【摘要】 背景:目前对雌激素β受体基因如何参与骨代谢的研究较少。目的:研究雌激素受体β对人成骨样细胞骨保护素、核因子kB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)表达的影响。方法:将先前含有最有效干扰序列及非特异性shRNA的反转录病毒分别感染人成骨样MG63细胞株后,筛选稳定克隆并扩大培养,以空白及非特异性shRNA作为对照,检测稳定抑制雌激素受体β的效率。分别向3组细胞即MG63细胞、雌激素受体βshRNA反转录病毒感染的MG63细胞、阴性对照shRNAnc反转录病毒感染的MG63细胞加入17β-雌二醇(E2)干预,检测人成骨样细胞株MG63的骨保护素、RANKL mRNA和蛋白表达情况。结果与结论:用pRNAT–H1.4/Retro-雌激素受体β-shRNA3进一步稳转人成骨样MG63细胞株,与空白及阴性病毒对照组相比,筛选出雌激素受体β表达稳定抑制的人成骨样细胞株,雌激素受体βmRNA抑制率为(88.17±1.17)%(P<0.05),蛋白抑制率为(89.01±1.22)%(P<0.05),证实实验成功建立了人成骨样细胞ERβ亚型基因敲低细胞模型。雌激素干预48 h后,显示雌激素受体β稳定抑制的MG63细胞较空白组及阴性对照组骨保护素mRNA及蛋白表达上调(P<0.05),RANKL mRNA和蛋白表达下调(P<0.05),骨保护素RANKL表达上调(P<0.05),提示雌激素受体β可能通过调节骨保护素/RANKL在骨代谢中发挥作用。更多还原

【Abstract】 BACKGROUND: Studies concerning how estrogen receptor β participates in bone metabolism are few now.OBJECTIVE: To investigate the effect of estrogen receptor β on the expression of osteoprotegerin and receptor activator of nuclear factor-κB ligand in human osteblast-like cells.METHODS: The retrovirus with the most effective interference sequence and non-specific short hairpin RNA was used to transfect human osteoblast-like cell MG63 in order to screen out the stable colon, and then amplified and cultured. The blank control and non-specific short hairpin RNA were used as control, and the stable inhibition rate of estrogen receptor β was detected. The 17β-estradiol was added into the cells in three groups, that were MG63cells, short hairpin RNA retrovirus estrogen receptor β-mediated MG63 cells and negative control short hairpin RNA retrovirus-medicated MG63 cells, in order to detect the expressions of osteoprotegerin and receptor activator of nuclear factor-κB ligand mRNA in human osteoblast-like cells.RESULTS AND CONCLUSION: The human osteoblast-like MG63 cell line was further stably transfected with pRNAT–H1.4/Retro-estrogen receptor β short hairpin RNA3, and then compared with the blank control andnegative control, and found that estrogen receptor β could express the stable inhibited human osteoblast-like cell line.The inhibition rate of estrogen receptor β mRNA was(88.17±1.17)%(P < 0.05), and the inhibition rate of estrogen receptor β protein was(89.01±1.22)%(P < 0.05), indicating that estrogen receptor β gene knockdown human osteoblast-like cell models were constructed successfully. After estrogen intervention for 48 hours, the inhibition of MG63 cells with estrogen receptor β could up-regulate the osteoprotegerin mRNA and protein expression in the blank control group and the negative control group(P < 0.05), down-regulate the receptor activator of nuclear factor-κB ligand mRNA and protein expression(P < 0.05), and up-regulate the osteoprotegerin receptor activator of nuclear factor-κB ligand expression. The results indicate that estrogen receptor β may play an important role in bone metabolism through regulating osteoprotegerin/receptor activator of nuclear factor-κB ligand ratio.更多还原