Smoothened慢病毒干扰载体的构建及其对人牙周膜干细胞增殖与分化的影响

Construction of lentiviral RNAi vectors containing human smoothened gene and its inhibitory effect on proliferation and differentiation in human periodontal ligament stem cells

目的体外构建及筛选人Smoothened(SMO)基因的慢病毒干扰载体,使用此载体下调人牙周膜干细胞(human periodontal ligament stem cells,PDLSCs)中SMO基因表达并检测其对细胞增殖与分化的影响。方法针对人SMO基因设计3条干扰序列,构建RNA干扰慢病毒载体,在293FT细胞中包装病毒液并转染293A细胞,通过实时荧光定量PCR检测干扰效率,得到具有最佳干扰效率的病毒后,转染PDLSCs,用Western blot检测其对PDLSCs中SMO基因的干扰效果及对细胞分化的影响,CCK-8法检测其对PDLSCs增殖的影响。结果成功构建3个SMO慢病毒干扰载体(pGP-SMO1、pGP-SMO2、pGP-SMO3),通过转染293A细胞并进行实时荧光定量PCR检测后,得到pGP-SMO1具有最佳干扰效率,转染后SMO mRNA水平下降到对照的(28.5±2.5)%。利用此病毒能够很好地转染体外培养的PDLSCs,并使PDLSCs中SMO蛋白水平下降80%。进一步检测细胞增殖发现,与未转染组比较,SMO下调后PDLSCs增殖变慢[(1.09±0.20)vs(1.86±0.21),P<0.01]。同时也导致成骨分化标志物Runx2的蛋白表达下调。结论利用慢病毒介导的干扰病毒载体成功干扰了PDLSCs SMO基因的表达,SMO基因的下调会导致PDLSCs增殖减慢,成骨分化能力减弱。

Objective To construct the lentiviral vector of human smoothened （SMO） gene which can down-regulate the SMO expression in human periodontal ligament stem cells （PDLSCs）. Methods According to the human SMO gene, 3 sequences were designed to build RNA interference lentiviral vector. All the 3 vectors were packaged in 293FT cells, and then transfected into the 293A cells. The interference efficiency was evaluated by quantitative PCR, and the optimal one were transfected into the PDLSCs. Western blotting were used to detect the interference effect of SMO gene and the effect on differentiation in PDLSCs. CCK-8 assay was used to test the effect of recombinant lentivirus on the proliferation of PDLSCs. Results Three SMO lentiviral RNAi vectors were constructed successfully and the lentiviral vector 1 was considered as the optimal one by quantitative PCR. The mRNA level of SMO was reduced by （28.5±2.5）% by this vector. The protein expression of SMO was decreased by 80% in the PDLSCs after transfected by the lentivector vector 1 when compared with the cells without transfection. This transfection also resulted in a decreased cell proliferation （1.090 9±0.199 2 vs 1.863 0±0.206 2, P〈0.01） and a decreased expression of bone differentiation marker, RUNX2. Conclusion A lentiviral vector of SMO is successfully constructed, and SMO interference suppresses the proliferation in PDLSCs, and inhibits the differentiation of PDLSC to osteoblasts.