人雌激素β受体RNAi反转录病毒载体在人成骨样MG63细胞中的表达

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。

Expression of the retroviral vector of RNA interference for human estrogen receptor beta in human MG63 osteoblast-like cells

BACKGROUND：There are many studies on how estrogen receptor（ER） α participates in bone metabolism at present.However,the studies of how ER β participates in bone metabolism are few.OBJECTIVE：To construct a retroviral expression vector of RNA interference（RNAi） for human ER β and to mediate its expression in human MG63 osteoblast-like cells via retrovirus.METHODS：Gene nucleotide sequence of ER β was retrieved from Genebank database.Three target small interfering RNA（siRNA） sequences were designed and converted into cDNA coding expression of small hairpin RNAs（shRNA） for ER β gene.The cDNA was synthesized and inserted into pRNAT-H1.4/Retro plasmid.The recombinant of shRNA eukaryotic expression vector-pRNΑT-H1.4/Retro-ERβ-shRNΑ was constructed and identified by restriction enzyme digestion and the sequence analysis.The recombinant vector was transfected into 293 cells by Lipofectamine 2000 and packaged as retrovirus.The blank and nonspecific shRNA of the packaged retrovirus served as controls.Human MG63 osteoblast-like cell strain was transfected.RESULTS AND CONCLUSION：Three retroviral vectors-ERβ-shRNA（pRNΑT-H1.4/Retro-ERβ-shRNΑ1,pRNΑT-H1.4/Retro-ERβ-shRNΑ2 and pRNΑT-H1.4/Retro-ERβ-shRNΑ3） were constructed.Enzyme digestion identification and sequence analysis had confirmed that the recombinant plasmid containing target sequence for ER β gene had been inserted into the site which was expected to meet the design requirements.The recombinant plasmid had been constructed successfully and was packaged as antivirus after transfected into 293 cells by Lipofectamine 2000.These findings suggest that retrovirus can transfect human MG63 osteoblast-like cell strain efficiently and stably.The transfecting rate was around 70%.Three kinds of ERβ-shRNA retrovirus：pRNΑT-H1.4/Retro-ERβ-shRNΑ1,pRNΑT-H1.4/Retro-ERβ-shRNΑ2 and pRNAT-H1.4/Retro-ERβ-shRNΑ3 all can transfect human MG63 osteoblast-like cells efficiently and stably,as well as inhibit the expression of ERβ remarkably,and ERβ-shRNA3 is the most efficient shRNA sequence.