健骨颗粒对ER表达沉默成骨细胞株ROS1728的分化的研究

Abstract

目的探讨健骨颗粒对成骨细胞中ERalpha介导的TERT信号通路的调控作用。方法采用雌激素受体拮抗剂ICI182780(Faslodex)阻断成; 骨细胞中雌激素受体alpha(ERalpha)的表达,建立ER抑制的大鼠成骨细胞株ROS1728细胞模型,采用酶联免疫吸附法(ELISA)检测成; 骨细胞液中碱性磷酸酶(alkaline phosphatase,ALP) 、骨钙素(osteocalcin,BGP); 、Ⅰ型胶原(collagen I,Col Ⅰ)的含量。采用实时荧光定量SYBR; GREEN法检测ERE、ERalpha、c-MYCmRNA的表达。采用Western Blot检测TERT、ERalpha、c-; MYC蛋白的表达。结果ELISA法检测结果显示:随着干预时间的延长,培养液中的ALP、BGP、ColⅠ的含量逐渐上升。其中对照组3种信号因子的含; 量最高血,雌激素组次之,健骨颗粒组再次之,模型组最低,各组比较差异有统计学意义(P < 0.05),; 4组TERT、ERalpha、c-MYCmRNA及蛋白表达量情况以对照组的蛋白表达含量最高,雌激素组次之,健骨颗粒组再次之,模型组最低。各组比较; 均有统计学意义(P < 0.05); 。结论雌激素介导TERT信号通路及其相关因子与成骨细胞分化的关系密切,而补肾健脾中药健骨颗粒可通过雌激素介导TERT信号通路促进成骨细胞分化。

Objective To investigate the effect of Strong-bone granules on the; regulation of TERT signaling pathway mediated by ER alpha in; osteoblasts. Methods The estrogen receptor antagonist ICI182780; (Faslodex) was used to inhibit the expression of ER in osteoblasts and; to establish the ER-silenced model of rat osteoblast cell line ROS1728.; Serum ALP,BGP,and Col I were determined using enzyme-linked; immunosorbent assay. The mRNA expression of ERE,ERalpha,and c-MYC was; determined using real time quantitative SYBR GREEN assay. The protein; expression of ERE,ERalpha,and c-MYC was detected using Western blotting.; Results The results of ELISA showed that the content of ALP,BGP,and Col; in the culture medium increased gradually with the prolonging of; intervention time. The levels were the highest in the control group,then; followed in estrogen group,Strong-bone granules group,and the model; group,and the difference among the groups was significant (P < 0.05).; The mRNA and protein expression of TERT,ER alpha,and c-MYC was the; highest in the control group,then followed in estrogen group,Strong-bone; granules group,and the model group,and the difference among the groups; was significant (P < 0.05). Conclusion The estrogen mediated TERT; signaling pathway and its related factors are closely related to the; differentiation of osteoblasts. Strong-bone granules promote osteoblast; differentiation through estrogen mediated TERT signaling pathway.