牛TXNIP基因的过表达载体构建及对乳腺上皮细胞增殖和凋亡的影响  

Construction of bovine TXNIP overexpression vector and its effect on proliferation and apoptosis of mammary epithelial cells

作　　者：吴晓婷 许贤吉 罗宗刚[1] 孙婷婷[1] 王玲[1] WU Xiaoting;XU Xianji;LUO Zonggang;SUN Tingting;WANG Ling(College of Animal Science and Technology,Southwest University,Rongchang,Chongqing 402460,China)

机构地区：[1]西南大学动物科学技术学院,重庆荣昌402460

出　　处：《中国兽医学报》2024年第12期2612-2618,共7页Chinese Journal of Veterinary Science

基　　金：重庆市自然基金面上资助项目(cstc2020jcyj-msxmX0436);贵州省农业农村厅黔财农资助项目(2024)。

摘　　要：为探究硫氧还蛋白相互作用蛋白(TXNIP)基因对牛乳腺上皮细胞增殖和凋亡的调控作用,本试验采用同源重组的方法构建了牛TXNIP基因过表达载体,通过酶切和测序进行鉴定,对得到的序列进行BLAST比对和系统进化分析;将TXNIP过表达质粒转染至牛乳腺上皮细胞,采用CCK-8和EdU检测细胞增殖、Annexin V-mCherry检测细胞凋亡水平,通过RT-qPCR检测细胞中凋亡相关基因mRNA的表达变化。结果表明,牛TXNIP基因CDS区1176bp,编码391个氨基酸,与羊的亲缘关系最近。与对照组相比,牛TXNIP基因过表达降低了乳腺上皮细胞活力,使细胞增殖数减少,促进细胞凋亡。过表达TXNIP基因导致牛乳腺上皮细胞中的凋亡相关基因Caspase-3、Caspase-9、Bax表达量显著上调(P<0.05),Caspase-8基因表达量无显著变化(P>0.05),抗凋亡基因Bcl-2表达极显著下调(P<0.01)、Bax/Bcl-2比值极显著升高(P<0.01)。结果表明,TXNIP基因可以抑制牛乳腺上皮细胞增殖,促进细胞凋亡,调控凋亡相关基因的表达,进而影响牛的乳腺发育。To investigate the regulatory effect of thioredoxin-interacting protein(TXNIP)on the proliferation and apoptosis of bovine mammary epithelial cells,a bovine TXNIP gene overexpression vector was constructed using the homologous recombination method.The overexpressed recombinant plasmid was verified through enzyme digestion and sequencing,and the obtained sequences were subjected to BLAST analysis and phylogenetic construction.The TXNIP overexpression plasmid was then transfected into bovine mammary epithelial cells.Cell proliferation was assessed using CCK-8and EdU assays,while apoptosis was evaluated using Annexin V-mCherry.Additionally,RT-qPCR was employed to measure changes in the mRNA expression of apoptosis-related genes in bovine mammary epithelial cells.The results showed that the CDS region of bovine TXNIP gene was 1176bp,encoded 391amino acids,which was closely related to sheep.Compared to the control group,overexpression of the TXNIP gene decreased the viability of mammary epithelial cells,reduced cell proliferation,and promoted apoptosis.Specifically,overexpression of the TXNIP gene led to a significant upregulation of apoptosis-related genes,including Caspase-3,Caspase-9,and Bax,in bovine mammary epithelial cells(P<0.05).In contrast,the expression of Caspase-8had no significant changes(P>0.05).Additionally,the expression of anti-apoptosis gene Bcl-2was significantly decreased(P<0.01),and the ratio of Bax/Bcl-2 was significantly increased(P<0.01).In conclusion,the TXNIP gene can inhibit the proliferation of bovine mammary epithelial cells,promote apoptosis,and regulate the expression of apoptosis-related genes,thereby influencing the development of the bovine mammary gland.

关 键 词：TXNIP基因 过表达重组质粒 乳腺上皮细胞 细胞增殖 细胞凋亡 牛 

分 类 号：S823[农业科学—畜牧学]