CAJ | 학술논문

目的：：构建人和鼠共打靶特异性 ERβ基因沉默慢病毒载体。方法：检索人和小鼠 ERβ基因转录本及其序列,应用 SiRNA Target Finder 软件设计人和小鼠共打靶 ERβ基因沉默 siRNA 和阴性对照siRNA,合成发夹状双链 shRNA,与线性化质粒 PLL3.7连接构建为重组质粒(ERβ-shRNA-PLL3.7和阴性对照质粒 ER-N-shRNA-PLL3.7),经鉴定后,转染人成骨肉瘤细胞 MG63和小鼠成骨细胞株MC3T3-E1,经 Realtime-PCR 和 western blot 法检测重组质粒对目的基因沉默效果。应用 SPSS16.0软件统计分析,检验水准为α=0.05。结果：经测序证明重组质粒中含有目的 shRNA 序列。分别转染小鼠成骨细胞株 MC3T3-E1和人成骨肉瘤细胞 MG63细胞后,无论 Realtime-PCR 检测 ERβ基因表达,还是western blot 法检测蛋白表达,均较空白对照组或阴性对照组显著降低,差异有显著性,P <0.05。结论：成功构建了人和小鼠共同高效打靶的 ERβ基因沉默慢病毒载体质粒,并包装成假病毒颗粒,为利用小鼠作为动物模型研究人基因功能或开发基因药物奠定了基础。
목적：：구건인화서공타파특이성 ERβ기인침묵만병독재체。방법：검색인화소서 ERβ기인전록본급기서렬,응용 SiRNA Target Finder 연건설계인화소서공타파 ERβ기인침묵 siRNA 화음성대조siRNA,합성발협상쌍련 shRNA,여선성화질립 PLL3.7련접구건위중조질립(ERβ-shRNA-PLL3.7화음성대조질립 ER-N-shRNA-PLL3.7),경감정후,전염인성골육류세포 MG63화소서성골세포주MC3T3-E1,경 Realtime-PCR 화 western blot 법검측중조질립대목적기인침묵효과。응용 SPSS16.0연건통계분석,검험수준위α=0.05。결과：경측서증명중조질립중함유목적 shRNA 서렬。분별전염소서성골세포주 MC3T3-E1화인성골육류세포 MG63세포후,무론 Realtime-PCR 검측 ERβ기인표체,환시western blot 법검측단백표체,균교공백대조조혹음성대조조현저강저,차이유현저성,P <0.05。결론：성공구건료인화소서공동고효타파적 ERβ기인침묵만병독재체질립,병포장성가병독과립,위이용소서작위동물모형연구인기인공능혹개발기인약물전정료기출。
Objective:To construct the silencing lentiviral vectors human and mice co-targeting ERβ-specific gene of human and mice.Methods:Retrieving the transcripts and sequence of the ERβgene of human and mice.Using the software SiRNA Target Finder to design human and mice co-targeting ERβ gene silencing siRNA and negative control,and compounding double stranded shRNA in shape of stem loop,being connected with linearized plasmid PLL3.7 by T4 DNA ligase to reconstruct as a recombinant plasmid (ERβ-shRNA-PLL3.7 and negative control plasmid ER-N-shRNA-PLL3.7), which were identified and transfected into human osteosarcoma cells MG63 and mouse osteoblastic cell line MC3T3-E1 cells. The testing ways of Realtime-PCR and western blot were used to test gene silencing effect of the recombinant plasmid.The SPSS1 6.0 software was used for statistical analysis,and the testing level was set at P <0.05.Results:It was confirmed by sequencing that the recombinant plasmid contained the shRNA sequence.After transfecting into mice osteoblastic cell line of MC3T3-E1 and human osteosarcoma cells of MG63,whether the ERβgene expression detected by Realtime-PCR,or Protein expression detected by the western blot,were decreased significantly compared with the control group or the negative control group,and there was a prominent difference,P <0.05.Conclusion:successfully constructed the ERβ-specific gene silencing lentiviral vectors of human and mice co-targeting effectively,and they were packaged into pseudoviral particles,which laid a foundation for the study of man gene function by using mouse as animal model or the development of gene-based drugs.