RNA干扰下调雌激素受体α亚基的人成骨样细胞模型的构建

Construction of human osteoblast-like cell model with the down-regulated estrogen receptor a subunit gene by RNA interference

目的构建雌激素受体α亚基（ERα）下调的人成骨样细胞模型。方法采用计算机辅助设计并合成ERα特异性小干扰RNA（siRNA）前体基因后，定向克隆人pSilencer4．1-CMV质粒中，构建重组的ERα siRNA表达载体并测序鉴定。由脂质体介导重组质粒稳定转染人成骨样细胞株MG63，潮霉素筛选阳性抗性克隆细胞。逆转录聚合酶链反应（RT-PCR）法检测ERα基因在人成骨样细胞株MG63内的表达，抗生物素蛋白-生物素-过氧化物酶复合体法（ABC）分析ERα蛋白在人成骨样细胞株MG63内的表达与定位。MTT法测ERα基因被特异性下调后对MG63细胞生长的影响。流式细胞术分析ERα siRNA对人成骨样细胞株MG63细胞生长周期的影响。采用改良Gomori氏钙钴法分析ERα siRNA对人成骨样细胞株MG63表达碱性磷酸酶的影响。结果构建表达ERα siRNA的重组真核表达载体，并成功地下调了人成骨样细胞株MG63的ERα亚基基因，其中MG63细胞的G1期为68．6％，G2期为17．6％，S期为13．8％；ERα siRNA MG63细胞的G1期为68.6％，G2期为16．8％，s期为14．6％。而ERα siRNA下调人成骨样细胞株MG63的ERα亚基对细胞的生长特性无明显影响。结论成功地构建了ERα下调的人成骨样细胞模型，该模型为进一步研究雌激素及其受体对骨代谢影响的分子机制提供了基础材料。

Objective To construct the human osteoblast-like cell model with estrogen receptor α（ERα） subunit gene knocked down. Methods According to the computer-aided design （CAD）, ERα-specific small interference RNA （siRNA） gene was synthesized and cloned into the expression vector pSilencer 4.1 CMV. The recombinant ERα siRNA plasmid was transfected into human osteoblast-like cell line MG63 by lipofectin, the cloned MG63 cells were selected by hygromycin, and the cloned MG63 cell was cultured more than 20 passages after transfection. The expression of ERα mRNA in MG63 cells was detected by reverse transcription-polymerase chain reaction （RT-PCR）. The expression and location of ERα protein were identified by immunocytochemistry. Compared with control groups, proliferation rate, growth cycle and expression did not show significant difference. Results The recombinant eukaryote plasmid vector was constructed. Furthermore, the recombinant plasmid knocked down ERα protein in human osteoblast-like cells. Conclusions The human osteoblast-like cell model with RNAi-knocked down ERα gene is constructed successfully. This model appears to be very useful for the future research on ERα biological characters and on molecular mechanism of bone metabolism.