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Objective To construct a complete chemical culture system based on small molecule compounds and study its role in promoting the reversal of senescence of mesenchymal stem cells (MSCs). Methods MSCs were transmitted to 20 generations (P20) continuously. The model of the replicative aging cells (P20-MSCs) was established, then divided into aging model group and small molecule treatment group, and the fifth generation of umbilical cord MSCs (P5-MSCs) was set as control group. The aging model group and control group were cultured in the whole chemical culture system for 7 days, and small molecular treatment group was cultured in the whole chemical culture system containing valproate and Repsox for 7 days. β-galactosidase staining was used to detect cell senescence, immunofluorescence staining was used to detect the protein expression levels of Ki-67, OCT4, Nanog,P16, and P21; RT-qPCR to detect the mRNA expression levels of OCT4, Nanog, P16 and P21. Flow cytometry was used to detect the anti-apoptotic ability of cells. Migration experiments, Transwell invasion experiments and clone formation experiments to detect small molecules effects on migration, invasion and self-cloning functions of senescent MSCs. Results The cell bodies of senescent mesenchymal stem cells were enlarged and presented dendritic processes. After incubation in the full chemical culture system, the cells return to a spindle shape or irregular triangle similar to young MSCs. SA-β-gal staining results showed that compared with aging model group, the positive rate of galactosidase in cells treated with small molecules was significantly reduced, and the difference was statistically significant (45.00%±1.23% vs. 84.80%±1.50%, P<0.001). The immunofluorescence results showed that compared with aging model group, the proportion of positive cells expressing Ki-67, OCT4 and Nanog increased (Ki-67: 89.00%±1.50%vs. 25.00%±2.00%, P<0.001; OCT4: 88.40%±0.80% vs. 25.40%±1.20%, P<0.001; Nanog: 76.30%±1.70% vs. 10.50%±0.60%,P<0.001), the proportion of positive cells expressing P16, P21 decreased (P16: 64.00%±3.20% vs. 98.00%±1.50%, P<0.05; P21:45.00%±1.10% vs. 82.00%±2.00%, P<0.05) in small molecule treatment group. RT-qPCR results showed that compared with aging model group, small molecule compounds could up-regulate the mRNA expression levels of OCT4 and Nanog in aging MSCs(P<0.001), down-regulate the mRNA expression levels of P16 and P21 (P<0.05). Compared with aging model group, the abilities of anti-apoptosis (21.60%±1.20% vs. 31.40%±0.80%), migration (49.30%±3.30% vs. 30.60%±4.40%), invasion [(90.00±12.00)cells vs. (34.00±9.00) cells] and self-cloning abilities [(144.00±10.00) cells vs. (68.00±7.00) cells] in small molecule treatment group were significantly increased, and the differences were statistically significant (P<0.05 or P<0.01). Conclusion The constructed small molecule full chemical culture system can inhibit and partially reverse the aging process of long-term cultured MSCs in vitro.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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