摘要
目的构建并鉴定特异性大鼠糖原合成酶激酶3β(GSK3β)基因siRNA腺病毒载体,观察其对肝卵圆细胞系WB-F344增殖的影响。方法用DNA重组技术将针对GSK3β基因不同部位所设计的2对shRNA序列克隆到高效RNAi真核表达质粒载体pGenesil-1.1中,构建shRNA表达载体pGenesil-1.1-GSK3β。分别酶切重组质粒及pDC312载体,转化连接,构建腺病毒载体pDC312-GSK3β,PCR扩增与鉴定。脂质体法介导腺病毒载体与骨架质粒pPE3-F11共转染293细胞,包装产生腺病毒颗粒AdD C3 12-GSK3β并测定滴度。取病毒上清感染WB-F34 4细胞,荧光显微镜观察细胞荧光含量,Western blotting检测GSK3β蛋白表达。CCK-8测定转染前后WBF-344增殖变化。结果经PCR酶切及Western blotting技术证实成功构建了针对大鼠GSK3β基因RNAi质粒pGenesil-1.1-GSK3β及GSK3β基因RNAi重组腺病毒载体AdDC312-GSK3β。Western blotting证实该重组腺病毒载体可抑制WBF-344细胞GSK3β的表达。CCK-8结果显示干扰GSK3β可促进WBF-344细胞增殖。结论成功构建了针对GSK3β基因RNAi的腺病毒载体,并在大鼠肝卵圆细胞系WBF-344中稳定表达,促进细胞增殖。
Objective To construct and identify the adenovirus vectors with the specific siRNA of rat GSK3 β gene,and observe those effects on the proliferation of hepatic oval cells WBF-344.Methods Two pairs of shRNA sequences against different parts of GSK3β gene were cloned into the RNAi plasmids vectors pGenesil-1.1 by recombinant DNA technology to construct shRNA expression vectors pGenesil-1.1GSK3 β.The vectors pDC312 and recombinant plasmids were digested respectively,transformed and connected.Adenovirus vectors pDC312-GSK3 β were then built and amplified and identified with PCR and Western blot.Adenovirus vectors and backbone plasmids pPE3-F11 were co-transfected to 293 cells by liposome means.Adenovirus particles AdDC312-GSK3β were packaged and produced.The titer of adenovirus was measured.After the viral supernatant infected WB-F344 cells,fluorescent levels were observed under fluorescence microscope,and the expression of GSK3 β was detected with Western blotting.The changes of cells proliferation were measured with CCK8.Results It was confirmed that RNAi plasmids pGenesil-1.1-GSK3 β and RNAi adenovirus vectors AdDC312-GSK3 β were successfully constructed with PCR and Western blot.The adenovirus vectors could interfere the expression of GSK3 β in WBF-344 cells.The results of CCK8 showed that interfering GSK3 β gene could promote the proliferation of WBF-344.Conclusion RNAi adenovirus vectors against GSK3β gene have successfully constructed,and they can steadily express in the hepatic oval cells line WBF-344,thus promote the cells＇ proliferation.
出处
《肝胆胰外科杂志》
CAS
2012年第2期123-127,共5页
Journal of Hepatopancreatobiliary Surgery
基金
国家自然科学基金资助项目(30700800)
