人孕酮受体膜组分1表达载体的构建及其重组蛋白对体外血管形成的诱导作用

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摘要 目的: 构建含人孕酮受体膜组分（progesterone receptor membrane component，PGRMC）1的重组表达质粒，转染人卵巢癌细胞，研究其调控卵巢癌细胞血管生成的作用。方法: 以卵巢癌细胞为模板，采用PCR技术扩增PGRMC1基因编码序列，插入真核载体pSecTag-2B构建重组表达载体；然后，将重组载体pSTPGRMC1分别转染人胚肾上皮细胞HEK293T和卵巢癌细胞SKOV-3，采用蛋白质印迹法检测PGRMC1蛋白的表达；实时荧光定量PCR检测卵巢癌细胞中VEGF mRNA的变化，用ELISA法检测上清液中VEGF的分泌情况。体外血管生成实验进一步观察PGRMC1促进体外血管生成的情况。结果： 重组表达载体pSTPGRMC1构建成功，重组蛋白PGRMC1在HEK293T和SKOV-3细胞中都能够表达；PGRMC1过表达可促使卵巢癌细胞合成VEGF，并且促进体外血管的形成。结论： 重组蛋白PGRMC1在促进卵巢癌细胞体外血管形成中起着重要作用，可能促进卵巢癌细胞的远处转移。

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关键词 ：人孕酮受体膜组分1, 质粒构建, 血管形成

Abstract：Objective： To construct the recombinant plasmid containing progesterone receptor membrane component 1(PGRMC) gene and investigate the effect of the recombinant protein product on angiogenesis by ovarian cancer cells. Methods：We constructed the recombinant plasmid pST-PGRMC1 from ovarian cancer cells by PCR. The pST-PGRMC1 plasmid was transfected to HEK293T cells and SKOV-3 cells, respectively. The protein level of PGRMC1 was detected by western blotting. The mRNA and protein level of VEGF was detected by real-time PCR and ELISA.And then,we investigated the effect of PGRMC1 on angiogenesis by ovarian cancer cells in vitro. Results：The recombinant pST-PGRMC1 plasmid was constructed successfully. The recombinant protein could be detected in HEK293T cells and SKOV-3 cells by western blotting.The overexpression of PGRMC1 promoted the synthesis of VEGF and microtubule formation by SKOV-3 in vitro. Conclusion：The recombinant protein PGRMC1 plays an important role in the microtubule formation of ovarian cancer cells in vitro, and promotes the metastasis of the ovarian cancer cells.

收稿日期: 2016-08-21

基金资助:

国家自然科学基金资助项目（81503368）

通讯作者: 朱小飞（通讯作者），主管技师，E-mail：zxfyxjy@163.com

作者简介: 季明德（1982—），男，主管技师，硕士；

引用本文:

季明德，朱小飞，杨学文，张春兵. 人孕酮受体膜组分1表达载体的构建及其重组蛋白对体外血管形成的诱导作用[J]. 江苏大学学报:医学版, 2016, 26(05): 391-394. JI Ming-de，ZHU Xiao-fei，YANG Xue-wen， HANG Chun-bing. Construction of recombinant plasmid containing human progesterone receptor membrane component 1 gene and effect of the corresponding protein product on angiogenesis in vitro. Journal of Jiangsu University(Medicine Edition), 2016, 26(05): 391-394.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2016/V26/I05/391

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