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目的探讨miR-20b过表达对P19细胞增殖和细胞分化的影响。方法将包含鼠miR-20b成熟序列的慢病毒载体及空载体分别感染P19细胞,通过嘌呤霉素筛选2周,建立稳定过表达鼠mR-20b的P19细胞系。用Real-time PCR的方法验证稳定细胞表达株。用CCK-8方法检测稳定感染miR-20b过表达慢病毒和空载体的P19细胞间细胞增殖的差别。将稳定感染的两组细胞以无血清培养液37℃孵育24小时使细胞停留于G0期,而后恢复血清培养,取不同时间点的细胞,使用流式细胞仪检测两组间细胞周期的差别,从而间接验证CCK-8的结果。以1%二甲基亚砜(DMSO)将两组稳定感染的P19细胞诱导分化为心肌细胞,Real-time PCR检测心肌分化相关标志基因的表达,并用光镜观察分化过程中的形态变化。结果建立了稳定表达miR-20b的P19细胞系,与空载体组相比,miR-20b过表达可抑制P19细胞的增殖,促进心肌分化相关标志基因的表达(P<0.05,P<0.01或P<0.001)。结论 miR-20b过表达可显著抑制P19细胞的增殖,并具有促进P19细胞向心肌细胞分化的作用。
Objective To investigate the effect of miR-20b overexpression on proliferation and differentiation of P19 cells. Methods P19 cells were infected with lentiviral vector and empty vector containing the mature sequence of mouse miR-20b respectively. The P19 cell line stably overexpressing mR-20b was established by puromycin selection for 2 weeks. Real-time PCR was used to verify stable cell-expressing strains. The difference of cell proliferation between P19 cells stably infected with miR-20b overexpression lentivirus and empty vector was detected by CCK-8 assay. The stable infected two groups of cells in serum-free medium incubated at 37 ℃ for 24 hours to stay in G0 cells, and then restore the serum culture, take cells at different time points, the use of flow cytometry to detect differences in cell cycle between the two groups, This indirectly verifies the result of CCK-8. Two groups of stable infected P19 cells were induced to differentiate into cardiomyocytes with 1% dimethylsulfoxide (DMSO). Real-time PCR was used to detect the expression of cardiomyocyte differentiation-related marker genes. Morphological changes during differentiation were observed by light microscopy. Results The P19 cell line stably expressing miR-20b was established. Compared with the empty vector group, miR-20b overexpression inhibited the proliferation of P19 cells and promoted the expression of myocardial marker genes (P <0.05, P <0.01 or P <0.001). Conclusion Overexpression of miR-20b can significantly inhibit the proliferation of P19 cells and promote the differentiation of P19 cells into cardiomyocytes.