慢病毒介导的RNA干扰下调p16的表达对hUC-MSCs衰老的影响韩 康,马珊珊,朱相展,孟 楠,王欣欣,
Effect of lentivirus-mediated RNAi targeting p16 on aging of hUC-MSCs

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(hUC-MSCs)中p16基因的表达,研究p16表达下调与hUC-MSCs衰老的关系。方法:实验分为第3代(P3)hUC-MSCs组(P3组)、P15 hUC-MSCs组(P15组)、LV-sip16感染P15 hUC-MSCs组(LV-sip16组)和LV-NC感染P15 hUC-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、pCNA、sirt1、sirt2、CyclinD1、CDK4 mRNA的表达。结果:LV-sip16感染能促进P15 hUC-MSCs增殖,降低衰老细胞率,促使细胞周期进入S期,抑制hUC-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pCNA、sirt1、sirt2、CyclinD1、CDK4 mRNA表达量升高(P均＜0.05)。结论:下调P15 hUC-MSCs中p16基因的表达能够延缓和改善hUC-MSCs的衰老。
Aim: To study the effect of lentivirus(LV)-mediated RNAi targeting p16 on the aging of hUC-MSCs.Methods: There were 4 groups,the 3rd generation hUC-MSCs group(P3 group), the 15th generation hUC-MSCs group(P15 group), P15 hUC-MSCs infected by LV-sip16 group(LV-sip16 group)and P15 hUC-MSCs infected by LV-NC group(negative control group). CCK-8 assay was performed to detect cell proliferation, β-galactosidase staining was used to detect aging cells, flow cytometry was used to detect cell cycle and apoptosis, and the expressions of p16,p21,p53,pCNA,sirt1,sirt2,CyclinD1 and CDK4 mRNAwere detected by qRT-PCR.Results: LV-sip16 infection could successfully promote the proliferation of P15 hUC-MSCs, reduce aging cell rate, induce S phase cells, and inhibit the early cell apoptosis(P＜0.05); meanwhile, the mRNA expressions of p16, p21, p53 were decreased, while the mRNA expressions of pCNA, sirt1, sirt2, CyclinD1 and CDK4 were increased(P＜0.05).Conclusion: Downregulating the expression of p16 in P15 hUC-MSCs could delay and improve the aging of hUC-MSCs