【摘要】 背景:生长因子作为体外细胞培养和体内细胞生长及增殖必需的调节因子,一直被广泛的关注。目的:探讨碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）与胰岛素样生长因子1（insulin-like growth facter,IGF-1）联合作用对小鼠精原干细胞增殖、凋亡的影响。方法:从6-8d龄昆明雄性小鼠睾丸内分离培养精原干细胞并进行鉴定。将精原干细胞接种于经丝裂霉素C处理过的胚胎成纤维细胞饲养层上,分组干预:对照组加入正常DMEM培养基进行培养;b FGF、IGF-1组分别加入含20μg/L bFGF、20μg/L IGF-1的DMEM培养基进行培养;b FGF+IGF-1组同时加入含20μg/L b FGF及20μg/L IGF-1的DMEM培养基进行培养。采用CCK-8、EDU染色法分别检测精原干细胞增殖活性,流式细胞仪检测精原干细胞生长周期和细胞凋亡情况,Western blot检测增殖和凋亡相关蛋白PCNA、Bax、Bcl-2的表达。结果与结论:①与对照组比较,b FGF组、IGF-1组、b FGF+IGF-1组吸光度值显著升高,与b FGF组、IGF-1组比较,b FGF+IGF-1组吸光度值进一步升高（P<0.05）,EDU染色得到与CCK-8实验一致的结论;②b FGF+IGF-1组S+G₂/M期细胞比例明显高于其他3组（P <0.05）,IGF-1组、b FGF组S+G₂/M期细胞比例高于对照组（P <0.05）;③与对照组比较,b FGF组、IGF-1组、b FGF+IGF-1组凋亡细胞降低;与b FGF组、IGF-1组比较,b FGF+IGF-1组凋亡细胞进一步降低;④与对照组比较,bFGF组、IGF-1组、b FGF+IGF-1组细胞中Bax蛋白相对表达水平显著下降（P <0.01）,Bcl-2和PCNA蛋白相对表达水平均显著升高（P <0.05）。与b FGF组、IGF-1组比较,b FGF+IGF-1组细胞中Bax蛋白相对表达水平进一步下降（P<0.01）,Bcl-2和PCNA蛋白相对表达水平进一步升高（P<0.05）;⑤结果表明,b FGF、IGF-1通过上调PCNA和Bcl-2蛋白的表达,下调Bax蛋白的表达,促进细胞增殖,抑制细胞凋亡,二者联合作用效果最佳。更多还原

【Abstract】 BACKGROUND: As a necessary regulatory factor of in vitro cell culture, and in vivo cell growth and proliferation, growth factors have attracted much attention. OBJECTIVE: To investigate the effects of basic fibroblast growth factor（bFGF） combined with insulin-like growth factor-1（IGF-1） on the proliferation and apoptosis of spermatogonial stem cells in mice. METHODS: Spermatogonial stem cells were isolated and cultured from the testis of 6-8 days old male Kunming mice and were identified. Spermatogonial stem cells were inoculated into the feeder layer of embryonic fibroblasts treated with mitomycin C, and were then divided into four groups. Control group was cultured with normal DMEM medium; bFGF and IGF-1 groups were cultured with DMEM medium containing 20 μg/L bFGF and 20 μg/L IGF-1, respectively; bFGF+IGF-1 group was cultured with DMEM medium containing 20 μg/L bFGF and 20 μg/L IGF-1. The proliferation activity of spermatogonial stem cells was detected by cell counting kit-8 assay and EDU staining. The growth cycle and apoptosis of spermatogonial stem cells were detected by flow cytometry. The expression levels of PCNA, Bax and Bcl-2 proteins were detected by western blot assay. RESULTS AND CONCLUSION: Compared with the control group, the absorbance values in the bFGF, IGF-1 and bFGF+IGF-1 groups were significantly increased. Compared with the bFGF and IGF-1 groups, the absorbance values in the bFGF+IGF-1 group were further increased（P < 0.05）. EDU staining results showed the same conclusion as cell counting kit-8 assay results. The proportion of S+G₂/M phase cells in the bFGF+IGF-1 group was significantly higher than that in the other three groups（P < 0.05）. The proportion in the IGF-1 and bFGF groups was significantly higher than that in control group（P < 0.05）. Compared with the control group, the number of apoptotic cells in the bFGF, IGF-1 and bFGF+IGF-1 groups was decreased. Compared with the bFGF and IGF-1 groups, the number of apoptotic cells in the bFGF+IGF-1 group was further decreased. Compared with the control group, the relative expression levels of Bax protein in the bFGF, IGF-1 and bFGF+IGF-1 groups were significantly decreased（P < 0.01）, and the expression levels of Bcl-2 and PCNA proteins were significantly increased（P < 0.05）. Compared with the bFGF and IGF-1 groups, the relative expression level of Bax protein in the bFGF + IGF-1 group was decreased further（P < 0.01）, and the relative expression levels of Bcl-2 and PCNA proteins were increased further（P < 0.05）. These results indicate that bFGF and IGF-1 can promote cell proliferation and inhibit cell apoptosis by up-regulating the expression of PCNA and Bcl-2 proteins and down-regulating the expression of Bax protein. The combination of bFGF and IGF-1 can achieve favorable effects.更多还原