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背景:目前已有较多关于雌激素α受体基因如何参与骨代谢的研究,而对雌激素β受体基因如何参与骨代谢的研究则相对较少。目的:构建人雌激素β受体RNAi反转录病毒表达载体,并通过病毒介导其在人成骨样MG63细胞中表达。方法:根据GeneBank数据库提供的雌激素β受体基因核苷酸序列,选择设计3条针对人雌激素β受体干扰靶序列,并与pRNAT-H1.4/Retro质粒定向连接,构建真核表达载体pRNAT-H1.4/Retro-雌激素β受体-shRNA,经限制性内切酶酶切和DNA测序进行鉴定。将pRNAT-H1.4/Retro-雌激素β受体-shRNA经脂质体转染至293细胞包装成反转录病毒。将包装好的反转录病毒,以空白及非特异性shRNA作为对照,感染人成骨样MG63细胞株。结果与结论:3个连接了雌激素β受体-shRNA的重组质粒经酶切鉴定分析证实目的序列己插入到预计位点,符合设计要求,测序鉴定表明重组质粒中含有针对雌激素β受体基因目的序列,表明重组质粒构建成功。并经脂质体转染至293细胞后成功包装成反转录病毒。反转录病毒载体能高效、稳定的感染人成骨样MG63细胞株,感染效率为70%左右。包装后的3种雌激素β受体-shRNA反转录病毒均能高效、稳定的感染人成骨样MG63细胞,并显著抑制雌激素β受体的表达。其中以雌激素β受体-shRNA3为最佳的干扰序列。
BACKGROUND: There are more studies on how estrogen α receptor genes participate in bone metabolism. However, there are relatively few studies on how estrogen β receptor genes participate in bone metabolism. OBJECTIVE: To construct human estrogen receptor β RNAi retroviral vector and express it in human osteoblast-like MG63 cells through virus-mediated. Methods: According to the nucleotide sequence of estrogen beta receptor gene provided by GeneBank database, three target sequences targeting human estrogen beta receptor were designed and targeted and ligated with pRNAT-H1.4 / Retro plasmid to construct eukaryotic expression vector The vector pRNAT-H1.4 / Retro-estrogen beta receptor-shRNA was identified by restriction enzyme digestion and DNA sequencing. The pRNAT-H1.4 / Retro-estrogen beta receptor-shRNA was liposome transfected into 293 cells and packaged into retroviruses. The packaged retroviruses were infected with human osteogenic MG63 cell line using blank and nonspecific shRNA as a control. RESULTS AND CONCLUSION: Three recombinant plasmids linked with estrogen receptor-shRNA were confirmed by restriction analysis and confirmed that the target sequence had been inserted into the predicted site, which was in line with the design requirements. Sequencing and identification showed that the recombinant plasmids contained the gene targeting estrogen receptor β Gene sequence, indicating that the recombinant plasmid was constructed successfully. And successfully packaged into retroviruses after being transfected into 293 cells by liposomes. The retroviral vector can efficiently and stably infect human osteoblast-like MG63 cell line, and the infection efficiency is about 70%. The packaged 3 estrogen beta receptor-shRNA retroviruses can efficiently and stably infect human osteoblast-like MG63 cells and significantly inhibit the expression of estrogen beta receptors. Which estrogen receptor β-shRNA3 as the best interference sequence.