Repair regeneration type stem cell for treating Parkinson's disease as well as preparation method and application of repair regeneration type stem cell

Abstract

The invention provides a repair and regeneration type stem cell for treating Parkinson's disease as well as a preparation method and application thereof, and belongs to the technical field of cell culture. Comprising the following steps: inoculating MSC into a culture medium 1, carrying out low-speed centrifugation, carrying out standing culture, and after cells are adhered to the wall, replacing a culture medium 2 for continuous culture so as to prepare MSC; on the basis of a DMEM culture medium, the culture medium 1 also comprises the following components: recombinant transferrin, a basic fibroblast growth factor, a platelet-derived growth factor, cell fibronectin and insulin; the culture medium 2 is prepared from the following components on the basis of a DMEM / F12 culture medium: geniposide, a basic fibroblast growth factor, a platelet-derived growth factor, resveratrol, SB431542, CHIR-99021 and sodium selenite. According to the invention, cell proliferation can be promoted, secretion of cell factors such as BDNF can be enhanced, cell aging or apoptosis can be reduced, and cell viability can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a repair and regeneration stem cell for treating Parkinson's disease, and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with the potential for self-renewal and multidirectional differentiation. They are widely found in tissues such as bone marrow, fat, umbilical cord, and dental pulp. Due to their unique biological properties, MSCs have shown tremendous potential for application in regenerative medicine, immunomodulation, tissue engineering, and disease treatment, becoming a hot topic in current biomedical research. They are effective in treating neurological diseases such as Parkinson's disease, Alzheimer's disease, and spinal cord injury. However, current MSC culture techniques often suffer from issues such as low proliferation efficiency, susceptibility to senescence, cell apoptosis, and unstable survival rates.

[0003] Parkinson's disease is a neurodegenerative disorder. The main cause of this disease is the degeneration and death of dopamine neurons in the substantia nigra, which is related to multiple factors such as genetics, environmental factors, and aging of the nervous system. It is generally recognized that aging is the most important factor in the development of Parkinson's disease, and the disease has a significant high incidence in the elderly. MSCs can mainly exert their therapeutic effects through the following pathways: 1. Secreting growth factors such as BDNF, GDNF, and NGF to promote the survival of dopamine neurons; 2. Reducing pro-inflammatory factors such as IL-6 and TNF-α; 3. Transferring healthy mitochondria to damaged neurons through exosomes; 4. Enhancing the autophagy pathway (LC3-II / Beclin-1 upregulation).

[0004] The present invention aims to provide a method for preparing repair and regenerative stem cells, improve the proliferation and survival ability of MSCs, promote the homing of MSCs in the body, increase the production of multiple growth factors, and thus enhance the therapeutic effect on neurodegenerative diseases such as Parkinson's disease. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing repair and regenerative stem cells, improve the proliferation and survival ability of MSCs, and promote the secretion of growth factors such as BDNF and GDNF.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing repair and regenerative stem cells for treating Parkinson's disease, comprising the following steps:

[0008] (1) MSCs were seeded into culture medium 1, centrifuged at low speed, and then cultured statically;

[0009] (2) After the cells adhered to the wall, medium 2 was replaced and cultured to obtain MSCs;

[0010] The culture medium 1 is based on the DMEM culture medium, and the following components are added: 8-12 mg / L recombinant transferrin, 6-10 μg / L basic fibroblast growth factor, 4-6 μg / L platelet-derived growth factor, 50-100 μg / mL cell fibronectin, and 8-12 mg / L insulin;

[0011] The culture medium 2 is based on DMEM / F12 culture medium, and is supplemented with the following components at the following concentrations: 15-20 μg / L geniposide, 6-10 μg / L basic fibroblast growth factor, 8-10 μg / L platelet-derived growth factor, 1-2 μM resveratrol, 3-5 μM SB431542, 1-2 μM CHIR-99021, and 1-1.5 μg / L sodium selenite.

[0012] Preferably, the initial density of MSC cells in step (1) is (0.5-1)×10 4 cells / mL.

[0013] Preferably, the low-speed centrifugation conditions in step (1) are: 200-300×g, 5-8 min.

[0014] Preferably, the static culture conditions in step (1) are 35-37° C. and 4-6% CO 2 .

[0015] Preferably, the process of continuing the culture in step (2) is: using culture medium 2 for culture, replacing fresh culture medium every day, culturing until the cells are 70-80% confluent, washing and digesting, collecting the single cell suspension, centrifuging, discarding the supernatant, and then using culture medium 2 for subculture to isolate mesenchymal stem cells.

[0016] More preferably, the centrifugal speed is 1400-1600 r / min and the time is 5 min.

[0017] More preferably, during subculture, the initial subculture ratio is 1:1-2, and the subsequent subculture ratio is 1:3-6, and the subculture conditions are: 37-39°C, 3-5% CO 2 .

[0018] The present invention also provides a repair and regeneration stem cell culture kit for treating Parkinson's disease, comprising a culture medium 1 and a culture medium 2.

[0019] The present invention also provides a repair and regeneration stem cell preparation for treating Parkinson's disease.

[0020] The present invention also provides the use of the stem cell preparation in preparing a medicine for treating Parkinson's disease.

[0021] The present invention can not only improve the proliferation and survival ability of MSCs, but also enhance the ability of MSCs to secrete BDNF and GDNF. Compared with Comparative Example 4, the contents of BDNF and GDNF in the cell supernatant are increased by about 38.77% and 32.05%, respectively.

[0022] The culture medium 1 of the present invention can promote cell proliferation and cell adhesion; the culture medium 2 can enhance the self-renewal ability of stem cells, promote cell proliferation, enhance the secretion of cytokines such as BDNF, etc. by adding cell growth factors, geniposide, resveratrol, and small molecule inhibitors such as SB431542 and CHIR-99021. It also has a certain antioxidant capacity, can reduce cell aging or apoptosis, and improve cell viability. DETAILED DESCRIPTION

[0023] The third generation hUC-MSCs isolated from Wharton's jelly tissue of the umbilical cord of naturally delivered newborns were used for the experiment.

[0024] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] A repair and regeneration stem cell culture kit for treating Parkinson's disease:

[0027] Medium 1

[0028] The following components were added to the DMEM medium: 10 mg / L recombinant transferrin, 10 μg / L basic fibroblast growth factor, 5 μg / L platelet-derived growth factor, 50 μg / mL cell fibronectin, and 10 mg / L insulin;

[0029] Medium 2

[0030] The following components were added to the DMEM / F12 medium: 15 μg / L geniposide, 10 μg / L basic fibroblast growth factor, 8 μg / L platelet-derived growth factor, 1 μM resveratrol, 5 μM SB431542, 1 μM CHIR-99021, and 1 μg / L sodium selenite.

[0031] Example 2

[0032] A repair and regeneration stem cell culture kit for treating Parkinson's disease:

[0033] Medium 1

[0034] The following components were added to the DMEM medium: 8 mg / L recombinant transferrin, 6 μg / L basic fibroblast growth factor, 4 μg / L platelet-derived growth factor, 60 μg / mL cell fibronectin, and 10 mg / L insulin;

[0035] Medium 2

[0036] The following components were added to the DMEM / F12 medium: 18 μg / L geniposide, 8 μg / L basic fibroblast growth factor, 8 μg / L platelet-derived growth factor, 1 μM resveratrol, 4 μM SB431542, 1.5 μM CHIR-99021, and 1 μg / L sodium selenite.

[0037] Example 3

[0038] A repair and regeneration stem cell culture kit for treating Parkinson's disease:

[0039] Medium 1

[0040] The following components were added to the DMEM medium: 12 mg / L recombinant transferrin, 10 μg / L basic fibroblast growth factor, 5 μg / L platelet-derived growth factor, 100 μg / mL cell fibronectin, and 12 mg / L insulin;

[0041] Medium 2

[0042] The following components were added to the DMEM / F12 medium: 20 μg / L geniposide, 10 μg / L basic fibroblast growth factor, 10 μg / L platelet-derived growth factor, 1 μM resveratrol, 5 μM SB431542, 2 μM CHIR-99021, and 1 μg / L sodium selenite.

[0043] Example 4

[0044] (1) MSCs were cultured at an initial density of 1×10 4 cells / mL was inoculated into the medium 1 described in Example 3, centrifuged at 200×g for 5 min, and then incubated at 37°C in 5% CO2;

[0045] (2) After the cells adhered to the wall, the medium 1 was discarded and an equal amount of the medium 2 described in Example 3 was added for culturing. The medium 2 was replaced every day. After the cells were cultured until 70% confluence, they were washed with PBS solution and digested for 3 min with 2 mL of 0.25% trypsin solution. The digestion was terminated with an equal amount of low-glucose DMEM medium. The single cell suspension was collected and centrifuged at 1500 r / min for 5 min. The supernatant was discarded and the medium 2 was added to adjust the cell density to 2 × 105 The cells were subcultured at a ratio of 1:1 for the first subculture and 1:5 for the next subculture. When the cell fusion degree reached 80%, MSCs were collected after digestion, neutralization, and washing according to the subculture requirements.

[0046] Comparative Example 1

[0047] The difference from Example 4 is that in step (1), the culture medium 1 has the following formula: based on the DMEM culture medium, 10 μg / L basic fibroblast growth factor and 5 μg / L platelet-derived growth factor are added.

[0048] Comparative Example 2

[0049] Different from Example 4, the culture medium 2 in step (2) is as follows: on the basis of DMEM / F12 culture medium, the following components are added at the following concentrations: 10 μg / L basic fibroblast growth factor, 10 μg / L platelet-derived growth factor, 2 μM CHIR-99021, and 0.8 μg / L sodium selenite.

[0050] Comparative Example 3

[0051] The difference from Example 4 is that the culture medium 2 in step (2) is as follows: on the basis of DMEM / F12 culture medium, the following components are added at the following concentrations: 20 μg / L total saponins of Panax notoginseng, 10 μg / L basic fibroblast growth factor, 10 μg / L platelet-derived growth factor, 1 μM L-ascorbic acid, 5 μM SB431542, 2 μM CHIR-99021 and 0.8 μg / L sodium selenite.

[0052] Comparative Example 4

[0053] Different from Example 4, the commercially available mesenchymal stem cell (MSC) serum-free culture medium purchased from Beijing Kelin Biotechnology Co., Ltd. was used in step (2).

[0054] Test Example 1

[0055] Evaluation of cell proliferation ability:

[0056] The P3 cells obtained by the methods of Example 4 and Comparative Examples 1 to 4 were taken and 2×10 4 The cells were seeded at a density of 100 cells / mL in a 12-well plate and divided into 4 groups, each with 12 wells. 1 mL of the culture medium 2 of the corresponding embodiment and comparative example was added to each well, and the plates were cultured in a 37°C, 5% CO2 incubator. After culturing for 7 days, the collected MSC cells were counted using a cell counter and 0.4% trypan blue staining method, and the cell activity was calculated. The results are shown in Table 1.

[0057] Table 1 Cell counting results

[0058] Example 4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 <![CDATA[Counting result (×10 6 pcs)]]> 4.26 3.64 3.21 3.85 2.14 Cell viability (%) 98.25 90.25 82.56 89.21 70.25

[0059] As shown in Table 2, the cell proliferation ability of Example 4 is significantly higher than that of Comparative Examples 1 to 4, indicating that the preparation method provided by the present invention can improve the proliferation ability and survival ability of MSCs.

[0060] Test Example 2

[0061] The MSCs collected from Example 4 and Comparative Examples 1 to 4 were collected at 8×10 4 Cells were seeded in 6-well plates at a density of 100 cells / mL. When the cells covered 90% of the well surface, the culture medium was aspirated and cultured for 72 hours with medium containing 1% FBS. The supernatant was collected, filtered through a 0.22 μm filter, and replaced with medium containing 1% FBS as a blank control. ELISA kits for BDNF (CB12019-Hu) and GDNF (CB10801-Hu) (Coab Biosciences) were used to measure the levels of BDNF and GDNF in the supernatant of each group and in the blank control, respectively. The results are shown in Table 2.

[0062] Table 2 Contents of BDNF and GDNF (pg / mL)

[0063]

[0064]

[0065] As shown in Table 2, the secretion levels of BDNF and GDNF in Example 4 were significantly higher than those in Comparative Examples 1 to 4. This indicates that the preparation method provided by the present invention can significantly enhance the ability of MSC cells to secrete BDNF and GDNF, promote the survival of dopamine neurons, and contribute to the subsequent treatment of neurodegenerative diseases such as Parkinson's disease.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing repair and regenerative stem cells for treating Parkinson's disease, characterized in that: The following steps are involved: (1) MSCs were seeded into culture medium 1, centrifuged at low speed, and then cultured statically; (2) After the cells adhered to the wall, medium 2 was replaced and cultured to obtain MSCs; The culture medium 1 is based on the DMEM culture medium and further includes the following components at the following concentrations: 8-12 mg / L recombinant transferrin, 6-10 μg / L basic fibroblast growth factor, 4-6 μg / L platelet-derived growth factor, 50-100 μg / mL cell fibronectin and 8-12 mg / L insulin; The culture medium 2 is based on DMEM / F12 culture medium and also includes the following components at the following concentrations: 15-20 μg / L geniposide, 6-10 μg / L basic fibroblast growth factor, 8-10 μg / L platelet-derived growth factor, 1-2 μM resveratrol, 3-5 μM SB431542, 1-2 μM CHIR-99021 and 1-1.5 μg / L sodium selenite.

2. The preparation method according to claim 1, wherein The initial density of MSC cells in step (1) is (0.5-1)×10 4 cells / mL.

3. The preparation method according to claim 2, wherein The low-speed centrifugation conditions in step (1) are: 200-300×g, 5-8 min.

4. The preparation method according to claim 3, wherein The static culture conditions in step (1) are 35-37° C. and 4-6% CO 2 .

5. The preparation method according to claim 4, wherein: The process of continuing the culture in step (2) is as follows: culture using culture medium 2, replacing fresh culture medium every day, culturing until the cells are 70-80% confluent, washing and digesting, collecting the single cell suspension, centrifuging, discarding the supernatant, and then using culture medium 2 for subculture to isolate mesenchymal stem cells.

6. The preparation method according to claim 5, characterized in that: The centrifugal speed is 1400-1600 r / min, and the time is 5 minutes.

7. The preparation method according to claim 6, characterized in that: During subculture, the initial subculture ratio is 1:1-2, and the subsequent subculture ratio is 1:3-6. The subculture conditions are: 37-39°C, 3-5% CO2.

8. A repair and regeneration stem cell culture kit for treating Parkinson's disease, characterized in that: The kit comprises the culture medium 1 and the culture medium 2 used in the preparation method according to any one of claims 1 to 7.

9. A repair and regeneration stem cell preparation for treating Parkinson's disease, prepared by the preparation method according to any one of claims 1 to 7.

10. Use of the stem cell preparation according to claim 9 in the preparation of a drug for treating Parkinson's disease.

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