siRNA联合顺铂抑制骨肉瘤细胞增殖的研究

目的构建人Survivin基因的siRNA真核表达载体,探讨其对骨肉瘤细胞MG63凋亡及顺铂化疗的增敏作用。方法应用pSilencer 3.0-H1 neo,构建Survivin特异性的RNA干涉载体,转染MG63细胞,G418筛选稳定转染的细胞系。半定量PCR和免疫荧光染色分别检测Survivin mRNA与蛋白的水平变化。Annexin V法检测细胞凋亡。MTT法检测顺铂对细胞的杀伤作用。结果构建Survivin基因siRNA真核表达载体PsiS。获得了稳定转染的细胞系。与MG63、阴性对照细胞比较,MG63/PsiS生长曲线则十分平缓。MG63/PsiS细胞中Survivin的mRNA及蛋白表达明显减少。MG63/PsiS凋亡率增加为17.8%(P<0.01)。1 mg/L顺铂作用下,MG63/PsiS死亡率是其他各组的2.3倍(P<0.01)。结论特异性siRNA能够明显阻断Survivin基因的表达促进MG63细胞凋亡,提高细胞对顺铂的敏感性。

Vector-based siRNA against Survivin coupling with cisplatin suppressing human osteosarcoma cell MG63 proliferation in vitro

Objective To explore the effect of siRNA on the expression of Survivin gene and leading apoptosis in osteosarcoma cell line MG63,and to investigate the chemotherapy sensitivity of MG63 treated with cisplatin.Methods The siRNA eukaryotic expression vector targeting Survivin genc was con- structed.MG63 cells were transfected with negative control vector or RNAi vectors and selected by G418. The expression of Survivin mRNA and protein in the stably transfected ceils was detected by RT-PCR and immunofluorescence microscopy respectively.The above cells were cultured,thus growth curve was drawn. Annexin V method was used to detect the apoptosis and cytotoxicity of ciplatin was measured by MTT as- say.Results The specific siRNA eukaryotic expression vector PsiS targeting Survivin gene was construc- ted successfully.The stable transfectants containing negative control vector and PsiS were obtained.The ex- pression of Survivin mRNA and protein was inhibited significantly in MG63/PsiS cells and apoptotic rate was increased to 17.8% (P<0.01).MTT assay revealed that MG63/PsiS cells were more sensitive to cisplatin than other transfectants.The number of apoptotic cells induced by 1 mg/L cisplatin was increased to 2.3 fold in MG63/PsiS as compared with controls.Conclusion Specific siRNA could obviously knock down the Survivin gene expression,promote apoptosis of MG63 cells and enhance the chemotherapy sensi- tivity of the cells to cisplatin significantly.