RNA干扰基因敲低雌激素受体β对人成骨样细胞生长的影响

Effect of estrogen receptorβ knocked down by RNA interference on human osteoblast-like cell

目的观察经特异性小干扰RNA(small interfere RNA,siRNA)下调人成骨样细胞株MG63的雌激素受体β(ERβ)后对其生长状况的影响。方法利用计算机辅助设计并合成ERβsiRNA前体基因后,定向克隆入pSilencer4.1-CMV质粒中,构建重组的ERβsiRNA表达载体并测序鉴定。由脂质体介导重组质粒稳定转染入人成骨样细胞株MG-63,潮霉素筛选阳性抗性克隆细胞。RT-PCR法检测ERβ基因在人成骨样细胞株MG-63的表达,抗生物素蛋白-生物素-过氧化物酶复合体法(ABC)分析ERβ蛋白在人成骨样细胞株MG-63内的表达与定位。MTT法测ERβ基因被特异性下调后对MG-63细胞生长的影响。流式细胞术分析ERβsiRNA对人成骨样细胞株MG-63细胞生长周期的影响。采用改良Gomori氏钙钴法分析ERβsiRNA对人成骨样细胞株MG-63表达碱性磷酸酶的影响。结果构建表达ERβsiRNA的重组真核表达载体,并成功地下调了人成骨样细胞株MG-63的ERβ基因,而ERβsiRNA下调人成骨样细胞株MG-63的ERβ对细胞的生长特性无明显影响。结论成功地构建了ERβ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 observe the growth of human osteoblast-like cell after estrogen receptor β subunit gene knocked down by RNA interference. Methods According to the computer aided design （ CAD）, ERβ specific small interference RNA （siRNA） gene was synthesized and cloned into the expression vector pSilencer 4. 1-CMV. The recombinant ERβ siRNA plasmid was transfected into human osteoblast-like cell line MG-63 by lipofectin, the cloned MG-63 cells were selected by hygromycin, and the cloned MG-63 cell was cultured more than 20 passages after transfection. The expression of ERβ mRNA in MG-63 cells was detected by RT-PCR. The expression and location of ERβ protein were identified by immunocytochemistry. The proliferation rate, growth cycle of MG-63 cell and alkaline phosphatase expression were observed by MTT, flow cytometry and modified Gomori method. Results The recombinant eukaryote plasmid vector was constructed. Furthermore, the recombinant plasmid knocked down ERβ protein in human osteoblast-like cells. Erβ siRNA downregulating MG-63 Erβ subgene had no obvious effect on cellular growth. Conclusions Human osteoblast-like cell model knocked down ERβ gene by RNAi is constructed successfully. This model appears to be very useful for the future research on ERβ biological characters and on molecular mechanism of bone metabolism.