摘要
目的构建雌激素受体β(ERβ)亚型沉寂表达载体,并观察其转染成骨细胞后抑制ERβ基因表达的效果。方法参考人ERβ基因mRNA序列,设计、合成ERβ-shRNA的DNA Oligo后行PCR扩增,获得的shRNA双链DNA片段与pLVTHM-GFP质粒双酶切后连接,转化大肠杆菌E.coliDH5α感受态细胞。取鉴定正确的pLVTHM-GFP/ERβ-shRNA重组质粒转染人成骨hMG63细胞,实验分空白载体组、空白对照组和ERβ-siRNA组,转染48h后于倒置荧光显微镜下观察各组细胞的转染效果,并采用流式细胞术检测转染效率。抽提各组细胞的总RNA,经RT-PCR生成cDNA,Realtime-PCR检测ERβ基因的抑制效率。结果倒置荧光显微镜下观察显示,ERβ-siRNA组hMG63细胞90%以上呈现绿色荧光。流式细胞术检测ERβ-shRNA转染hMG63细胞的转染效率为92.3%。Realtime-PCR检测结果表明,ERβ-siRNA组ERβ表达量(0.17±0.01)仅约为空白载体组(1.00±0.01)的17.3%(P<0.001),ERβ-siRNA对hMG63细胞ERβ基因的抑制效率约为83%。结论成功建立了人ERβ沉寂表达的成骨细胞模型,慢病毒载体介导的ERβ-siRNA能有效抑制成骨细胞ERβ基因的表达,满足了研究成骨细胞中ERβ相关功能的需要。
Objective To construct silence expression vector for beta-estrogen receptor （ERβ）, and evaluate the inhibition efficiency of ERD in hMG63 cells.Methods According to the mRNA sequence of human estrogen receptor beta （ERβ）,the DNA Oligo of ERβ-shRNA was designed, synthesized, PCR amplification and inserted into the vector of pLVTHM-GFP, which was integrated into the expression plasmid of pLVTHM-GFP/ERβ-shRNA. After correctly identified from the competent cell of E. coli DH5α, the pLVTHM-GFP/ERβ-shRNA finally was transfected into hMG63 cells. The transfection efficiency was evaluated by the inverted fluorescence microscope and flow cytometry for ERβ-siRNA group, blank vector group and control group. The inhibition efficiency of ERβ gene was determined by Realtime-PCR.Results Inverted fluorescence microscope showed that 90% of hMG63 cells in the ERβ-siRNA group appeared green fluorecent. The transfection efficiency measured by FCM for hMG63 cells transfected with ERβ-shRNA was 92.3%. The relative expression value of ERβ-siRNA group （0.17 ± 0.01） was 17.3% of the blank vector group （1.00 ± 0.01） （P 〈 0.001）. The inhibition efficiency of ERβ-siRNA for ERβ gene in the hMG63 cells was 83%.Conclusion The cell model of ERβ silence was successfully developed in hMG63 cells. Lentiviral vector-mediated ERβ-siRNA could effectively inhibit the expression of ERβ in hMG63 cells, which meets the research for ERβ function in hMG63 cells.
出处
《中国医药生物技术》
CSCD
2010年第5期353-356,共4页
Chinese Medicinal Biotechnology
基金
国家自然科学基金(30801230)
上海市科委"创新行动计划"重大基础研究项目子课题(09DJ1400404)
上海市卫生局基金(2008001)
上海市卫生局青年基金(044Y06)
