatory factors. Therefore, the drugs described in the present invention provide a more effective direction for the treatment of nervous system diseases, especially the treatment method of mesenchymal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the cell morphology of the working bank cells;

[0036] Figure 2 This is the result of mesenchymal stem cell proliferation;

[0037] Figure 3 This is the result of mesenchymal stem cell differentiation;

[0038] Figure 4 The effect of hUC-MSC on novel object recognition in AD mice (±SEM), compared with the normal control group: ###P<0.001; compared with the model control group: ***P<0.001;

[0039] Figure 5 The effect of hUC-MSC on the escape latency of AD mice (±SEM), compared with the normal control group: ###P<0.001; compared with the model control group, ***P<0.001;

[0040] Figure 6 The effect of hUC-MSC on the number of crossing the platform in AD mice (±SEM), compared with the normal control group: ###P<0.001; compared with the model control group, **P<0.01, ***P<0.001;

[0041] Figure 7 The effect of hUC-MSC on TNF-α concentration in brain tissue homogenate of AD mice (±SD), compared with the normal control group: ###P<0.001; compared with the model control group, ***P<0.001;

[0042] Figure 8 The effect of hUC-MSC on the IL-1β concentration in brain tissue homogenate of AD mice (±SD), compared with the normal control group: ###P<0.001; compared with the model control group, ***P<0.001;

[0043] Figure 9 The effect of hUC-MSC on MDA in brain tissue homogenates of AD mice (±SD), compared with the normal control group: ###P<0.001; compared with the model control group, ***P<0.001;

[0044] Figure 10 The expression percentage of Aβ in brain tissue (±SD) is compared with the normal control group: ###P<0.001; compared with the model control group, *P<0.05;

[0045] Figure 11The expression percentage of Tau in brain tissue (±SD) is compared with the normal control group: ###P<0.001; compared with the model control group, *P<0.05, **P<0.01;

[0046] Figure 12 The effect of hUC-MSC on the neurological function score in the acute phase of ischemic stroke in rats (±SEM). Compared with the sham operation group: ###P<0.001; compared with the model control group: *P<0.05, **P<0.01, ***P<0.001;

[0047] Figure 13 The effect of hUC-MSC on the cerebral infarction range in the acute phase of ischemic stroke in rats (±SEM), compared with the sham operation group: ###P<0.001; compared with the model control group: *P<0.05, **P<0.01, ***P<0.001;

[0048] Figure 14 The effect of hUC-MSC on the oxidative stress factor MDA in the hippocampus of rats in the acute phase of ischemic stroke (±SEM). Compared with the sham operation group: ###P<0.001; compared with the model control group: *P<0.05, **P<0.01, ***P<0.001;

[0049] Figure 15 The effect of hUC-MSC on the oxidative stress factor SOD in the hippocampus of rats in the acute phase of ischemic stroke (±SEM). Compared with the sham operation group: ###P<0.001; compared with the model control group: *P<0.05, ***P<0.001;

[0050] Figure 16 The effect of hUC-MSC on BBB score after spinal cord injury in rats: compared with the sham operation group: ###P<0.001; compared with the model control group: *P<0.05, **P<0.01;

[0051] Figure 17 The effect of hUC-MSC on the extent of spinal cord lesions after spinal cord injury in rats, compared with the sham operation group: ###P<0.001; compared with the model control group: **P<0.01, ***P<0.001;

[0052] Figure 18 The effect of hUC-MSC on inflammatory factor IL-1β after spinal cord injury in rats (±SD), compared with the sham operation group: ###P<0.001; compared with the model control group: ***P<0.001;

[0053] Figure 19The effect of hUC-MSC on inflammatory factor IL-6 after spinal cord injury in rats (±SD), compared with the sham operation group: ###P<0.001; compared with the model control group: ***P<0.001;

[0054] Figure 20 The effect of hUC-MSC on inflammatory factor IFN-γ after spinal cord injury in rats (±SD), compared with the sham operation group: ###P<0.001; compared with the model control group: ***P<0.001;

[0055] Figure 21 The effect of hUC-MSC on inflammatory factor TNF-α after spinal cord injury in rats (±SD), compared with the sham operation group: ###P<0.001; compared with the model control group: *P<0.05, **P<0.01, ***P<0.001;

[0056] Figure 22 The secretion of HGF, TNFR1, BDNF, and GDNF by hUC-MSCs after siRNA treatment;

[0057] Figure 23 The expression of HGF, TNFR1, BDNF, and GDNF in hUC-MSCs was inhibited by siRNA;

[0058] Figure 24 The results of novel object recognition in mice (±SEM), compared with the normal control group: ##P<0.01;

[0059] Figure 25 The results are for the escape latency of mice (±SEM), compared with the normal control group: ###P<0.001; compared with the model control group: **P<0.01; compared with the hUC-MSC treatment group: &P<0.05;

[0060] Figure 26 The results are the number of times mice crossed the platform (±SEM), compared with the normal control group: ##P<0.01; compared with the model control group: *P<0.05; compared with the hUC-MSC treatment group: &P<0.05;

[0061] Figure 27 The results are the time mice stayed in the target quadrant (±SEM), compared with the model control group: *P<0.05, compared with the hUC-MSC treatment group: &P<0.05;

[0062] Figure 28The results are for the concentration of TNF-α in mouse brain tissue homogenate (±SD). Compared with the normal control group: ##P<0.01; compared with the model control group: ***P<0.001; compared with the hUC-MSC treatment group: &&P<0.01, &&&P<0.001;

[0063] Figure 29 The results are the IL-6 concentrations in mouse brain tissue homogenates (±SD). Compared with the normal control group: ###P<0.001; compared with the model control group: **P<0.01, ***P<0.001; compared with the hUC-MSC treatment group: &P<0.05, &&P<0.01;

[0064] Figure 30 The results are the IL-1β concentrations in mouse brain tissue homogenates (±SD). Compared with the normal control group: #P<0.05; compared with the model control group: *P<0.05;

[0065] Figure 31 The results are the concentrations of Aβ40 in mouse brain tissue homogenates (±SD). Compared with the normal control group: ###P<0.001; compared with the model control group: **P<0.01, ***P<0.001; compared with the hUC-MSC treatment group: &P<0.05, &&P<0.01;

[0066] Figure 32 The results are for the concentration of Tau in mouse brain tissue homogenate (±SD). Compared with the normal control group: ###P<0.001; compared with the model control group: ***P<0.001; compared with the hUC-MSC treatment group: &P<0.05, &&P<0.01;

[0067] Figure 33 The results are the MDA concentration in mouse brain tissue homogenate (±SD), compared with the normal control group: #P<0.05;

[0068] Figure 34 The results of neurological function scores in the acute phase of ischemic stroke in rats (±SEM): compared with the sham operation group: ###P<0.001; compared with the model control group: *P<0.05; compared with the hUC-MSC treatment group: &P<0.05;

[0069] Figure 35 The results are the results of cerebral infarction range of rats with ischemic stroke (±SEM). Compared with the sham operation group: ###P<0.001; compared with the model control group: **P<0.01; compared with the hUC-MSC treatment group: &P<0.05. DETAILED DESCRIPTION

[0070] The invention provides a culture medium for culturing mesenchymal stem cells, which uses DMEM / F12 culture medium as a basic culture medium and further comprises 8-20% v / v FBS, 4-15 μg / L bFGF and 5-10 μg / L glutamine.

[0071] The culture medium of the present invention uses DMEM / F12 culture medium as the basal culture medium, and there is no special limitation on the source of the basal culture medium. Conventional commercially available DMEM / F12 culture medium in the art can be used.

[0072] The culture medium of the present invention contains FBS, and the content of the FBS is 8-20% v / v, which can be 8% v / v, 9% v / v, 10% v / v, 11% v / v, 12% v / v, 13% v / v, 14% v / v, 15% v / v, 16% v / v, 17% v / v, 18% v / v, 19% v / v or 20% v / v. In one embodiment, 12% v / v is used as an example for illustration, but it cannot be considered as the entire protection scope of the present invention. In the present invention, the content can be a listed number or a decimal value between any two values.

[0073] The culture medium of the present invention contains bFGF, and the concentration of bFGF is 4 to 15 μg / L, such as 4 μg / L, 5 μg / L, 6 μg / L, 7 μg / L, 8 μg / L, 9 μg / L, 10 μg / L, 11 μg / L, 12 μg / L, 13 μg / L, 14 μg / L, or 15 μg / L. In one embodiment, 7.5 μg / L is used as an example for illustration, but this should not be considered the only bFGF in the entire scope of the present invention.

[0074] The culture medium of the present invention contains glutamine at a concentration of 5 to 10 μg / L, such as 5 μg / L, 6 μg / L, 7 μg / L, 8 μg / L, 9 μg / L, or 10 μg / L. In one embodiment, 5 μg / L is used as an example for illustration, but this alone cannot be considered the entire scope of protection of the present invention.

[0075] The culture medium of the present invention may further contain hydrogen, and the concentration of the hydrogen is 1 to 1.8 μg / mL, such as 1 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.7 μg / mL, or 1.8 μg / mL. In one embodiment, 1.5 μg / mL is used as an example for illustration, but it cannot be considered as the only concentration within the entire scope of the present invention.

[0076] In the culture medium of the present invention, FBS can provide basic nutrients for the growth and proliferation of mesenchymal stem cells; bFGF can bind to specific receptors on the cell surface to promote proliferation and maintain stemness; glutamine is one of the important energy substances in cells and plays a key role in the energy metabolism of cells; hydrogen can neutralize hydroxyl free radicals and regulate cell signaling pathways, has antioxidant and anti-apoptotic effects, and can reduce the damage of oxidative stress to cells.

[0077] The present invention also provides a method for culturing mesenchymal stem cells, comprising the following steps: incubating a tissue containing mesenchymal stem cells in a cell-free culture medium for a period of time, placing the tissue in the above-mentioned culture medium for culturing, and digesting the tissue after the cell confluence reaches 70-90% to obtain P0 mesenchymal stem cells;

[0078] The P0 mesenchymal stem cells were collected after centrifugation and subcultured using the above culture medium.

[0079] The types of mesenchymal stem cells that can be cultured using the methods of the present invention include any of the following: adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells. The tissue containing these mesenchymal stem cells can be adipose tissue, umbilical cord, placenta, umbilical cord blood, bone marrow, or dental pulp. In one embodiment, umbilical cord-derived mesenchymal stem cells were cultured as an example.

[0080] The present invention requires that the tissue be broken into tissue blocks and then incubated twice in a non-cell culture medium environment, such as cutting the umbilical cord tissue into small segments with a diameter of 1 to 1.5 mm without washing in order to preserve the original environment of the tissue; then directly inoculating each umbilical cord tissue block into a T75 culture bottle for the first incubation, and no cell culture medium is added during the first incubation; after the first incubation is completed, a second incubation is performed, and no cell culture medium is added during the second incubation. The temperature of the first incubation of the present invention is 38 to 39 ° C, the CO2 concentration is 4 to 6%, the O2 concentration is 2 to 20%, and the time is 1 to 2 hours; the temperature of the second incubation of the present invention is 36 to 37 ° C, the CO2 concentration is 4 to 6%, the O2 concentration is 18 to 20%, and the time is 5 to 7 hours.

[0081] After the second incubation, the culture medium is added to the culture flask and cultured until the cell confluence reaches 80%, followed by digestion to obtain P0 mesenchymal stem cells. During the culture process, the culture medium needs to be replaced every 3 to 5 days, and the culture parameters include: a temperature of 36 to 37°C, a CO2 concentration of 4 to 6%, an O2 concentration of 2 to 20%, and a culture time of 48 to 72 hours.

[0082] The present invention centrifuges the P0 mesenchymal stem cells and discards the supernatant. The centrifugation includes centrifugation at 300-400g for 5 minutes. The culture medium is added to the cell pellet and subcultured until the P5 generation, thereby obtaining a working bank of cells for cryopreservation. The subculture parameters of the present invention include a temperature of 37°C, a CO2 concentration of 4-6%, an O2 concentration of 18-20%, and a subculture time of 48-72 hours per generation.

[0083] The present invention also provides an injection of mesenchymal stem cells, comprising mesenchymal stem cells cultured using the above method and a cell preservation solution, wherein the content of mesenchymal stem cells in the injection is (1-10)×10 6 / mL;

[0084] The cell preservation solution contains the following components at the following concentrations: a compound electrolyte injection solution of 10-20 mg / mL human serum albumin and 60-80 IU / mL heparin.

[0085] The cell preservation solution of the present invention contains 10-20 mg / mL human serum albumin, such as 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL or 20 mg / mL.

[0086] The cell preservation solution of the present invention is a compound electrolyte injection containing 60-80 IU / mL of heparin, such as 60 IU / mL, 61 IU / mL, 62 IU / mL, 63 IU / mL, 64 IU / mL, 65 IU / mL, 66 IU / mL, 67 IU / mL, 68 IU / mL, 69 IU / mL, 70 IU / mL, 71 IU / mL, 72 IU / mL, 73 IU / mL, 74 IU / mL, 75 IU / mL, 76 IU / mL, 77 IU / mL, 78 IU / mL, 79 IU / mL or 80 IU / mL.

[0087] The compound electrolyte injection of the present invention includes the following components in the following concentrations: 5.26 g / L of sodium chloride, 5.02 g / L of sodium gluconate, 3.68 g of sodium acetate (C2H3NaO2·3H2O), 0.37 g / L of potassium chloride, and 0.30 g / L of magnesium chloride (MgCl2·6H2O).

[0088] In the cell preservation solution of the present invention, human serum albumin provides cells with necessary nutrients and stabilizes the structure and function of cell membranes; the addition of heparin can prevent cells from aggregating; sodium gluconate can provide an energy source for cell metabolism; and other injection solution components can regulate cell signal transduction and maintain acid-base balance and cell osmotic pressure balance.

[0089] The present invention also provides a method for preparing the above-mentioned injection, comprising the following steps: culturing the mesenchymal stem cells obtained by the above-mentioned method to 80% cell confluence and then digesting them, collecting the cells by centrifugation and washing them, and resuspending them in a cell preservation solution to prepare the injection of the mesenchymal stem cells.

[0090] In one embodiment of the present invention, the injection solution is prepared using frozen working bank cells. The frozen working bank cells are first revived. The method for resuscitating the cells is not particularly limited, and mesenchymal stem cells can be revived using conventional methods in the art. The cells are then washed using the compound electrolyte injection solution, for example, three times.

[0091] The present invention also provides the use of mesenchymal stem cells cultured by the above method in preparing medicines for treating nervous system diseases.

[0092] In a preferred embodiment of the present invention, the neurological disease includes at least one of the following: Alzheimer's disease, ischemic stroke, Parkinson's disease, multiple sclerosis, spinal cord injury, epilepsy, dementia, depression, neuropathic pain, nervous system infection, demyelinating disease of the nervous system, movement disorders and mental disorders, such as Alzheimer's disease, ischemic stroke or spinal cord injury.

[0093] The present invention also provides a medicine for treating nervous system diseases, comprising mesenchymal stem cells cultured by the above method and pharmaceutically acceptable excipients.

[0094] In a preferred embodiment of the present invention, the dosage form of the drug includes any of the following: drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, powders, emulsions, solutions, pills, pills, buccal tablets, freeze-dried powder injections, gels, suppositories, and aerosols. The present invention does not specifically limit the content of the excipients, and the excipients corresponding to the drug dosage form in the art can be used. The present invention requires that the number of mesenchymal stem cells in the drug reaches (1 to 10) × 10 6 / mL or (1~10)×10 6 / g.

[0095] The present invention also provides a method for detecting the biological efficacy of mesenchymal stem cells in treating nervous system diseases, comprising detecting the ability of mesenchymal stem cells to secrete sTNFR1, HGF, BDNF and / or GDNF;

[0096] The mesenchymal stem cells include any one of the following sources: mesenchymal stem cells cultured by the above method, the above injection solution and the above medicine.

[0097] Before using the mesenchymal stem cells or mesenchymal stem cell products of the present invention, especially before using them for the treatment of nervous system diseases, it is necessary to test the biological efficacy of the mesenchymal stem cells in treating nervous system diseases. The judgment criteria for the biological efficacy of the mesenchymal stem cells in treating nervous system diseases of the present invention include at least one of the following: per 1×10 6 The amount of sTNFR1 secreted by mesenchymal stem cells within 48 hours was 200-1000 pg; 6 The amount of HGF secreted by mesenchymal stem cells in 48 hours is 100-300 ng; 6 The amount of BDNF secreted by mesenchymal stem cells in 48 hours is 300-1200 pg; 6 The amount of GDNF secreted by each mesenchymal stem cell within 48 hours is 300-800 pg. The present invention has no particular limitation on the detection method of the above four proteins. For example, an ELISA kit is used for detection in the examples.

[0098] To further illustrate the present invention, the culture method, application and detection method of mesenchymal stem cells for treating nervous system diseases provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0099] In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each example are the same.

[0100] Table 1 Experimental materials

[0101]

[0102] Example 1

[0103] 1. Human umbilical cord tissue was obtained from healthy fetuses born by cesarean section at a cooperative obstetrics and gynecology hospital. Umbilical cord tissue from healthy donors exceeding 15 cm was selected and cut into small segments with a diameter of 1-1.5 mm without washing to preserve the tissue's original environment. Each umbilical cord tissue fragment was then directly plated and inoculated into a T75 culture flask and incubated in a 38°C incubator with 5% CO2 and 2% O2 for 1 hour without the addition of cell culture medium. Following incubation, each flask containing umbilical cord tissue was incubated in a 37°C incubator with 5% CO2 and 20% O2 for an additional 5 hours without the addition of cell culture medium. Following incubation, 5 mL of complete medium-1 was added to each flask. Following overnight incubation, 10 mL of complete medium-1 was added. Thereafter, complete medium-1 was replaced every 3-5 days. When the cells reached 80% confluency, they were digested with trypsin to obtain P0 human umbilical cord mesenchymal stem cells.

[0104] 2. Centrifuge the P0 human umbilical cord mesenchymal stem cells obtained in step 1 at 300-400 g for 5 minutes, discard the supernatant, add complete culture medium-1, and place them in an incubator at 37°C in a 5% CO2, 20% O2 environment for subculture for 72 hours, which is recorded as P1.

[0105] 3. Centrifuge the P1 human umbilical cord mesenchymal stem cells obtained in step 2 at 400 g for 5 minutes, discard the supernatant, add complete culture medium-1, and incubate in a 37°C incubator with 5% CO2 and 20% O2 for 72 hours, designated as P2. Centrifuge the P2 human umbilical cord mesenchymal stem cells at 400 g for 5 minutes, discard the supernatant, and collect the cells for use as a seed bank.

[0106] 4. The seed bank cells obtained in step 3 were cultured in complete medium-1 to passage P5 and frozen to obtain working bank cells (denoted as W1). The culture conditions were: complete medium-1, 37°C, 20% CO2.

[0107] The components of the complete culture medium-1 include: DMEM / F12 culture medium containing 12% v / v FBS, 7.5 μg / L bFGF and 5 μg / L glutamine.

[0108] Example 2

[0109] The only difference from Example 1 is that the culture of umbilical cord tissue, separation, acquisition and culture of P0 cells are all placed in a 5% CO2, 2% O2 incubator at 37°C to obtain working bank cells (denoted as W2).

[0110] Example 3

[0111] The only difference from Example 1 is that the culture of umbilical cord tissue, the expansion and culture of cells from generation P0 to generation P5 all use complete culture medium-2 to obtain working bank cells (denoted as W3).

[0112] The complete culture medium-2 comprises the following components: DMEM / F12 culture medium containing 12% v / v FBS, 7.5 μg / L bFGF, 5 μg / L glutamine and 1.5 μg / mL hydrogen.

[0113] Example 4

[0114] The only difference from Example 3 is that the culture of umbilical cord tissue, separation, acquisition and culture of P0 cells are all placed in a 5% CO2, 2% O2 incubator at 37°C to obtain working bank cells (denoted as W4).

[0115] Comparative Example 1

[0116] The only difference from Example 1 is that no bFGF was added to the complete culture medium 1, and working bank cells (denoted as W5) were obtained.

[0117] Comparative Example 2

[0118] The only difference from Example 1 is that the FBS in the complete culture medium 1 is 20%, and working bank cells (denoted as W6) are obtained.

[0119] Comparative Example 3

[0120] The only difference from Example 1 is that the bFGF in the complete culture medium 1 is 15 μg / L, and working bank cells (denoted as W7) are obtained.

[0121] Experimental Example 1

[0122] The working bank cells (W1-W7) prepared in Examples 1 to 4 and Comparative Examples 1 to 3 were tested, and the test items were as follows:

[0123] (1) Safety testing for bacteria, mycoplasma, viruses, etc.;

[0124] Sterility testing was performed according to the sterility inspection method in 1101 of the General Rules of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; mycoplasma testing was performed according to the first method in 3301 of the General Rules of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; according to the instructions of the kit, the q-PCR method was used to complete the working library cell human Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV) and other viral tests. All q-PCR kits used were products of Daan Gene Co., Ltd.

[0125] The test results showed that the working library cells W1-W7 were all negative for bacteria, mycoplasma and viruses.

[0126] (2) Cell morphology;

[0127] Use an inverted microscope to observe the cell status of the cells in the cell status working library. Figure 1 As shown, it can be seen that except for W5, the cells in other groups are in good condition, with single cells in a spindle shape and fused growth in a spindle shape; W5 cells show aging.

[0128] (3) cell proliferation;

[0129] The working bank cells were obtained and seeded into a 96-well plate at 10,000 cells per well. Cell proliferation was determined by CCK-8 assay at 0 h, 24 h, 48 h, 72 h, and 96 h after seeding.

[0130] The results of the test are as follows Figure 2As shown, the cell proliferation ability of W1-W7 was strong, among which W4 had the strongest proliferation ability compared with the other 6 groups, while W5 had the worst proliferation ability.

[0131] (4) flow phenotyping;

[0132] Take the working bank cells and perform the following operations: collect 5×10 6 The cells were evenly divided into 10 centrifuge tubes and centrifuged at 300 g for 5 minutes, then the supernatant was discarded. The following antibodies were added respectively: PE-IgG1, PE-CD73, PE CD90, PE-CD105, PE-CD45, PE-HLA-DR, PE-CD11b, FITC-IgG1, FITC-CD19 and FITC-CD34 (antibodies from BD Biosciences, USA). Cell surface antigens were detected by BD Calibur flow cytometer.

[0133] The measurement results are shown in Table 2: The working library cells (W1-W7) all highly expressed CD73, CD90 and CD105, and did not express CD19, CD34, CD45, CD11b and HLA-DR.

[0134] Table 2 Flow cytometry phenotypes of working library cells

[0135]

[0136]

[0137] (5) osteogenic and adipogenic differentiation capacity;

[0138] Take working bank cells W1-W7 and perform the following operations:

[0139] Each group of cells was 1×10 4 / well inoculated into 24-well plates, and after the cell confluence reached about 80%, the adipogenic and osteogenic induction medium (Gibco products) was replaced every 2 to 3 days. On the 21st day of induction culture, the cells were fixed with 4% paraformaldehyde solution at room temperature for 30 minutes, and then stained with 10% Oil Red-O and observed under a microscope for adipogenic differentiation, and stained with Alizarin Red solution and observed under a microscope for osteogenic differentiation. The results are shown in Figure 2. Figure 3 , W1-W7 mesenchymal stem cells all have the ability to differentiate into osteoblasts and adipocytes, among which W4 has the strongest differentiation ability, followed by W3 and W2, while W5 has the weakest differentiation ability.

[0140] (6) Biological efficacy (sTNFR1, HGF, BDNF, GDNF) detection.

[0141] Working bank cells were seeded into 6-well plates and cultured for 48 hours. The supernatant was collected and digested for cell counting. ELISA was used to determine the levels of sTNFR1, HGF, BDNF, and GDNF in the supernatant.

[0142] The test results are shown in Table 3: W1-W7 working library cells can express and secrete sTNFR1, HGF, BDNF, and GDNF. Among them, the factor secretion levels of the four groups W4 are higher than those of the other six groups, and the levels of sTNFR1, HGF, BDNF and GDNF secreted by group W5 are the lowest.

[0143] Table 3 Contents of sTNFR1, HGF, BDNF, and GDNF

[0144]

[0145] Example 5

[0146] The working bank cells W1 prepared above were revived and cultured to 80% cell confluency. The cells were then trypsinized, centrifuged, washed three times with compound electrolyte injection, and resuspended in cell preservation solution to prepare a mesenchymal stem cell injection solution, designated P1. The injection volume was 20 mL, and the cell count was 1E+7.

[0147] The cell preservation solution is a compound electrolyte injection containing 10 mg / mL human serum albumin and 65 IU / mL heparin.

[0148] Example 6

[0149] The working bank cells W2 prepared above were taken, revived and cultured to 80% cell confluence, then trypsinized, centrifuged, washed three times with compound electrolyte injection, and resuspended in cell preservation solution to prepare mesenchymal stem cell injection, recorded as P2.

[0150] The cell preservation solution is a compound electrolyte injection containing 10 mg / mL human serum albumin and 65 IU / mL heparin.

[0151] Example 7

[0152] The working bank cells W3 prepared above were taken, revived and cultured to 80% cell confluence, then trypsinized, centrifuged, washed three times with compound electrolyte injection, and resuspended in cell preservation solution to prepare mesenchymal stem cell injection, recorded as P3.

[0153] The cell preservation solution is a compound electrolyte injection containing 10 mg / mL human serum albumin and 65 IU / mL heparin.

[0154] Example 8

[0155] The working bank cells W4 prepared above were taken, revived and cultured to 80% cell confluence, then trypsinized, centrifuged, washed three times with compound electrolyte injection, and resuspended in cell preservation solution to prepare mesenchymal stem cell injection, recorded as P4.

[0156] The cell preservation solution is a compound electrolyte injection containing 10 mg / mL human serum albumin and 65 IU / mL heparin.

[0157] Example 9

[0158] The working bank cells W5 prepared above were taken, revived and cultured to 80% cell confluence, then trypsinized, centrifuged, washed three times with compound electrolyte injection, and resuspended in cell preservation solution to prepare mesenchymal stem cell injection, which was recorded as P5.

[0159] The cell preservation solution is a compound electrolyte injection containing 10 mg / mL human serum albumin and 65 IU / mL heparin.

[0160] Experimental Example 2: Testing the Mesenchymal Stem Cell Injections Prepared in Examples 5 to 9

[0161] 2.1 Inspection of visible foreign matter

[0162] Visible foreign matter was detected according to the Visible Foreign Matter Inspection Method in accordance with the General Chapter 0904 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0163] 2.2 Insoluble particle inspection

[0164] The insoluble particles were inspected according to the first method of Part III 0903 of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0165] 2.3 Filling quantity inspection

[0166] The filling quantity is checked in accordance with General Rule 0102 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0167] 2.4 Osmotic pressure test

[0168] The osmotic pressure molar concentration was determined according to General Rule 0632 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0169] 2.5 Cell number and cell viability

[0170] A cell counter was used to count cells, and the stem cell preparation to be tested was stained with 0.04% trypan blue (1:1), and the cell number was read and the viability was calculated.

[0171] 2.6 Sterility testing

[0172] Sterility testing was performed according to the sterility test method in General Chapter 1101 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0173] 2.7 Mycoplasma Detection

[0174] Mycoplasma testing was performed according to the first method in General Chapter 3301 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0175] 2.8 Endotoxin detection

[0176] Endotoxin testing was performed according to the gel method in General Chapter 1143 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0177] 2.9sTNFR1, HGF, BDNF, GDNF results

[0178] The obtained mesenchymal stem cell injection (P1-P5) cells were divided into 1×10 4 / cm 2 The cells were inoculated into T75 culture flasks and cultured for 48 h. The supernatant was harvested, the cells were digested and counted, and the contents of sTNFR1, HGF, BDNF and GDNF in the samples were detected according to the instructions of the sTNFR1, HGF, BDNF and GDNF kits.

[0179] Pharmacopeia standards for visible foreign matter, insoluble particles, volume variation, osmotic pressure, cell count, cell viability, sterility, mycoplasma testing, and endotoxin testing are shown in Table 4. The test results for mesenchymal stem cell injections (P1-P5) are shown in Table 5, and all tested qualified. The test results for sTNFR1, HGF, BDNF, and GDNF are shown in Table 6.

[0180] Table 4 Pharmacopoeia standards

[0181]

[0182]

[0183] Table 5 Test results of mesenchymal stem cell injection

[0184]

[0185] Table 6 Results of NGF, HGF, BDNF and GDNF in mesenchymal stem cell injection

[0186]

[0187] Experimental Example 3 Effect of Mesenchymal Stem Cell Injection on AD Mice

[0188] The experiment was conducted using 32-week-old male APP / PS1 Alzheimer's disease mouse AD model.

[0189] AD mice were randomly divided into 6 groups: AD model control group, mesenchymal stem cell injection group (P1-P5), 12 mice in each group, and a normal control group of 12 C57BL / 6J mice.

[0190] APP / PS1 mice were dosed starting at 8 months of age, with a 2-week interval for a total of 3 doses, namely 0, 2, and 4 weeks. The endpoint of the study was the end of the 6-week post-dose period (day 42). The test drug was injected into the tail vein of each group with mesenchymal stem cell injection at a dose of 2×10 6 cells / mouse, volume 0.2 mL / mouse; the normal control group and the model control group were injected with equal volume of normal saline into the tail vein.

[0191] 3.1 New Object Recognition and Detection

[0192] Alzheimer's disease (AD) mice were subjected to environmental adaptation training and novel object recognition test before administration and 38-39 days after the first administration (10-11 days after the last administration).

[0193] The results are as follows Figure 4 As shown in the results, compared with the normal control group, the model control group mice had a significantly lower degree of exploration of novel objects (P < 0.001). Compared with the model control group, three intravenous injections of mesenchymal stem cell injections (P1-P5) significantly enhanced the AD mice's ability to explore novel objects (P < 0.001), with the P4 group having the best effect in improving novel object recognition.

[0194] 3.2 Water maze test

[0195] AD mice were trained and tested in water maze before administration and 40-42 days after the first administration (12-14 days after the last administration).

[0196] The results are as follows Figure 5 and Figure 6 As shown, compared with the normal control group, the model control group mice showed obvious symptoms of Alzheimer's disease: memory and behavioral impairment, manifested by a significant increase in escape latency and a significant decrease in the number of platform crossings (P<0.001). Compared with the model control group, three treatments with mesenchymal stem cell injections (P1-P5) significantly shortened the escape latency and increased the number of platform crossings in AD mice (P<0.01-0.001), with the P4 treatment group showing the best effect, followed by the P3 treatment group.

[0197] 3.3 Brain oxidative stress, ELISA inflammatory factor detection and immunohistochemistry detection

[0198] At the end of the experiment, 42 days after the first dose (14 days after the last dose), the animals were dissected and brain tissue homogenates were obtained. MDA levels in the brain were measured using a kit; IL-1β and TNF-α levels in the brain were measured using ELISA; and Aβ and Tau protein expression in brain tissue was determined using immunohistochemistry.

[0199] The results are as follows Figure 7-11 As shown, compared with the normal control group, the concentrations of TNF-α and IL-1β in brain tissue homogenates of mice in the model control group were significantly increased (P<0.001). Three intravenous injections of mesenchymal stem cell injections (P1-P5) significantly reduced TNF-α and IL-1β concentrations in brain tissue of AD mice compared with the model control group (P<0.001). Mesenchymal stem cell injections in group P4 had the greatest effect on reducing TNF-α and IL-1β, followed by group P3. Compared with the normal control group, the MDA content in brain tissue homogenates of mice in the model control group was significantly increased (P<0.001). Three intravenous injections of mesenchymal stem cell injections (P1-P5) significantly reduced MDA content compared with the model control group (P<0.001). Immunofluorescence / histochemistry assays for the expression of Aβ and Tau proteins in brain tissue revealed that Aβ and Tau expression in the model control group was significantly increased compared with the normal control group (P<0.001). Among them, three intravenous injections of mesenchymal stem cell injection in the P4 and P3 groups could significantly reduce the expression of Aβ, Tau, etc. in the brain tissue of AD mice.

[0200] Experimental Example 4 Effect of Mesenchymal Stem Cell Injection on MACO Mice

[0201] An ischemic stroke rat model (MACO stroke model) was established using the silk suture method. First, the internal carotid artery was exposed, and cerebral blood flow to the middle cerebral artery was blocked using a fiber suture to induce transient occlusion. Two hours after the initiation of MCAO, the suture was carefully removed from the internal carotid artery lumen, and the internal carotid artery was allowed to reperfuse for 24 hours to complete the model.

[0202] Two hours after cerebral ischemia-reperfusion, the animals were blindly scored for the degree of neurological impairment. Those with obvious neurological deficits (≥8 points) were considered successful. The rats with successful modeling were randomly divided into 6 groups according to the scores, namely the model control group and the mesenchymal stem cell injection group (P1-P5), with 10 rats in each group; another 10 rats were set as the sham operation group. The experimental endpoint was 3 days after the drug. The drug was administered 1 day after cerebral ischemia-reperfusion, with a single tail vein injection. The mesenchymal stem cell injection group (P1-P5) was given 2×10 hUC-MSCs, respectively. 7 / kg; the model control group and sham operation group were given equal volume of normal saline.

[0203] 4.1 Neurological function score

[0204] Neurological function scores were evaluated before administration and 1, 2, and 3 days after administration (total score is 16 points).

[0205] Table 7 Scoring criteria for neurological impairment in MCAO rats

[0206]

[0207]

[0208] The results are as follows Figure 12 As shown in the results, compared with the sham-operated group, rats with ischemic stroke showed significant neurological dysfunction, manifested by forelimb flexion and circling toward the hemiplegic side (P<0.001). Compared with the model control group, a single injection of mesenchymal stem cells (P1-P5) improved the neurological function scores of the model rats to varying degrees. The P4 group showed the best improvement in neurological function scores 3 days after the mesenchymal stem cell injection, followed by the P3 group, and the P5 group had the worst therapeutic effect.

[0209] 4.2 Total Trace Column (TTC)

[0210] Three days after administration, rats were anesthetized with isoflurane gas, bled, and sacrificed. Brains were then frozen at -20°C and sliced ​​from front to back, each 2 mm thick. Brain sections were incubated in 2% tetrazolium tetrazolium (TTC) solution at 37°C for 5 minutes. Infarcted tissue appears white, while non-infarcted tissue appears red. Image J software was used to measure infarct area, and the percentage of infarcted area to total brain area was calculated.

[0211] Percentage of cerebral infarction area (%) = cerebral infarction area / total brain area × 100%.

[0212] The results are as follows Figure 13 As shown in the results, compared with the sham-operated group, rats with ischemic stroke developed significant cerebral infarction (P<0.001). Compared with the model control group, a single injection of mesenchymal stem cells (P1-P5) reduced the size of cerebral infarction in the model rats to varying degrees (P<0.05-0.001). The P4 group showed the best improvement in cerebral infarction size 3 days after administration (P<0.001), followed by the P3 group, and the P5 group had the worst therapeutic effect.

[0213] 4.3 Hippocampal oxidative stress factors (MDA, SOD)

[0214] Three days after administration, the rats were anesthetized with isoflurane gas, blood was collected, and the rats were killed to remove the brain. The hippocampus tissue was removed and a 10% tissue homogenate was prepared with normal saline using an ultrasonic disruptor. The mixture was centrifuged at 3000 rpm for 10 min and the supernatant was collected. The levels of MDA and SOD were detected using kits.

[0215] The results are as follows Figure 14-15 As shown in the results, compared with the sham operation group, the MDA level in the hippocampus of the stroke model control group was significantly increased (P<0.001), while the SOD level was significantly decreased (P<0.001). Compared with the model control group, mesenchymal stem cell injection (P1-P5) could reduce the MDA level in the hippocampus of rats with ischemic stroke to varying degrees (P<0.05-0.001) and increase the SOD level in the hippocampus of the model rats (P<0.05-0.001). The mesenchymal stem cell injection in the P4 group had the best improvement effect, followed by the P3 group, and the P5 group had the worst treatment effect.

[0216] Experimental Example 5 Effect of Mesenchymal Stem Cell Injection on Rat Spinal Cord Injury Model

[0217] A spinal cord injury model in SD rats was established using the Allen's method. This model has good clinical relevance and is suitable for spinal cord injury research. After anesthesia, the rats were immobilized in the prone position. The dorsal incision was clipped and disinfected with iodine. A prominent dorsal protuberance, representing the spinous process of the T10 vertebra, was observed. A 3-cm incision was made around this protuberance. The subcutaneous fascia and muscle were then dissected layer by layer to expose the spinous process, lamina, and dura mater of the spinal cord. The rats were then secured under a JK052 impactor. A 10g impactor was used to impact the T10 thoracic spinal cord with a 5-cm free-fall impactor. After impact, the impactor was removed. Immediately after impact, the rats were observed. Sterile gauze was applied to the wound to stop bleeding. The wound was repeatedly irrigated with sterile saline. The exposed spinal cord area was covered with superficial fascia tissue. The wound was sutured layer by layer, and the dorsal skin was disinfected again with iodine. 1 mL of 4 U / mL penicillin was injected intramuscularly into the leg muscles of each rat for three consecutive days. After the blow, the spinal cord at the injury site becomes congested and edematous, the rat's tail swings spasmodically, and both hind limbs and trunk retract and flutter. After waking up from anesthesia, both lower limbs become flaccid, indicating that the model is successfully established.

[0218] The rats with successful modeling were divided into 6 groups: model control group, mesenchymal stem cell injection group (P1-P5), 10 rats in each group, and 10 rats in sham operation group. A single drug treatment was given 24 hours after modeling. The mesenchymal stem cell injection group (P1-P5) was given a dose of 2×10 7 / kg, and the model control group and sham operation group were given equal volume of normal saline.

[0219] 5.1 Behavioral scoring of rat spinal cord injury model

[0220] The rats' motor function was analyzed using the BBB behavioral scoring method. See the BBB scoring table for rat spinal cord injury. Animals were blindly evaluated before surgery (normal values), before cell injection, and 3, 7, 14, and 28 days after cell injection.

[0221] Table 8 BBB score table for spinal cord injury in rats

[0222]

[0223]

[0224]

[0225] The results are as follows Figure 16 As shown, the BBB scores of rats in the sham-operated group were normal, while those in the model group with spinal cord injury were significantly reduced (P < 0.001), indicating successful model establishment. Three days after injection, the BBB scores of rats in the mesenchymal stem cell injection groups (P1-P5) were significantly improved (P < 0.05-0.01). Specifically, P4 mesenchymal stem cells significantly improved the BBB scores of rats with spinal cord injury (P < 0.01).

[0226] 5.2 Spinal cord lesion extent

[0227] Three days after cell injection, magnetic resonance imaging (MRI) was used to observe the spinal cord structure and injury location of the rats at the coronal position.

[0228] Three days after cell injection, magnetic resonance imaging (MRI) was used to observe the spinal cord structure and injury location of the rats at the coronal position. Figure 17 As shown, the spinal cord injury lesions in the model control group were significantly greater (P < 0.001), indicating successful modeling. Three days after injection, the mesenchymal stem cell injection groups (P1-P5) significantly reduced the extent of the spinal cord lesions (P < 0.01-0.001). The mesenchymal stem cell injection had the greatest improvement in the P4 group, followed by the P3 group, and the least effective in the P5 group.

[0229] 5.3 Factor Detection

[0230] Blood was collected from the inner canthus 1, 3, and 7 days after cell injection, and the levels of TNF-α, IL-1β, IL-6, and IFN-γ in serum were detected by Luminex.

[0231] 1 day, 3 days, and 7 days after mesenchymal stem cell injection, 200 μL of blood was collected from the inner canthus of the eye and centrifuged at 7500 rpm for 15 minutes to collect serum. The levels of TNF-α, IL-1β, IL-6, and IFN-γ in the serum were detected by Luminex. Figures 18-21As shown, serum levels of inflammatory factors such as IFN-γ, TNF-α, IL-6, and IL-1β in the model control group were higher than those in the sham-operated group (P < 0.001), indicating a more severe inflammatory response in the model group. Mesenchymal stem cell injection significantly reduced serum levels of inflammatory factors such as IFN-γ, TNF-α, IL-6, and IL-1β at 1, 3, and 7 days of administration in the mesenchymal stem cell injection groups (P1-P5) (P < 0.001). This suggests that hUC-MSCs have pharmacological activity by inhibiting the release of inflammatory factors and antagonizing inflammatory responses. Mesenchymal stem cell injection in group P4 showed the best inhibitory effect on inflammatory factors, followed by group P3, and the worst effect in group P5.

[0232] Experimental Example 6 Mechanism of action of mesenchymal stem cell injection on AD mice

[0233] The experiment was conducted using 32-week-old male APP / PS1 Alzheimer's disease mouse AD model.

[0234] AD mice were randomly divided into six groups: AD model control group, mesenchymal stem cell injection group (hUC-MSC), HGF knockdown mesenchymal stem cell injection group (siHGF-MSC), TNFR1 knockdown mesenchymal stem cell injection group (siTNFR1-MSC), BDNF knockdown mesenchymal stem cell injection group (siBDNF-MSC), and GDNF knockdown mesenchymal stem cell injection group (siGDNF-MSC), with 9 mice in each group. A normal control group of 9 C57BL / 6J mice was also set up. HGF siRNA was synthesized by Suzhou Hongxun (SEQ ID No. 1: CCAAUGUGCUAAUAGAUGUdTdT, SEQ ID No. 2: ACAUCUAUUAGCACAUUGGdTdT); TNFR1 siRNA was purchased from Thermo, catalog number s14266; BDNF siRNA was purchased from Thermo, catalog number s1964; and GDNF siRNA was purchased from Thermo, catalog number s5694.

[0235] APP / PS1 mice were dosed starting at 8 months of age, with dosing intervals of 2 weeks for a total of 3 doses, namely, 0, 2, and 4 weeks. The endpoint of the study was the end of the 6th week after dosing (day 42). The test drugs were injected into the tail vein of each group with hUC-MSC, siHGF-MSC, siTNFR1-MSC, siBDNF-MSC injection, and siGDNF-MSC injection, respectively, at a dose of 2×10 6 cells / mouse, volume 0.2 mL / mouse; the normal control group and the model control group were injected with equal volume of normal saline into the tail vein.

[0236] HGF, TNFR1, BDNF, GDNF siRNA were used to silence the genes of hUC-MSCs, respectively. The concentrations of HGF, TNFR1, BDNF, and GDNF in the supernatant of hUC-MSCs were detected by ELISA. Figure 22-23 As shown, the four siRNFs can significantly reduce the secretion levels of HGF, TNFR1, BDNF, and GDNF by hUC-MSC (p < 0.001), and the inhibition efficiency of HGF, TNFR1, BDNF, and GDNF expression is more than 80%.

[0237] New object recognition and detection

[0238] AD mice were subjected to environmental adaptation training and novel object recognition test before administration and 38 to 39 days after the first administration (10 to 11 days after the last administration).

[0239] The results are as follows Figure 24 As shown, the model control group mice showed reduced exploration of novel objects (P<0.01), while the hUC-MSC treatment group significantly improved the exploration of novel objects in AD mice (P=0.054). Compared with the hUC-MSC group, the other treatment groups showed a reduced improvement in novel object recognition in AD mice, and there were no significant differences compared with the model control group (P>0.05).

[0240] Water maze test

[0241] AD mice were trained and tested in water maze before administration and 40-42 days after the first administration (12-14 days after the last administration).

[0242] The results are as follows Figures 25-27 As shown, compared with the normal control group, mice in the model control group exhibited significant memory and behavioral impairments consistent with Alzheimer's disease: escape latency was significantly prolonged (P<0.001) and the number of platform crossings was significantly decreased (P<0.01). Compared with the model control group, the hUC-MSC group significantly shortened the escape latency of AD mice (P<0.01), increased the number of platform crossings (P<0.05), and increased the time mice spent in the target quadrant (P<0.05). Compared with the hUC-MSC group, the therapeutic effects of the siHGF-MSC, siTNFR1-MSC, siBDNF-MSC, and siGDNF-MSC groups were significantly reduced (P<0.05).

[0243] Brain oxidative stress, ELISA inflammatory factor detection and immunohistochemistry detection

[0244] Forty-two days after the first dose (14 days after the last dose) marked the experimental endpoint. The animals were dissected and brain tissue homogenates were obtained. Brain MDA levels were measured using a kit, and ELISA was used to measure brain levels of inflammatory factors such as IL-6, IL-1β, TNF-α, and Aβ40 and Tau proteins.

[0245] The results are as follows Figures 28-30 As shown in the results, compared with the normal control group, the concentrations of inflammatory factors TNF-α, IL-6, and IL-1β in the brain tissue homogenates of mice in the model control group were significantly increased (P<0.05-0.001). Compared with the model control group, the hUC-MSC group significantly reduced the levels of inflammatory factors such as TNF-α (P<0.001), IL-6 (P<0.001), and IL-1β (P<0.05) in the brain tissues of AD mice. Compared with the hUC-MSC group, the TNF-α concentration in the brain tissues of AD model mice in the siHGF-MSC group, siTNFR1-MSC group, siBDNF-MSC group, and siGDNF-MSC group was significantly increased (P<0.001), approaching that of the model control group. The IL-6 concentration in the siHGF-MSC group, siTNFR1-MSC group, siBDNF-MSC group, and siGDNF-MSC group was significantly increased compared with the hUC-MSC group (P<0.05-0.01).

[0246] The results are as follows Figures 31-32 As shown in the results, compared with the normal control group, the Aβ40 and Tau concentrations in the model control group were significantly increased (P<0.001). Compared with the model control group, the hUC-MSC group significantly reduced the Aβ40 and Tau contents in the brain tissue of AD mice (P<0.01-0.001), while the siHGF-MSC group, siTNFR1-MSC group, siBDNF-MSC group, and siGDNF-MSC group significantly decreased the effects of reducing Aβ40 and Tau contents (P<0.05-0.01).

[0247] The results are as follows Figure 33 As shown in the figure, compared with the normal control group, the MDA content in the model control group was significantly increased (P<0.05). Compared with the model control group, hUC-MSC reduced the MDA content; while the MDA levels in the siHGF-MSC group, siTNFR1-MSC group, siBDNF-MSC group, and siGDNF-MSC group increased, with the MDA level in the siTNFR1-MSC group approaching the level of the model group.

[0248] Experimental Example 7 Mechanism of action of mesenchymal stem cell injection on MACO mice

[0249] An ischemic stroke rat model (MACO stroke model) was established using the silk suture method. First, the internal carotid artery was exposed, and cerebral blood flow to the middle cerebral artery was blocked using a fiber suture to induce transient occlusion. Two hours after the initiation of MCAO, the suture was carefully removed from the internal carotid artery lumen, and the internal carotid artery was allowed to reperfuse for 24 hours to complete the model.

[0250] Two hours after cerebral ischemia-reperfusion, the animals were blindly scored for the degree of neurological impairment. Those with obvious neurological deficits (≥8 points) were considered successful. The rats with successful modeling were randomly divided into 6 groups according to the scores, namely model control group, hUC-MSC group, siHGF-MSC group, siTNFR1-MSC group, siBDNF-MSC group, and siGDNF-MSC group, with 10 rats in each group; another 10 rats were set as sham operation group. The experimental endpoint was 3 days after the drug. The drug was administered 1 day after cerebral ischemia-reperfusion, with a single tail vein injection. Each mesenchymal stem cell injection group was given the corresponding hUC-MSC 2×10 7 / kg; the model control group and sham operation group were given equal volume of normal saline.

[0251] Neurological function score

[0252] According to the scoring criteria in Table 7, neurological function scores were performed before administration and 1, 2, and 3 days after administration.

[0253] The results are as follows Figure 34 As shown, compared with the sham-operated group, rats in the model group exhibited significant neurological dysfunction, such as forelimb flexion and circling toward the hemiplegic side (P<0.001). hUC-MSC treatment improved the neurological function scores of model rats compared with the model control group (P<0.05). Compared with the hUC-MSC group, the neurological improvement effect of each siRNA gene silencing group was significantly attenuated 3 days after treatment (P<0.05). This suggests that the neurological improvement effect of hUC-MSC is correlated with HGF, TNFR1, BDNF, or GDNF.

[0254] TTC

[0255] Three days after administration, rats were anesthetized with isoflurane gas, bled, and sacrificed. Brains were then frozen at -20°C and sliced ​​from front to back, each 2 mm thick. Brain sections were incubated in 2% tetrazolium tetrazolium (TTC) solution at 37°C for 5 minutes. Infarcted tissue appears white, while non-infarcted tissue appears red. Image J software was used to measure infarct area, and the percentage of infarcted area to total brain area was calculated.

[0256] Percentage of cerebral infarction area (%) = cerebral infarction area / total brain area × 100%.

[0257] The results are as follows Figure 35 As shown, compared with the sham-operated group, rats in the model group developed significant cerebral infarction (P<0.001). Compared with the model control group, hUC-MSC reduced the size of cerebral infarction (P<0.01). There were no significant differences between the siRNA gene silencing groups and the model control group (P>0.05). Compared with the hUC-MSC group, the effect of siRNA gene silencing on reducing cerebral infarction size was significantly attenuated in all groups (P<0.05). This suggests that the effect of hUC-MSC on reducing cerebral infarction size is correlated with HGF, TNFR1, BDNF, or GDNF.

[0258] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A culture medium for culturing mesenchymal stem cells, characterized in that The basic culture medium is DMEM / F12 culture medium, and further comprises 8-20% v / v FBS, 4-15 μg / L bFGF and 5-10 μg / L glutamine.

2. The culture medium according to claim 1, characterized in that The culture medium also includes hydrogen, and the concentration of the hydrogen is 1 to 1.8 μg / mL.

3. A method for culturing mesenchymal stem cells, characterized in that: The following steps are involved: After incubating the tissue containing mesenchymal stem cells in a cell-free culture medium for a period of time, the tissue is placed in the culture medium according to claim 1 or 2 for culture, and digested after the cell confluence reaches 70-90%, thereby obtaining P0 mesenchymal stem cells; The P0 mesenchymal stem cells are collected after centrifugation and subcultured using the culture medium according to claim 1 or 2.

4. The method according to claim 3, characterized in that The incubation includes breaking the tissue into tissue blocks, directly inoculating them into a culture device, and sequentially performing a first incubation for 1 hour and a second incubation for 5 hours; The first incubation temperature is 38-39°C, the CO2 concentration is 4-6%, the O2 concentration is 2-20%, and the time is 1-2 hours; The second incubation temperature is 36-37° C., the CO 2 concentration is 4-6%, the O 2 concentration is 18-20%, and the time is 5-7 hours.

5. The method according to claim 3, characterized in that: The culture temperature is 36-37°C, the CO2 concentration is 4-6%, the O2 concentration is 2-20%, and the culture time is 48-72 hours; The temperature of the subculture is 37° C., the CO 2 concentration is 4-6%, the O 2 concentration is 18-20%, and the subculture time of each generation is 48-72 hours.

6. A mesenchymal stem cell injection, characterized in that: Comprising mesenchymal stem cells cultured by the method according to any one of claims 3 to 5 and a cell preservation solution; The content of mesenchymal stem cells in the injection is (1-10)×10 6 / mL; The cell preservation solution contains the following components at the following concentrations: a compound electrolyte injection solution of 10-20 mg / mL human serum albumin and 60-80 IU / mL heparin.

7. The method for preparing the injection according to claim 6, characterized in that: The method comprises the following steps: culturing mesenchymal stem cells obtained by the method according to any one of claims 3 to 5 to 80% cell confluence, digesting the cells, collecting the cells by centrifugation, washing the cells, and resuspending the cells in a cell preservation solution to prepare an injection of the mesenchymal stem cells.

8. Use of mesenchymal stem cells cultured by the method according to any one of claims 3 to 5 in the preparation of drugs for treating nervous system diseases.

9. A stem cell drug for treating nervous system diseases, characterized in that: The method comprises mesenchymal stem cells cultured by the method according to any one of claims 3 to 5 and pharmaceutically acceptable excipients.

10. A method for detecting the biological efficacy of mesenchymal stem cells in treating nervous system diseases, characterized in that: This includes testing the ability of mesenchymal stem cells to secrete sTNFR1, HGF, BDNF, and / or GDNF; The sources of the mesenchymal stem cells include any one of the following: mesenchymal stem cells cultured using the method according to any one of claims 3 to 5, the injection according to claim 6, and the medicine according to claim 9.

11. The method according to claim 10, characterized in that: The criteria for judging the biological efficacy of the mesenchymal stem cells in treating nervous system diseases include at least one of the following: Every 1×10 6 The amount of sTNFR1 secreted by mesenchymal stem cells within 48 hours was 200-1000 pg; Every 1×10 6 The amount of HGF secreted by each mesenchymal stem cell within 48 hours is 100-300 ng; Every 1×10 6 The amount of BDNF secreted by mesenchymal stem cells within 48 hours is 300-1200 pg; Every 1×10 6 The amount of GDNF secreted by mesenchymal stem cells within 48 hours is 300-800 pg.

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