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目的 :检测RhoC在胃癌细胞系中的表达 ,并构建其小干扰RNA(siRNA)表达载体。方法 :以Westernblot方法检测RhoC在多株胃癌细胞系中的表达。利用计算机辅助设计RhoC特异性siRNA ,体外合成siRNA基因并将其定向克隆入真核表达载体mU6pro。以脂质体法转染RhoC siRNA载体至高转移的人胃癌细胞系AGS中 ,以Westernblot方法鉴定RhoC siRNA对RhoC表达的作用。结果 :RhoC蛋白在具有高转移潜能的胃癌细胞系中表达增强。利用BbsⅠ和XbaⅠ双酶切法鉴定重组载体后 ,DNA测序证实合成的siRNA基因序列正确并已被准确克隆入mU6pro载体。RhoC siRNA载体可特异性抑制AGS细胞中RhoC的表达。结论 :RhoC特异性siRNA表达载体成功构建 ,为后期基因治疗的研究奠定了基础
Objective: To detect the expression of RhoC in gastric cancer cell lines and to construct its expression vector for small interfering RNA (siRNA). Methods: The expression of RhoC in multiple gastric cancer cell lines was detected by Western blot. Using computer-aided design of RhoC-specific siRNA, in vitro synthesis of siRNA gene and its directional cloning into the eukaryotic expression vector mU6pro. The RhoC siRNA vector was transfected into the highly metastatic human gastric cancer cell line AGS by lipofectamine. The effect of RhoC siRNA on RhoC expression was identified by Western blot. Results: The expression of RhoC protein was enhanced in gastric cancer cell lines with high metastatic potential. After identification of the recombinant vector by double enzyme digestion with Bbs I and Xba I, DNA sequencing confirmed that the synthetic siRNA gene sequence was correctly cloned into the mU6pro vector. RhoC siRNA vector specifically inhibits RhoC expression in AGS cells. Conclusion: The successful construction of RhoC specific siRNA expression vector laid the foundation for the study of gene therapy in the later stage