雌激素受体β基因沉默对人成骨细胞转化生长因子β1和骨形态发生蛋白2表达的影响  

Silencing of estrogen receptor beta gene influences the expressions of transforming growth factor beta1 and bone morphogenetic protein 2 in human osteoblasts

作　　者：邓盎[1] 张宏其[1] 郭超峰[1] 王昱翔[1] 高琪乐[1] 唐明星[1] 刘少华[1] 刘金洋[1] 

机构地区：[1]中南大学湘雅医院脊柱外科,湘雅脊柱外科中心,湖南省长沙市410008

出　　处：《中国组织工程研究》2016年第29期4261-4268,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81271940,81472145)~~

摘　　要：背景:雌激素受体β基因参与骨代谢的研究较少,其对骨代谢的具体调节机制仍不清楚。目的:分析雌激素受体β基因沉默对人成骨细胞转化生长因子β1和骨形态发生蛋白2表达的影响。方法:实验分3组:空白对照组(即h FOB 1.19,未感染任何反转录病毒)、阴性对照组(即含无效干扰片断雌激素受体β-sh RNA-nc)、最佳RNAi组(即ERβ-sh RNA-3)。将前期最佳RNAi组的雌激素受体β-sh RNA反转录病毒载体感染人成骨细胞,通过抗性筛选并扩大培养,利用MTT法检测雌激素受体β稳定抑制后对细胞增殖的影响;随后在雌激素干预下,通过Western blot技术检测雌激素受体β蛋白表达的稳定抑制效率,并使用半定量RT-PCR和Western blot技术检测雌激素受体β稳定抑制后对转化生长因子β1和骨形态发生蛋白2表达的影响。结果与结论:(1)成功筛选出稳定感染ERβ-shR NA-3反转录病毒载体的人成骨细胞,MTT法检测显示雌激素受体β稳定抑制后对细胞的增殖没有明显影响(P>0.05);(2)在雌激素干预下,雌激素受体β蛋白的抑制率为(93.11±0.57)%(P<0.05),且雌激素受体β稳定抑制后对转化生长因子β1 mR NA和蛋白的上调率分别为(26.65±3.81)%和(23.79±3.76)%,骨形态发生蛋白2 mR NA和蛋白的上调率分别为(16.62±1.71)%和(18.08±3.20)%(均P<0.05);(3)结果提示雌激素受体β可能通过调控转化生长因子β1和骨形态发生蛋白2的表达在骨代谢中发挥作用。BACKGROUND：There are few studies concerning estrogen receptorβgene, and its mechanism of regulating the bone metabolism is stil unclear now. OBJECTIVE：To analyze the effect of estrogen receptorβ（ERβ） silencing on the expressions of transforming growth factorβ1 （TGF-β1） and bone morphogenetic protein 2 （BMP-2） in human osteoblasts METHODS：There were three groups：blank control group （hFOB 1.19 uninfected with any retrovirus）;negative control group （containing invalid interference fragment ERβ-shRNA-nc）;optimal RNAi group （ERβ-shRNA-3）. ERβ-shRNA retroviral vectors in the optimal RNAi group were used to transfect human osteoblasts fol owed by resistance screening and cel expansion. MTT assay was used to detect the proliferative activity of ERβ-silenced osteoblasts. Then under estrogen intervention, the stable inhibition rate of ERβwas determined using western blot assay, and the expressions of TGF-β1 and BMP-2 in human osteoblasts after ERβsilencing were detected by RT-PCR technology and western blot assay. RESULTS AND CONCLUSION：Human osteoblasts that were stably transfected by ERβ-shRNA-3 retroviral vector was selected successful y, and ERβsilencing had no significant influence on the cel proliferation （P〉0.05）. Under the interference of estrogen, the silencing efficiency of ERβprotein was （93.11±0.57）%（P〈0.05）, and after ERβsilencing, the expressions of TGF-β1 and BMP-2 were increased by （26.65±3.81）%and （16.62±1.71）%at mRNA level, and increased by （23.79±3.76）%and （18.08±3.20）%at protein level （both P〈0.05）. In conclusion, ERβmay play an important role in bone metabolism by regulating the expressions of TGF-β1 and BMP-2.

关 键 词：雌激素受体Β 成骨细胞 转化生长因子β1 骨形态发生蛋白2 组织工程 组织构建 骨细胞 人成骨细胞 RNA干扰 基因沉默 骨代谢 雌激素 反转录病毒 细胞模型 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]