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目的:利用慢病毒(LV)介导的RNA干扰下调老龄人脐带间充质干细胞(h UC-MSCs)中p16基因的表达,研究p16表达下调与h UC-MSCs衰老的关系。方法:实验分为第3代(P3)h UC-MSCs组(P3组)、P15 h UC-MSCs组(P15组)、LV-sip16感染P15 h UC-MSCs组(LV-sip16组)和LV-NC感染P15 h UC-MSCs组(空病毒组)。采用CCK-8法检测细胞增殖,β-半乳糖苷酶染色计算衰老细胞率,流式细胞术检测细胞周期及凋亡,qRT-PCR检测衰老相关基因p16、p21、p53、p CNA、sirt1、sirt2、Cyclin D1、CDK4 mRNA的表达。结果:LV-sip16感染能促进P15 h UCMSCs增殖,降低衰老细胞率,促使细胞周期进入S期,抑制h UC-MSCs的早期凋亡(P均<0.05);同时,LV-sip16组细胞中p16、p21、p53 mRNA表达量较P15组降低,而p CNA、sirt1、sirt2、Cyclin D1、CDK4 mRNA表达量升高(P均<0.05)。结论:下调P15 h UC-MSCs中p16基因的表达能够延缓和改善h UC-MSCs的衰老。
OBJECTIVE: To study the relationship between the downregulation of p16 expression and the senescence of h UC-MSCs by lentivirus (LV) -mediated RNAi knockdown of p16 gene in human umbilical cord mesenchymal stem cells (h UC-MSCs). Methods: The experiment was divided into the third generation (P3) h UC-MSCs group (P3 group), the P15 h UC-MSCs group (P15 group), LV-sip16 infected P15 h UC-MSCs group -NC infected P15 h UC-MSCs group (empty virus group). The cell proliferation was detected by CCK-8 assay, the percentage of senescent cells was calculated by β-galactosidase staining, the cell cycle and apoptosis were detected by flow cytometry, the expression of p16, p21, p53, p CNA, sirt1, sirt2, Cyclin D1, CDK4 mRNA expression. Results: LV-sip16 infection could promote the proliferation of P15 h UCMSCs, decrease the rate of senescent cells, promote the cell cycle into S phase and inhibit the early apoptosis of h UC-MSCs (all P <0.05). Meanwhile, p16 , P21, p53 mRNA expression decreased compared with P15 group, while p CNA, sirt1, sirt2, Cyclin D1, CDK4 mRNA expression increased (P all <0.05). Conclusion: The down-regulation of p16 gene expression in UC-MSCs can delay and improve the senescence of h UC-MSCs.