表皮生长因子对牦牛卵丘细胞低氧诱导因子-1α表达的影响及与凋亡的关联性分析  

The Effect of Epidermal Growth Factor on the Expression of Hypoxia Inducible Factor-1α in Cumulus Cells of Yak(Bos grunniens)and Its Correlation Analysis with Apoptosis

作　　者：张译夫[1] 潘阳阳[1] 温泽星[1] 余四九[1] 

机构地区：[1]甘肃农业大学动物医学院甘肃省牛羊胚胎工程技术研究中心,兰州730070

出　　处：《畜牧兽医学报》2016年第6期1154-1161,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31272616;31472244)

摘　　要：旨在研究EGF是否通过调控HIF-1α抑制牦牛卵丘细胞凋亡。本研究在牦牛卵丘细胞体外培养时加入不同浓度的EGF,运用qRT-PCR和免疫荧光技术检测HIF-1α、Bcl-2和Bax的表达,用一步法TUNEL检测不同处理组卵丘细胞的凋亡情况。结果表明:(1)牦牛卵丘细胞体外培养液中添加不同浓度的EGF后,卵丘细胞HIF-1α和Bax mRNA的相对表达量降低,Bcl-2 mRNA的相对表达量增加,且具有浓度依赖性;当EGF浓度为50ng·mL^(-1)时,HIF-1α和Bax mRNA的相对表达量最低,Bcl-2mRNA的相对表达量最高。(2)向其体外培养液中添加不同浓度的EGF后,卵丘细胞HIF-1α和Bax蛋白的相对表达量降低,Bcl-2蛋白的相对表达量增加,且具有浓度依赖性;当EGF浓度为50ng·mL^(-1)时,HIF-1α和Bax蛋白的相对表达量最低,Bcl-2蛋白的相对表达量最高。(3)TUNEL凋亡检测表明,对照组中卵丘细胞凋亡率最高,当EGF浓度为25ng·mL^(-1)时,细胞凋亡率显著降低(P<0.05);当EGF浓度为50ng·mL^(-1)时,细胞凋亡率最低(P<0.05),但随着EGF浓度的增加,卵丘细胞的凋亡率又升高。本研究结果表明,EGF可通过调控HIF-1α抑制卵丘细胞凋亡,其作用可能与线粒体介导的Bax和Bcl-2凋亡途径相关。The present study was carried out to verify whether EGF inhibited the apoptosis of cumulus cells of yak by regulating the expression of HIF-1α.The cumulus cells of yak were cultured in vitro with the supplement of different concentration of EGF.The expression levels of HIF-1α,Bax and Bcl-2 in different groups were detected by qRT-PCR and immunofluorescence.TUNEL was used to evaluate apoptosis rate.Results showed that：（1）After the addition of different concentration of EGF to the cumulus cells,there would be found a declined mRNA relative expression of HIF-1α and Baxand an increased mRNA relative expression of Bcl-2,with a characteristic manner of concentration-dependent.When the concentration of EGF was as high as 50ng·mL^-1,the mRNA relative expressions of HIF-1αand Bax were the lowest,while that of Bcl 2was the highest.（2）The protein relative expressions of HIF-1αand Bax were decreased and that of Bcl-2was increased,with a characteristic manner of concentration-dependent.When the concentration ofEGF was as high as 50ng·mL^-1,the protein relative expressions of HIF-1αand Bax were the lowest,while that of Bcl-2was the highest.（3）Apoptosis detection shown that the apoptosis rate from control group was the highest.It was extremely declined when the concentration of EGF was25ng·mL^-1（P0.05）,and down to its lowest level when EGF was 50ng·mL^-1（P0.05）,while rose again when EGF was continues to increase.It is concluded from this study that EGF can inhibit the apoptosis of cumulus cells by regulating HIF-1αand may be related to mitochondrial Bax and Bcl-2apoptotic pathway.

关 键 词：表皮生长因子(EGF) 低氧诱导因子-1α(HIF-1α) 凋亡 卵丘细胞 牦牛 

分 类 号：S823.8[农业科学—畜牧学]