低氧对体外培养鸡原始生殖细胞凋亡的影响

Effects of Hypoxia on Apoptosis of Chicken Primordial Germ Cells in Vitro

本研究的目的是通过低氧处理体外培养的鸡原始生殖细胞(Primordial germ cell)优化培养系统,降低细胞凋亡率,提高细胞活力,为高效生产转基因鸡提供科学依据。研究采用低氧气体(1%O2)分别处理鸡PGCs 6 h、12 h、24 h,随后用q-PCR检测凋亡相关基因Caspase3的表达。与常氧组(21%O2)相比,1%O2-24h低氧组Caspase3显著下调(p<0.05)。进一步检测1%O2-24 h低氧组p53和Bcl2的表达情况,结果显示p53表达显著下调(p<0.05),Bcl2表达无显著差异。采用流式细胞术检测细胞凋亡比例,低氧组((13.5±0.8)%)凋亡比例低于常氧组((21.8±2.1)%)。采用q-PCR,免疫荧光染色,细胞迁移鉴定低氧处理后的鸡PGCs生物学特性,发现其仍保持生殖细胞的生物学特性。本研究表明,在体外培养条件下,1%O2低氧处理鸡PGCs 24 h能抑制细胞凋亡,且不改变其生物学特性。

The aim of th e present study was to optimize the in vitro culture system of Primordial germ cells(PGCs),in order to reduce apoptosis,improve their livability and subsequently enhance efficiency of transgenic chicken production.The chicken PGCs were cultured under hypoxic environment(1%O2)for 6 h,12 h and 24 h respectively,and then the expression of apoptosis-related gene Caspase3 was detected through using quantitative PCR.It was revealed that PGCs cultured under hypoxia for 24 h had significantly down-regulated(p<0.05)expression of Caspase 3 as compared to PGCs cultured under normoxic condition(21%O2)and other hypoxia treated groups.Moreover,expression of p53 and Bcl2 was also detected in 1%O2-24 h hypoxic group and it was disclosed that p53 was significantly down regulated in 1%O2-24 h hypoxic group as compared to normoxic group,while Bcl2 had similar expression between both the groups.The percentage of apoptosis in PGCs was determined through flow cytometry and it was divulged that hypoxia treated group((13.5±0.8)%)had lower number of apoptotic cells as compared to normoxic group((21.8±2.1)%).The results of q-PCR,immunofluorescence staining and cell migration assay showed that hypoxia does not affect germ cell related biological characteristics of the cells.Therefore,it is concluded from the present study that hypoxia(1%O2)treatment of in vitro cultured chicken PGCs for 24 h reduces the cellular apoptosis without hampering its biological characteristics.