【摘要】 目的研究siRNA表达载体对骨肉瘤MG63细胞KDR蛋白表达的抑制作用。方法构建pSuper质粒,化学合成2段编码发卡RNA序列、靶向KDR基因的寡核苷酸,克隆进pSuper的pol IIIH I启动子的下游,重组构建抑制KDR表达的siRNA质粒,并转染入骨肉瘤MG63细胞,W estern b lot检测转染后KDR基因在蛋白水平表达的变化。结果酶切后电泳鉴定正确的重组质粒小量扩增后测序,证实成功构建了KDR siRNA表达质粒,转染入细胞后,有效抑制了KDR基因在蛋白水平的表达。结论成功构建的siRNA表达载体有效抑制了骨肉瘤MG63细胞KDR蛋白的表达,为进一步分析骨肉瘤中KDR基因的功能及肿瘤治疗奠定了研究基础。更多还原

【Abstract】 Objective To construct expressing vector of siRNA in order to inhibit KDR gene in osteosarcoma cell.Methods First,construct pSuper vector,then Oligos for hairpin RNA expression which targeted KDR gene were chemically synthesized,annealed and inserted into the downstream of treated pSuper’s pol III HI promoter to construct RNAi plasmid(pSuper-KDR),then transfected into the cultured MG63 cell.The expression of KDR protein was dectected by Western blotting.Results pSuper-KDR siRNA expression vector was successfully constructed.Gene transfection of pSuper-KDR siRNA expression vector into MG63 cell down-regulated the protein expression level of KDR gene,as compared with control group.Conclusion pSuper-KDR RNAi system has been constructed successfully.This will facilitate the study of KDR activity inhibition.Based on the results,RNAi synthesis we can be developed in vivo.This technique not only facilitates a wide range of gene functional analysis and cell culture but also offers therapeutic means for treatment of tumors.更多还原