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目的:构建GPER-shRNA载体并研究其对血管内皮细胞增殖的影响。方法:化学合成靶向GPER的短发夹RNA的寡核苷酸序列,退火与线性化pSUPER质粒连接,行双酶切及测序鉴定;脂质体法转染血管内皮细胞(VEC),Western Blot鉴定重组质粒的功能,MTT法测定其对VEC增殖情况的影响。结果:重组质粒经酶切及测序分析,表明目的核苷酸序列成功插入了预计位点且序列完全一致;重组质粒成功转染VEC,WesternBlot检测显示GPER基因蛋白表达明显降低,pSUPER-GPER-2组抑制效果明显,其抑制率为84.2%,与对照组相比差异有统计学意义(P<0.05),并显著抑制了VEC的增殖。结论:GPER-shRNA载体构建成功,并可抑制VEC的增殖。
Objective: To construct GPER-shRNA vector and study its effect on the proliferation of vascular endothelial cells. METHODS: The oligonucleotide sequence of short hairpin RNA targeting GPER was chemically synthesized and annealed to the pSUPER plasmid. The double-stranded DNA was digested and sequenced. Lipofectamine 2000 was transfected into vascular endothelial cells (VECs) and Western Blot Identification of recombinant plasmid function, MTT assay of VEC proliferation. Results: The recombinant plasmids were successfully digested with restriction endonucleases and sequenced. The target sequences were successfully inserted into the predicted sites and the sequences were exactly the same. The recombinant plasmids were successfully transfected into VEC. Western blot analysis showed that GPER gene protein expression was significantly reduced. PSUPER-GPER-2 The inhibitory rate was 84.2%, which was significantly lower than that of the control group (P <0.05), and significantly inhibited the proliferation of VEC. Conclusion: GPER-shRNA vector was successfully constructed and inhibited the proliferation of VEC.