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Objective To investigate the effect of oleanolic acid (OA) in the maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) through switching pyruvate kinase type M isoforms. Methods HiPSC-CMs were derived from human induced pluripotent stem cells (hiPSCs), through sequential activation and inhibition of the Wnt signaling pathway. On day 19 hiPSC-CMs were divided into control group (RPMI 1640+B27), DMSO group (RPMI 1640+B27+2 μl DMSO), and 10 μmol/L OA group (RPMI 1640+B27+2 μl 5 mmol/L OA) and treated for 7 days, and the medium was changed every 2 days. Immunofluorescence or RT-qPCR were used to detect the expression of stem factor in hiPSCs and cardiac specific markers in hiPSC-CMs. Western blotting was used to detect the expression of PK and mitofusin 2 (MFN2). Mito-Tracker staining showed the morphology of mitochondria, and transmission electron microscope (TEM) showed the ultrastructure of mitochondria. EDU staining was used to detect cell proliferation, and flow cytometry was used to detect cell cycle. Results Immunofluorescence showed that hiPSCs expressed Nanog and Sox2, and hiPSC-CMs expressed myocardial specific markers cTnT, Cx43 and α-actinin.RT-qPCR showed that, compared with hiPSCs, significant differences (P<0.05 or P<0.01) existed in hiPSC-CMs expressed troponin I3 (TNNI3), myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). Immunofluorescence showed that, compared with control group and DMSO group, the cell area increased, sarcomere length increased, and the cell circularity decreased in hiPSC-CMs cultured with OA, and the differences were statistically significant (P<0.05); Western blotting showed that, compared with control group and DMSO group, the ratio of PKM1/PKM2 and the expression of MFN2 increased obviously, and the differences were statistically significant (P<0.05 or P<0.01), the fusion lengthening of mitochondria was observed under electron microscope, and Mito-Tracker staining showed that mitochondria formed more mature structure; EDU staining showed a decreased cell proliferation in hiPSC-CMs cultured with OA group compared with control group and DMSO group, all differences were statistically significant(P<0.05). The flow cytometry results indicated that the number of coenocytes increased in hiPSC-CMs cultured with OA group compared with control group and DMSO group. Conclusion OA can promote the maturation of cell structure, mitochondrial structure and morphology, and also can promote cell multinucleation. OA can promote hiPSC-CMs' maturation by switching pyruvate kinase type M isoforms.
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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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