过表达雌激素相关受体α干预沉默Bak1、Bcl2重组腺病毒载体转染MG63细胞的增殖与分化

Proliferation and differentiation of MG63 cells transfected with recombinant adenovirus vector overexpressing estrogen-related receptor alpha after silencing Bak1 and Bcl2

背景:雌激素缺乏是绝经后骨质疏松症的主要发病机制,而关于雌激素相关受体α(estrogen-relatedreceptor alpha,ERRα)与骨质疏松症的相关性研究较少,ERRα在骨质疏松症中的具体作用及其机制在目前尚不明确。目的:研究过表达ERRα对沉默Bak1、Bcl2重组腺病毒载体转染的MG63细胞和相关蛋白的影响。方法:构建过表达ERRα和沉默Bak1、Bcl2腺病毒载体,将培养好的MG63细胞分成空载病毒组、Ad-shBak1组、Ad-sh Bcl2组、Ad-shBak1+shBcl2组,过表达ERRα进行干预。MTT法检测细胞增殖,考马斯亮蓝蛋白定量检测碱性磷酸酶活性,流式法测定钙离子浓度,Western blot法分析骨调节蛋白(骨形态发生蛋白4、结缔组织生长因子、骨桥蛋白、Runt相关转录因子2、肿瘤坏死因子α)的表达。结果与结论:(1)与空载病毒组对比,Ad-shBak1组的细胞活性、碱性磷酸酶活性均升高,钙离子浓度降低,各骨调节蛋白除肿瘤坏死因子α降低外,其余均升高;Ad-shBcl2组细胞活性、碱性磷酸酶活性、结缔组织生长因子均降低,但无显著性意义(P> 0.05),钙离子浓度、骨形态发生蛋白4、Runt相关转录因子2均降低,骨桥蛋白、肿瘤坏死因子α显著升高(P <0.01);Ad-shBak1+shBcl2组的细胞活性、碱性磷酸酶活性稍升高,钙离子浓度稍降低,各骨调节蛋白水平均升高,但差异不明显(P> 0.05);(2)与Ad-sh Bcl2组比较,Ad-shBak1组与Ad-shBak1+shBcl2组细胞活性、碱性磷酸酶活性升高,钙离子浓度显著降低,除肿瘤坏死因子α水平显著降低外,其余骨调节蛋白水平显著升高(P <0.01或P <0.05);(3)结果提示,过表达ERRα可提高重组腺病毒Bak1转染后的MG63细胞增殖和碱性磷酸酶活性,降低钙离子浓度,且对骨调节蛋白有一定影响作用。

BACKGROUND: Estrogen deficiency is the main pathogenesis of osteoporosis in postmenopausal women, but there are few researches on the correlation between estrogen-related receptor α(ERRα) and osteoporosis. Little is known about their mechanisms of action. OBJECTIVE: To investigate the effect of adenoviral overexpression of ERRα and silencing of Bak1/Bcl2 in MG63 cells. METHODS: Adenovirus vector overexpressing ERRα and silencing of Bak1/Bcl2 was constructed. MG63 cells were divided into blank control, Ad-shBak1, Ad-shBcl2, and Ad-shBak1+shBcl2 groups. MG63 cells of different groups were infected by ERRα, Bak1, and Bcl2 overexpressing recombinant adenovirus. The cell proliferation was detected by MTT assay. The alkaline phosphatase activity was measured by Coomassie brilliant blue method. The Ca2+ concentration was detected by flow cytometry. The expression levels of related bone-regulating proteins (bone morphologic protein 4, connective tissue growth factor, osteopontin, Runt 2 and tumor necrosis factor α) were tested by western blot assay.. RESULTS AND CONCLUSION: Compared with the blank control group, the cell viability and alkaline phosphatase activity were significantly increased and Ca2+ concentration was significantly decreased, bone morphologic protein 4, connective tissue growth factor, osteopontin and Runt 2 were increased, and tumor necrosis factor α was decreased in the Ad-shBak1 group. The cell viability, alkaline phosphatase activity and connective tissue growth factor level were decreased, but not significant in the Ad-shBcl2 group (P > 0.05). In the Ad-shBcl2 group, the Ca2+ concentration, bone morphologic protein 4, and Runt 2 were significantly decreased, osteopontin and tumor necrosis factor α were significantly increased (P < 0.01). In the Ad-shBak1+shBcl2 group, the cell viability and alkaline phosphatase activity were increased, Ca2+ concentration was decreased, and level of each protein was increased (P > 0.05). Compared with the Ad-shBcl2 group, in the Ad-shBak1 and Ad-shBak1+shBcl2 groups, the cell viability and alkaline phosphatase activity were significantly increased, Ca2+ concentration was significantly decreased, bone morphologic protein 4, connective tissue growth factor, osteopontin and Runt 2 were increased, and tumor necrosis factor α was significantly decreased (P < 0.01 or P < 0.05). Our findings suggest that the overexpression of ERRα can increase the MG63 cell proliferation and alkaline phosphatase activity after transfection by Bak1 recombinant adenovirus, decrease Ca2+ concentration, and also has certain effects on related bone regulating proteins.