添加小分子化合物CHIR99021维持水牛精原细胞的体外培养

Addition of the Small Molecuar Compound CHIR99021 Maintains the Culture of Buffalo Spermatogonial Cells in vitro

目前,水牛精原干细胞(spermatogonial stem cells,SSCs)体外培养系统因尚无法支持水牛(Bubalus bubalis)SSCs体外长久培养而限制了水牛雄性遗传资源的高效利用。通过添加合适小分子化合物优化水牛SSCs体外培养系统是一个可选方案。本研究推测GSK3β特异性抑制剂CHIR99021能促进水牛精原细胞的体外增殖和自我更新。为了验证此假说,本研究探索了添加不同浓度CHIR99021对水牛精原细胞体外培养效果的影响。分别将不同浓度CHIR99021(0,5,10,20μmol/L)添加到水牛精原细胞体外培养细胞培养液中,设置4个试验组,体外培养7 d后收集细胞,进行实时定量PCR(real-time quantitative PCR,RT-qPCR)和免疫荧光染色细胞计数。RT-qPCR结果表明,添加10、20μmol/L的CHIR99021显著提高了SSCs特异性表达基因(PLZF,Nanos2)和增殖相关基因(CCND1,CCNE1)的表达水平;显著降低了分化相关基因(DMRT1,DAZL)和氧化相关基因(CAT,SOD3)的表达水平。通过免疫荧光染色进行DDX4^(+)和PGP9.5^(+)细胞计数,发现添加10μmol/L CHIR99021显著增加了精原细胞数量。以上结果说明,在体外培养系统中添加CHIR99021能有效维持水牛精原细胞的SSCs分子特征,促进其体外增殖,显著改善了水牛精原细胞体外培养条件。本研究能为利用小分子化合物优化水牛SSCs体外培养系统提供重要参考。

At present,the in vitro culture system of buffalo(Bubalus bubalis)spermatogonial stem cells(SSCs)cannot support the long-term culture of buffalo SSCs in vitro,which limits the efficient utilization of buffalo male genetic resources.Optimization of buffalo SSCs in vitro culture system by addition of suitable small mole-cular compounds is an option.This research hypothesized that buffalo spermatogonial cells were promoted proli-feration and self-renewal with GSK3βspecific inhibitor CHIR99021 in vitro.To verify this hypothesis,the CHIR99021(0,5,10,20μmol/L)was added into the culture medium of buffalo spermatogonial cells in vitro,set as four experimental groups.After 7 days in vitro culture,the cells were collected and carried out by real-time quantitative PCR(RT-qPCR)and cell count based on immunofluorescence staining.The results of RT-qPCR showed that additional of 10μmol/L and 20μmol/L CHIR99021 significantly increased the expression of SSCs specific genes(PLZF,Nanos2)and proliferation-related genes(CCND1,CCNE1);significantly decreased differentiation related genes(DMRT1,DAZL)and oxidation-related genes(CAT,SOD3).DDX4^(+)and PGP9.5^(+)cells were counted by immunofluorescence staining.It was found that addition of 10μmol/L CHIR99021 significantly increased the spermatogonial cells number.These results indicated that additional of CHIR99021 could effectively maintain the SSCs molecular characteristics of buffalo spermatogonial cells,promote their proliferation in vitro,thus improve the in vitro culture conditions of buffalo spermatogonia.This study can provide important reference for optimizing the in vitro culture system of buffalo SSCs with small molecular compounds.