牦牛bta-miR-146a对卵巢卵泡颗粒细胞自噬的影响研究  

Effect of Yak bta-miR-146a on Autophagy of Ovarian Follicular Granulosa Cells

作　　者：徐业芬 王运路 孟朝轶 刘博华 昝瑞隆 XU Yefen;WANG Yunlu;MENG Zhaoyi;LIU Bohua;ZAN Ruilong(College of Animal Science,Xizang Agricultural and Animal Husbandry College;Provincial Key Laboratory of Xizang Plateau Animal Epidemic Disease Research,Linzhi,Xizang 860000)

机构地区：[1]西藏农牧学院动物科学学院 [2]西藏高原动物疫病研究自治区高校重点实验室,西藏林芝860000

出　　处：《高原农业》2024年第4期367-375,共9页Journal of Plateau Agriculture

基　　金：国家自然科学基金(31960661,31460604);西藏自治区科学技术厅区域科技协同创新专项(QYXTZX-NQ2022-04);西藏自治区科技厅重点研发计划项目(XZ202301ZYO003N04)。

摘　　要：自噬是细胞广泛存在的程序性死亡机制,通过对细胞内衰老细胞器、长寿命蛋白以及外源病原微生物进行吞噬、降解,以维持细胞平衡。在卵巢中,卵泡颗粒细胞自噬对卵泡发育起着关键作用,失衡会导致闭锁。为探究miR-106a对牦牛卵泡颗粒细胞自噬的影响,本试验通过体外分离培养牦牛卵泡颗粒细胞后,分别转染bta-miR-146a模拟物(bta-miR-146a mimics)和bta-miR-146a抑制剂(bta-miR-146a inhibitor),利用荧光定量RT-PCR法、Western Blot和激光共聚焦法检测细胞的自噬相关基因mRNA和蛋白表达情况。结果显示,转染bta-miR-146a mimic组与mimic NC组相比激光共聚焦可检测到LC3B蛋白荧光强度降低(P<0.01);荧光定量RT-PCR检测发现LC3B、Atg-5和Beclin-1自噬指示基因mRNA表达量均极显著降低(P<0.01);bta-miR-146a inhibitor组与bta-miR-146a inhibitor NC组相比LC3B、Atg-5和Beclin-1极显著升高(P<0.01),bta-miR-146a mimic组与bta-miR-146a inhibitor组相比,LC3B、Atg-5和Beclin-1自噬指示基因m RNA表达量极显著降低(P<0.01)。Western Blot检测发现bta-miR-146a inhibitor组自噬指示蛋白LC3B、Atg-5和Beclin-1表达最高,bta-miR-146a mimic组表达最低;bta-miR-146a mimic组与bta-miR-146a mimic NC组相比,LC3B、Atg-5和Beclin-1蛋白表达极显著降低(P<0.01),bta-miR-146a inhibitor组与bta-miR-146a inhibitor NC相比LC3B、Atg-5和Beclin-1蛋白表达极显著升高(P<0.01),bta-miR-146a mimic组与bta-miR-146a inhibitor组相比,自噬指示蛋白LC3B、Atg-5和Beclin-1的表达量极显著降低(P<0.01)。上述结果表明bta-miR-146a mimic抑制牦牛卵泡颗粒细胞的自噬,而bta-miR-146a inhibitor促进牦牛卵泡颗粒细胞的自噬。Autophagy is a mechanism of programmed cell death,which can maintain cell balance by phagocytosis and degradation of senescent organelles,long-lived proteins and exogenous pathogenic microorganisms.In the ovary,the autophagy of follicular granulosa cells plays a key role in follicular development,and imbalance can lead to atresia.In order to explore the effect of miR-106a on autophagy of yak follicular granulosa cells(GCs),yak follicular GCs was isolated and cultured in vitro and transfected with bta-miR-146a mimic(bta-miR-146a mimics)and bta-miR-146a inhibitor(bta-miR-146a inhibitor).The expression of autophagy related gene mRNA and protein in GCs cells was detected by fluorescence quantitative RT-PCR,WesternBlot and laser confocal method.The results showed that the fluorescence intensity of LC3B protein in bta-miR-146a mimic group was lower than that in mimic NC group(P<0.01);and the expression of LC3B,Atg-5 and Beclin-1 autophagy indicator gene mRNA was significantly decreased by fluorescence quantitative RT-PCR(P<0.01).The expression of LC3B,Atg-5 and Beclin-1 in bta-miR-146a inhibitor group was significantly higher than that in bta-miR-146a inhibitor NC group(P<0.01),while the expression of LC3B,Atg-5 and Beclin-1 autophagy indicator gene mRNA in bta-miR-146a mimic group was significantly lower than that in bta-miR-146a inhibitor group(P<0.01).WesternBlot detection showed that the expression of autophagy indicator proteins LC3B,Atg-5 and Beclin-1 was the highest in bta-miR-146a inhibitor group,and the lowest in bta-miR-146a mimic group.The expression of LC3B,Atg-5 and Beclin-1 in bta-miR-146a mimic group was significantly lower than that in bta-miR-146a mimic NC group(P<0.01),while the expression of LC3B,Atg-5 and Beclin-1 in bta-miR-146a inhibitor group was significantly higher than that in bta-miR-146a inhibitor NC group(P<0.01).The expression of autophagy indicator proteins LC3B,Atg-5 and Beclin-1 in bta-miR-146a mimic group was significantly lower than that in bta-miR-146a inhibitor group(P<0.01).These r

关 键 词：牦牛 bta-miR-146a 颗粒细胞 自噬 

分 类 号：S8[农业科学—畜牧兽医]