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

摘要

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,re-spectively.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 an-giogenesis 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 SK-OV-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.%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。