摘要
目的探讨特异性MUC5AC-siRNA沉默MUC5AC基因后对人肝内胆管癌细胞株HCCC-9810生长能力的影响。方法设计合成三对特异性siRNA,构建了三个稳定表达质粒,pRNAT-U6.1/Neo-MUC5AC-siRNA1/2/3,应用脂质体转染技术分别将三个稳定表达的质粒和对照质粒(空质粒对照)转染HCCC-9810,RT-PCR检测MUC5AC基因mRNA水平;SABC免疫组化染色技术检测MUC5AC粘蛋白的表达;MTT检测细胞生长增殖情况。结果基因测序表明成功构建质粒;转染后RT-PCR结果表明在mRNA水平,三个质粒都可抑制MUC5AC基因的表达,并可使MUC5AC的表达下降,MTT结果对细胞的生长有一定的抑制作用。结论构建pRNAT-U6.1/Neo-MUC5AC-siRNA1/2/3质粒明显抑制了MUC5AC基因在mRNA水平及蛋白的表达,并可抑制肿瘤细胞的生长增殖,为探讨MUC5AC在MUC5AC相关肿瘤的基因治疗方面奠定了一定的理论基础。
Objective To investigate the effects on the proliferation of human intrabepatic cholangiocarcinoma cell line HCCC-9810 after silencing the MUC5AC gene expression with specific siRNAs. Methods Three pairs of specific MUC5AC-siRNA was designed and synthesized through transcription in vitro. The the three different siRNAs expression plasmids （ pRNAT-U6. 1/Neo- MUC5AC-siRNA1/2/3 ） were constructed by gene recombination. Then the three stable expression plasmids and the comparison plasmid （ empty plasmid - transfeeted control） were transfected into HCCC-9810 by liposome-mediated transfection. MUC5AC-mRNA level was detected by RT-PCR. Expression of MUC5AC was investigated by immunohistochemical SABC method. Cell proliferation were analyzed by MTT. Results The results of gene sequencing indicated that the pRNAT-U6. 1/Neo-MUC5AC-siRNA1/2/3 were successfully constructed ;The results of RT-PCR and immunocytoehemistry showed that constructed plasmids down-regulated mRNA and protein of MUC5AC at 48 h after the transfection . The results of MTT indicated that the growth of HCCC-9810 could be obviously inhibited after silencing the MUC5AC gene. Coulusion Constructed three different stable expression plasmids of siRNA specific for MUC5AC gene could obviously inhibit the expression at MUC5AC-mRNA and protain level. The blockage of MUC5AC gene expression in HCCC-9810 cells shows significant effect on cell proliferation. These findings suggest that this study may provide theoried basis for the applications of MUC5AC in the gene therapy of MUC5AC related tumors.
出处
《肝胆外科杂志》
2009年第2期143-146,共4页
Journal of Hepatobiliary Surgery
基金
安徽医科大学附属省立医院2007年度院科研基金资助课题
