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

doi:10.3969/j.issn.2095-4344.0570

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Osteoporosis, Postmenopausal, Estrogen Receptor alpha, Adenoviridae, Tissue Engineering

CLC Number:

Huang Hong1, Huang Jiachun2, Huang Hongxing3, Wang Jili2, Liu Shaojin2, Wang Yuedong2 . Proliferation and differentiation of MG63 cells transfected with recombinant adenovirus vector overexpressing estrogen-related receptor alpha after silencing Bak1 and Bcl2[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(7): 1041-1045.

Figures/Tables 1

2.1 Ad-ERRα对各组MG63细胞增殖、碱性磷酸酶活性、钙离子浓度的影响与空载病毒组比较，Ad-shBak1组细胞活性、碱性磷酸酶活性明显升高，钙离子浓度明显降低，差异有显著性意义(P < 0.01)；与空载病毒组比较， Ad-shBcl2组细胞活性、碱性磷酸酶活性均降低，但降低幅度不明显(P > 0.05)，钙离子浓度明显升高，差异有显著性意义(P < 0.01)；与空载病毒组比较，Ad-shBak1+shBcl2组的细胞活性、碱性磷酸酶活性稍有升高，钙离子浓度稍有降低，但差异无显著性意(P > 0.05)；与Ad-shBcl2组比较，Ad-shBak1组与Ad-shBak1+shBcl2组碱性磷酸酶活性、Ad-shBak1组的细胞活性升高，钙离子浓度明显降低，差异有显著性意义(P < 0.01)， Ad-shBak1+shBcl2组细胞活性显著升高(P < 0.05)；与Ad-shBak1组比较， Ad-shBak1+shBcl2组的细胞活性、碱性磷酸酶活性显著降低，钙离子浓度明显升高(P < 0.01)，见图1。 2.2 Ad-ERRα干预Bak1、Bcl2重组腺病毒转染的各组MG63细胞中骨调节蛋白的影响与空载病毒组比较， Ad-shBcl2组骨形态发生蛋白4、Runt相关转录因子2均降低，而骨桥蛋白、肿瘤坏死因子α升高，差异有显著性意义(P < 0.01)，结缔组织生长因子虽然也降低，但无显著性意义(P > 0.05)；与空载病毒组比较，Ad-shBak1组骨形态发生蛋白4、结缔组织生长因子、骨桥蛋白、Runt相关转录因子2均升高，而肿瘤坏死因子α降低，差异有显著性意义(P < 0.01)；Ad-shBak1+ shBcl2组骨桥蛋白、Runt相关转录因子2、骨形态发生蛋白4、结缔组织生长因子、肿瘤坏死因子α均升高。差异有显著性意义(P < 0.01)。与Ad-shBcl2组比较，Ad-shBak1组和shBcl2组骨形态发生蛋白4、结缔组织生长因子、骨桥蛋白、Runt相关转录因子2均升高，而肿瘤坏死因子α降低，差异有显著性意义(P < 0.01)；与Ad-shBak1组比较，Ad-shBak1+shBcl2组骨形态发生蛋白4、结缔组织生长因子、骨桥蛋白均降低，肿瘤坏死因子α升高(P < 0.01)，Runt相关转录因子2也升高，但差异不明显(P > 0.05)，见图2。"

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