CAJ | 학술논문

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.
배경:목전이유교다관우자격소α수체기인여하삼여골대사적연구,이대자격소β수체기인여하삼여골대사적연구칙상대교소.목적:구건인자격소β수체RNAi반전록병독표체재체,병통과병독개도기재인성골양MG63세포중표체.방법:근거GeneBank수거고제공적자격소β수체기인핵감산서렬,선택설계3조침대인자격소β수체간우파서렬,병여pRNAT-H1.4/Retro질립정향련접,구건진핵표체재체pRNAT-H1.4/Retro-자격소β수체-shRNA,경한제성내절매매절화DNA측서진행감정.장pRNAT-H1.4/Retro-자격소β수체-shRNA경지질체전염지293세포포장성반전록병독.장포장호적반전록병독,이공백급비특이성shRNA작위대조,감염인성골양MG63세포주.결과여결론:3개련접료자격소β수체-shRNA적중조질립경매절감정분석증실목적서렬기삽입도예계위점,부합설계요구,측서감정표명중조질립중함유침대자격소β수체기인목적서렬,표명중조질립구건성공.병경지질체전염지293세포후성공포장성반전록병독.반전록병독재체능고효、은정적감염인성골양MG63세포주,감염효솔위70%좌우.포장후적3충자격소β수체-shRNA반전록병독균능고효、은정적감염인성골양MG63세포,병현저억제자격소β수체적표체.기중이자격소β수체-shRNA3위최가적간우서렬.
BACKGROUND:There are many studies on how estrogen receptor (ER)αparticipates in bone metabolism at present. However, the studies of how ERβparticipates in bone metabolism are few. OBJECTIVE:To construct a retroviral expression vector of RNA interference (RNAi) for human ERβand to mediate its expression in human MG63 osteoblast-like cel s via retrovirus. METHODS:Gene nucleotide sequence of ERβwas retrieved from Genebank database. Three target smal interfering RNA (siRNA) sequences were designed and converted into cDNA coding expression of smal hairpin RNAs (shRNA) for ERβgene. The cDNA was synthesized and inserted into pRNAT-H1.4/Retro plasmid.The recombinant of shRNA eukaryotic expression vector-pRNΑT-H1.4/Retro-ERβ-shRNΑwas constructed and identified by restriction enzyme digestion and the sequence analysis. The recombinant vector was transfected into 293 cel s by Lipofectamine 2000 and packaged as retrovirus. The blank and nonspecific shRNA of the packaged retrovirus served as controls. Human MG63 osteoblast-like cel 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 ERβgene had been inserted into the site which was expected to meet the design requirements. The recombinant plasmid had been constructed successful y and was packaged as antivirus after transfected into 293 cel s by Lipofectamine 2000. These findings suggest that retrovirus can transfect human MG63 osteoblast-like cel 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 al can transfect human MG63 osteoblast-like cel s efficiently and stably, as wel as inhibit the expression of ERβremarkably, and ERβ-shRNA3 is the most efficient shRNA sequence.