Nrf2重组慢病毒对C2C12细胞增殖、凋亡和分化的影响

Effects of lentivirus Nrf2 on proliferation,differentiation,and apoptosis of C2C12 cells

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 study the effects of Nrf2 on proliferation,apoptosis and differentiation of C2C12 cells during osteoblastic differentiation. Methods：The recombination lentivirus of nuclear factor erythroid 2 p45 related factor 2（Nrf2）and its siRNA were constructed in order to investigate the function of gene Nrf2 on apoptosis during osteogenic differentiation of C2C12 cells. The plasmids p WPT-GFP-Nrf2,p WPT-GFP-si NRF2 and p WPT-GFP-siATF4 were co-transfected respectively with other two lentivirus packaging plasmids into 293 T cells to produce Lv-Nrf2,Lv-si Nrf2 and Lv-siATF4 virus particles. Different treatment groups of lentivirus were set under the induction of bone morphogenetic protein 2（BMP2）,the proliferation,apoptosis and expression of osteogenic-differentiation-related marker proteins were detected respectively. Results：FCM analysis showed that the apoptosis rate in BMP2＋Lv-Nrf2 group was lower than that in BMP2＋Lv-GFP group;the apoptosis rate in BMP2＋Lv-si Nrf2 group was higher than that in BMP2＋Lv-GFP group;the apoptosis rate in BMP2＋Lv-Nrf2＋Lv-siATF4 group was lower than that in BMP2＋Lv-siATF4 group;the results of Western blotanalysis were consistent with those of FCM. Cell immunochemistry staining showed that the expression levels of osteocalcin and alkaline phosphatasewere higher in BMP2＋Lv-Nrf2 group than in control group BMP2＋Lv-GFP,lower in BMP2＋Lv-si Nrf2 groupthan in control group,lower in combined treatment group BMP2 ＋Lv-Nrf2 ＋Lv-siATF4 than in control group and single treatment group BMP2＋Lv-Nrf2. Conclusion ：Under the induction of BMP2,gene Nrf2 could inhibit the apoptosis and promote the osteogenic differentiation of C2C12 cells. Additionally,gene Nrf2 could rescue the increased cell apoptosis level,but could not rescue the decreased osteogenic differentiation level both caused by ATF4 deficiency.