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目的:研究Nrf2对C2C12细胞向成骨细胞分化过程增殖、凋亡和分化的影响。方法:分别构建和包装Nrf2(nuclear factor erythroid 2 p45 related factor 2)过表达及靶向Nrf2和活性转录因子4(activating transcription factor 4,ATF4)的siRNA重组慢病毒颗粒;在骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)诱导下,设立不同病毒感染组处理C2C12细胞,检测各处理组C2C12细胞的增殖、凋亡及成骨相关标志基因的表达。结果:FCM结果显示,BMP2+Lv-Nrf2组细胞凋亡率低于BMP2+Lv-GFP组(P<0.05),BMP2+Lv-si Nrf2组细胞凋亡率高于BMP2+Lv-GFP组(P<0.05),BMP2+Lv-Nrf2+Lv-siATF4组细胞凋亡率低于BMP2+Lv-GFP组(P<0.05)并同时低于BMP2+Lv-siATF4组(P<0.05);蛋白免疫印迹检测结果与FCM凋亡结果一致;免疫细胞化学检测结果显示,实验组BMP2+Lv-Nrf2组中骨钙蛋白(osteocalcin,OCN)与碱性磷酸酶(alkaline phosphatase,ALP)表达增高,而BMP2+Lv-si Nrf2组中两种蛋白表达均降低(P<0.05),联合处理组BMP2+Lv-Nrf2+Lv-siATF4中两种蛋白表达低于对照组和BMP2+Lv-Nrf2组(P<0.05)。结论:上述结果表明,在BMP2诱导C2C12细胞成骨分化过程中,Nrf2可抑制C2C12细胞的凋亡,促进其向成骨分化,同时,可挽回因ATF4蛋白减少而产生的细胞凋亡,但对C2C12细胞的骨向分化没有影响。
Objective: To investigate the effect of Nrf2 on the proliferation, apoptosis and differentiation of C2C12 cells during osteoblast differentiation. METHODS: siRNA recombinant lentiviral particles overexpressing Nrf2 and targeting Nrf2 and activating transcription factor 4 (ATF4) were constructed and packaged respectively. In bone morphogenetic protein 2 ( bone morphogenetic protein 2 and BMP2, C2C12 cells were treated with different virus infection groups to detect the proliferation, apoptosis and expression of osteogenic marker genes in C2C12 cells. Results: FCM showed that the apoptosis rate of BMP2 + Lv-Nrf2 group was lower than that of BMP2 + Lv-GFP group (P <0.05), and the apoptosis rate of BMP2 + Lv-si Nrf2 group was higher than that of BMP2 + Lv- The apoptosis rate of BMP2 + Lv-Nrf2 + Lv-siATF4 group was lower than that of BMP2 + Lv-GFP group (P <0.05) and lower than that of BMP2 + Lv-siATF4 group The result of immunocytochemistry showed that the expression of osteocalcin (OCN) and alkaline phosphatase (ALP) in BMP2 + Lv-Nrf2 group were increased in experimental group, while the expression of BMP2 + Lv-si Nrf2 group (P <0.05). The expression of two proteins in BMP2 + Lv-Nrf2 + Lv-siATF4 group was lower than that in control group and BMP2 + Lv-Nrf2 group (P < 0.05). CONCLUSION: The above results indicate that Nrf2 can inhibit the apoptosis of C2C12 cells during BMP2-induced osteogenic differentiation of C2C12 cells and promote the osteogenic differentiation of C2C12 cells. At the same time, Nrf2 can reverse the apoptosis of C2C12 cells induced by reduced ATF4 protein, C2C12 cells had no effect on bone differentiation.