使用慢病毒载体干扰人牙周膜干细胞中 - 第三军医大学学报.doc

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 张洁1，常慧君3，舒绍兵1, 2罗金英1，徐志玲汤玲重庆大学生物医学工程学院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 ）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基因设计三条干扰序列，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，转染人牙周膜干细胞，用western blot检测其对干细胞中SMO基因的干扰效果及对细胞分化的影响。CCK-8法检测其对干细胞增殖的影响。结果：成功构建三个SMO慢病毒干扰载体，通过转染293A细胞并进行定量PCR检测后，得到1#干扰载体具有最佳干扰效率，在mRNA水平达到70%。SMO基因的表达。进一步检测细胞增殖发现SMO下调后牙周膜干细胞增殖变慢。成骨分化标志物Runx-2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的SMO基因的表达，SMO基因的下调会导致牙周膜干细胞增殖减慢，成骨分化能力减弱。为研究干细胞中Hedgehog信号通路提供了有效的实验工具。 【关键词】：Smoothened；RNAlentiviral RNA interference vectors containing human smoothened gene and its inhibitory effect on proliferation and differentiation in human periodontal ligament stem cells Zhang Jie1,Chang Huijun3,Shu Shaobing1,2,Luo Jinying1,Xu Zhiling4,Tang ling4,Yang Yanchun1,Zhou Jixiang1 (Department of Stomatology, Southwest Hospital, Third Military Medical University, Chongqing, 400038; 1 Department of Stomatology，Authority Clinic of Chongqing Armed Police Force , Chongqing，401147;2 Department of Stomatology,Out-patient Department of Sichuan Military Command,Chengdu,Sichuan Province,610041; 3 College of Biomedical Engineering, Chongqing University , Chongqing, 400044;4) [Abstract] objective: to constructe and screen the lentiviral vector of human smoothened (SMO) gene which can downregulate the SMO gene expression in human periodontal ligament stem cells (PDLSCs). Methods: according to the human SMO gene, three sequences were designed to bulid RNA interference lentiviral vector. Then all the three vectors have been packaged in 293FT and transfected into the 293A cells. The interference efficiency has been evaluated by quantitative PCR and the optimal one were transfected into the PDLSCs. At last, western blot were used to detect the interference effect of SMO gene and the differentiation in PDLSCs . CCK-8 assay was used to test the effect of recombinant lentiviruses on the proliferation of