膜联蛋白A5重组质粒的构建及其过表达对SiHa细胞增殖的影响

目的 构建膜联蛋白A5(ANXA5)重组质粒并探讨过表达ANXA5对宫颈癌SiHa细胞的增殖产生的影响. 方法 将ANXA5基因克隆入含His标签的pcDNA3.1质粒载体,构建pcDNA3.1-ANXA5重组质粒;用磷酸钙共沉淀法将重组质粒和pcDNA3.1空质粒分别转染宫颈癌细胞系SiHa细胞,G418加压培养,免疫印迹法筛选过表达ANXA5的细胞克隆,免疫组织化学筛选已转染pcDNA3.1空质粒的细胞克隆;将过表达ANXA5蛋白的细胞、载有pcDNA3.1空质粒的细胞以及未处理的SiHa细胞分别进行四甲基偶氮噻唑蓝(MTT)实验和CCK-8实验检测细胞增殖情况. 结果 成功构建了peDNA3.1-ANXA5重组质粒,测序证实ANXA5基因序列正确;普通未处理的SiHa细胞和转染了pcDNA3.1空质粒的SiHa细胞生长速度较快,两组同无显著性差异(P>0.05);过表达ANXA5的SiHa细胞其增殖速度比前两组显著降低(P<0.05). 结论 膜联蛋白A5可能对宫颈癌细胞系SiHa细胞的增殖具有抑制作用.

Construction of recombinant plasmid pcDNA3.1-ANXA5 and the affection to the proliferation of the SiHa cells by overexpressed annexin A5

Objective To construct the recombinant plasmid pcDNA3.1-ANXA5 so as to study the affection of the overexpressed annexin A5(ANXA5) protein to the proliferation of uterine cervical carcinomal SiHa cells. Methods The ANXA5 cDNA fragment was cloned into pcDNA3.1 vector to construct the recombinant plasmid pcDNA3.1-ANXA5; The pcDNA3.1-ANXA5 plasmid and pcDNA3.1 vector were transfected into the uterine cervical carcinoma SiHa cells respectively and screened by G418. Western blotting was used to identify cell clones which overexpress ANXA5 and immunohistochemistry was applied to identify cells transfected with pcDNA3.1 vector which has a His-tag. Cells overexpressed ANXA5, cells transfected with pcDNA3.1 vector and cells without any treatment were tested by MTT and CCK-8 method to examine whether overexpressed ANXA5 protein can affect the proliferation of the SiHa cells. Results The recombinant plasmid pcDNA3.1-ANXA5 was successfully constructed and the ANXA5 gene sequence was proved correct; The proliferation of the SiHa cells without treatment and those transfected with pcDNA3.1 vector was fast and there was no difference（P>0.05）between the two kinds of cells；while the proliferation of the SiHa cells overexpressed ANXA5 was very slow. The difference from the other two kinds of cells was significant (P<0.05). Conclusion The ANXA5 protein maybe affect the