中文摘要：

为研究胰岛素样生长因子（insulin-like growth factor,IGF-1）是否通过调控热休克蛋白70 （heat shock protein 70,HSP70）抑制牦牛（Bos grunniens）卵丘细胞凋亡,本研究在牦牛卵丘细胞体外培养时分别加入0、50、100和200 ng/mL IGF-1,运用实时荧光定量PCR（quantitative Real-time PCR,qRT-PCR）、蛋白免疫印迹（Western blot,WB）和免疫荧光技术从基因和蛋白水平检测HSP70的表达;qRT-PCR分析IGF-1对凋亡相关基因B-细胞淋巴瘤/白血病-2原癌基因（B-cell lymphoma/leukemia-2,Bcl-2）和B细胞淋巴瘤/白血病基因伴随蛋白x（B-cell lymphoma/leukemia associated x protein,Bax）的调节作用,流式细胞技术统计不同处理组卵丘细胞凋亡率.结果显示,（1） IGF-1浓度为100 ng/mL时,卵丘细胞的HSP70 mRNA和蛋白相对表达量最高,显著高于IGF-1浓度为50和200 ng/mL组（P＜0.05）,对照组HSP70 mRNA的相对表达量最低（P＜0.05）,卵丘细胞的细胞质和细胞核均可表达HSP70蛋白;（2）加入IGF-1,Bax mRNA相对表达量显著下降（P＜0.05）,1 00 ng/mL时最低,Bcl-2 mRNA相对表达量显著增加,100 ng/mL时最高;（3）卵丘细胞的凋亡率在100 ng/mL IGF-1最低,仅为（6.32±0.84）％,其他处理组凋亡率均显著增加（P＜0.05）,对照组凋亡率最高,为（26.14±3.16）％.上述结果表明,IGF-1可调节卵丘细胞的HSP70、Bax和Bcl-2表达,降低细胞凋亡率,其最佳作用浓度为1 00 ng/mL.本研究为阐明IGF-1抑制细胞凋亡的分子机制提供了新的依据,为进一步揭示IGF-1在卵母细胞成熟及早期胚胎发育过程中的作用机制提供理论依据,有助于牦牛胚胎体外发育技术改进.

英文摘要：

The aim of this study is to verify whether the expression of HSP70 （heat shock protein, HSP） and apoptosis of yak （Bos grunniens） cumulus cells were regulated by IGF-1 （insulin-like growth factor, IGF-1）. The yak cumulus cells were cultured in vitro supplemented with different concentrations of IGF-1 （0, 50, 100 and 200 ng/mL）. The relative expression levels of HSP70 mRNA and protein in different groups were detected by Real-time quantitative PCR （qRT-PCR） and Western blot （WB）, and location of HSP70 protein was detected by immunofluorescence. Furthermore, relative expression levels of Bax （B-cell lymphoma/leukemia associated x protein, Bax） and Bcl-2 （B-cell lymphoma/Leukemia-2, Bcl-2） mRNA in different groups were also evaluated by qRT-PCR, flow cytometry was used to evaluate the rate of apoptosis. The result showed that： （1） IGF-1 improved the relative levels of HSP70 mRNA and protein; these improvements were highest in the 100 ng/mL IGF-1 group, followed by 200 ng/mL and 50 ng/mL groups, the relative levels of HSP70 mRNA and protein were lowest in control group （0 ng/mL IGF-1）; （2） Detection by immunofluorescence showed that HSP70 protein could be observed in yak cumulus cells cytoplasm as well as within nuclei; （3） Relative level of Bax was down-regulated by IGF- 1, while Bcl-2 was up-regulated. The Bax mRNA was lowest in groups with 100 ng/mL IGF-1, where the Bcl-2 was the highest. In 200 and 50 ng/mL IGF-1 groups, the relative expression levels of Bax was decreased compared with that of in 100 ng/mL group, while relative expression levels of Bcl-2 was increased. Bax expression in the groups with IGF-1 was lower than the group without IGF- l（control group）, while Bcl-2 expression was higher in groups with IGF-1 than without IGF-1; （4） IGF-1 inhibited apoptosis of yak cumulus cells, and the rate of apoptosis in cumulus cells was （6.32±0.84）% in 100 ng/mL IGF-1 group, which was lowest among these groups. In 200 and 50 ng/mL IGF-1 groups, the apopt