摘要
本研究旨在揭示肌抑素(MSTN)与基质金属蛋白酶(MMPs)的调控关系,探明MSTN对PK15细胞MMP-2/7/9表达的影响。对MSTN单等位基因敲除猪背膘组织进行转录组测序分析,复苏前期制备的MSTN基因敲除PK15细胞系:单等位基因敲除的PK3108细胞系和双等位基因敲除的L18细胞系,通过实时荧光定量PCR和Wesrern blotting分别检测PK15、PK3108和L18细胞系中MSTN、MMP-2/7/9基因的mRNA和蛋白表达水平。结果发现,与野生型猪相比,MSTN单等位基因敲除猪背膘组织转录因子C/EBPδ、MMP-2/7基因mRNA表达量均极显著下调(P<0.01);细胞外基质中纤连蛋白(FN)和层连接蛋白(LN)含量均极显著增加(P<0.01)。复苏的PK3108和L18细胞呈现绿色荧光。实时荧光定量PCR结果显示,PK3108和L18细胞中MSTN、MMP-2/7/9的mRNA表达量均极显著低于PK15细胞(P<0.01);Western blotting结果显示,PK3108和L18细胞中MSTN、MMP-2/7/9的蛋白表达量均明显低于PK15细胞。本研究结果表明,在MSTN基因敲除的PK15细胞中,MSTN功能缺失能显著降低MMP-2/7/9的表达,且MMP-2/7/9蛋白表达降低的趋势与MSTN蛋白表达的趋势相一致。
The study was aimed to reveal the regulation relationship between myostation(MSTN)and matrix metalloproteinase(MMPs),and explore the effects of MSTN on the expression of MMP-2/7/9 in PK15 cells.The backfat tissues of MSTN single allele knockout pig was analyzed by transcriptome sequencing.MSTN knockout PK15 cells line of MSTN single allele knockout PK3108 cells and MSTN double allele knockout L18 cells were prepared,the mRNA and protein expression of MSTN and MMP-2/7/9 in PK15,PK3108 and L18 cells were performed by Real-time PCR and Western blotting,respectively.The results showed that compared with the wild type pigs,the levels of transcription factor C/EBPδand MMP-2/7 mRNA were extremely significantly reduced(P<0.01),and the main components of extracellular matrix laminin(LN)and fibronectin(FN)were extremely significantly increased in MSTN single allele knockout pigs(P<0.01).The revived PK3108 and L18 cells showed green fluorescence.Real-time PCR results showed that the mRNA expression of MSTN and MMP2/7/9 in PK3108 and L18 cells were extremely significantly reduced than that in PK15 cells(P<0.01).Western blotting results indicated that the protein expression of MSTN and MMP2/7/9 in PK3108 and L18 cells were significantly reduced than that in PK15 cells.The results revealed that the expression of MMP-2/7/9 were significantly decreased by MSTN deletion in PK15 cells,and the expression trend of MMP-2/7/9 protein were consistent with the trend of MSTN protein expression.
作者
朱喆
张剑英
华再东
任红艳
肖红卫
张立苹
郑新民
毕延震
卢晟盛
ZHU Zhe;ZHANG Jianying;HUA Zaidong;REN Hongyan;XIAO Hongwei;ZHANG Liping;ZHENG Xinmin;BI Yanzhen;LU Shengsheng(Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding,Institute of Animal Science and Veterinary Medicine,Hubei Academy of Agricultural Sciences,Wuhan 430064,China;Faculty of Animal Science and Technology,Guangxi University,Nanning 530004,China)
出处
《中国畜牧兽医》
CAS
北大核心
2020年第2期345-351,共7页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31772577)
湖北省杰出青年(2018CFA043)
湖北省自然科学基金创新群体(2018CFA014)
湖北省农业科学院领军人才培养计划(L2018015)
湖北省农业科技创新中心(2019-620-004-001)
