维生素D对小鼠精原干细胞增殖和凋亡的影响及机制研究

Effects of vitamin D on proliferation and apoptosis of mouse spermatogonial stem cells and its related mechanism

目的研究维生素D对小鼠精原干细胞增殖和凋亡的影响及其机制。方法体外培养小鼠精原干细胞C18-4细胞,采用CCK-8法检测不同浓度和作用时间1α,25-(OH)_(2)D_(3)处理对C18-4细胞活力和增殖的影响,采用流式细胞仪(AnnexinV/PI染色)检测1α,25-(OH)_(2)D_(3)处理对C18-4细胞凋亡的影响,采用qRT-PCR方法检测1α,25-(OH)_(2)D_(3)处理C18-4细胞后Bcl-2、Bax、Vdr和Pcna基因表达的情况。结果与对照组相比较,10^(-7)M浓度1α,25-(OH)_(2)D_(3)处理24h和48h后细胞增殖率显著降低(P<0.05)。10^(-7)M浓度1α,25-(OH)_(2)D_(3)处理48h后细胞的早期凋亡率显著增高(P<0.05)。10^(-7)M浓度1α,25-(OH)_(2)D_(3)处理48h后细胞的Bax基因和Vdr基因表达显著上调(P<0.05),而Bcl-2基因和Pcna基因表达显著下调(P<0.05)。结论较高浓度1α,25-(OH)_(2)D_(3)通过抑制Pcna的表达而抑制小鼠精原干细胞的增殖,通过上调Bax和下调Bcl基因表达而促进小鼠精原干细胞的凋亡。

Objective To investigate the effectsof vitamin D3 on mouse spermatogonial stem cells and its related mechanism.Methods Mouse spermatogonial stem cells(C18-4)were treated with 1α,25-(OH)_(2)D_(3)at different concentrations and durations of action,and their effects on cell viability and proliferation were detected by the CCK-8 method.The effect of1α,25-(OH)_(2)D_(3)with a concentration of 10^(-7)Mon the apoptosis of C18-4 cells was measuredby Flow cytometry(AnnexinV/PI staining),and its effectson m RNA expression of Bcl-2,Bax,Vdr and Pcna genes in C18-4 cells were analyzed by real-time q PCR method.Results Compared with that of the control cells,the proliferation rates of C18-4 cells after 1α,25-(OH)_(2)D_(3)treatment for 24h and 48h were all significantly decreased(P<0.05),the percentage of early apoptosis of C18-4 cells was significantly increased after 1α,25-(OH)_(2)D_(3)treatment for 48h(P<0.05),the m RNA expression of Bax and Vdr was significantly upregulated(P<0.05),whereas the m RNA expression of Bcl-2 and Pcna was significantly downregulated(P<0.05).Conclusion High concentration of 1α,25-(OH)_(2)D_(3)could inhibit the proliferation of mouse spermatogonial stem cells by downregulating Pcna expression,and induce the apoptosis of mouse spermatogonial stem cells by upregulating Bax expression and downregulating Bcl expression.