冷应激条件下猪睾丸细胞中RNA结合基序蛋白3过表达对Caspase 3表达的影响

Effect of RNA binding motif protein 3 overexpression on Caspase 3 expression in swine testiclar cell under cold exposure

目的:探讨冷应激(32℃)条件下,猪睾丸(ST)细胞系中RNA结合基序蛋白3(RBM3)过表达时凋亡蛋白半胱天冬酶3(Caspase 3)表达量变化情况。方法:利用本实验室成功构建的携带绿色荧光蛋白(GFP)的RBM3过表达慢病毒载体pLenti6/V5-GW/EmGFP-RBM3和空病毒载体pLenti6/V5-GW/EmGFP-DEST分别感染ST细胞,作为过表达病毒(OEV)组和空载体病毒(EVV)组,此外还设立野生型细胞(WTC)组作对照,利用实时荧光定量RCR(qPCR)法和Western blot分析法检测各组中RBM3 mRNA和蛋白的表达情况。然后对各组细胞进行37℃以及32℃(2 h、4 h,8 h)的冷应激处理,利用酶联免疫吸附实验(ELISA)法检测各组Caspase 3表达量的变化。结果:OEV组RBM3基因和蛋白相对表达量高于EVV组和WTC组。在37℃以及32℃冷应激实验(2 h、4 h,8 h)中,OEV组Caspase 3表达量显著低于EVV组和WTC组。结论:冷应激ST细胞中RBM3过表达能够显著地降低Caspase 3的表达程度,为RBM3基因具有抵抗低温诱导ST细胞凋亡作用提供实验依据。

Objective： To investigate the change of apoptosis protein （Caspase 3） expression when RNA binding motif protein 3 （RBM3） overexpression in swine testicle （ST） cell line under cold stress （32℃） condition. Methods： In present study, RBM3 overexpression lentiviral vector （pLenti6/V5-GW/EmGFP-RBM3） and empty viral vector （pLenti6/V5-GW/EmGFP-DEST） with green fluorescent protein （GFP） that were successfully constructed in our laboratory were transfected into ST cells as overexpression virus group （OEV）, empty vector virus （EVV） group and establishment of wildtype cell （WTC） group as the control group. The real-time fluorescence quantitative RCR （qPCR） and Western blot were used to detect expression of RBM3 mPuNA and protein in each group, then cells in each group were cultured at 37℃ or 32℃ （2 h, 4 h, 8 h）, the changes of Caspase 3 expression in each group were detected by enzyme linked immunosorbent assay （ELISA）. Results： Relative RBM3 gene and protein expression in OEV group were significantly higher than those in EVV group and WTC group. At the temperature of 37℃ and at 32℃（2 h, 4 h, 8 h） in cold stress experiment, Caspase 3 expression in OEV group was significantly lower than those in EVV group and WTC group. Conclusion： RBM3 overexpression in ST cell line exposured to cold can significantly decreased the level of Caspase 3 expression, this study provides an experimental basis of RBM3 resistance against apoptosis of ST cells induced by low temperature.