脂多糖对牛乳腺上皮细胞自噬和凋亡的影响  

Effects of Lipopolysaccharide on Autophagy and Apoptosis of Bovine Mammary Epithelial Cells

作　　者：张兴 高英魁 郝中华 刘静静 张华强 归荣 张珂菲 周广薇 邓立新[1] 王学兵[1,2] 童超 ZHANG Xing;GAO Ying-kui;HAO Zhong-hua;LIU Jing-jing;ZHANG Hua-qiang;GUI Rong;ZHANG Ke-fei;ZHOU Guang-wei;DENG Li-xin;WANG Xue-bing;TONG Chao(School of Veterinary Medicine,Henan Agricultural University,Zhengzhou,Henan,450002,China;International Joint Research Center for Animal Immunology,Henan Agricultural University,Zhengzhou,Henan,450046,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450002 [2]河南农业大学动物免疫学国际联合研究中心,河南郑州450046

出　　处：《动物医学进展》2023年第9期69-75,共7页Progress In Veterinary Medicine

基　　金：河南省科技攻关项目(212102110095,212102110352);国家肉牛牦牛产业技术计划资助系统项目(CARS-37)。

摘　　要：为探究脂多糖(lipopolysaccharide,LPS)对牛乳腺上皮细胞(MAC-T)自噬和凋亡的影响,以MAC-T细胞为试验对象,用CCK-8和乳酸脱氢酶(LDH)法检测不同浓度LPS(100、150、200、250μg/mL),检测细胞的存活率和损伤,从上述分组中挑选浓度为0(对照)、50、100、200μg/mL的LPS建立试验组,Hoechst33342检测细胞核形态,qPCR法检测MAC-T细胞中自噬和凋亡基因(LC3B、p62、BAX、BCL-2、ATG5、ATG7、Beclin-1、caspase-3)mRNA的表达量,Western blot检测MAC-T细胞中自噬和凋亡(LC3B、p62、BAX、BCL-2、ATG5、ATG7、Beclin-1、caspase-3)相关蛋白表达。结果显示,MAC-T细胞的活力会随着LPS的浓度和时间的推移下降,细胞上清中的LDH随着LPS浓度上升而增加,Hoechst33342染色后可以发现细胞核形态出现异型性,染色质碎裂、固缩。qPCR和Western blot检测结果显示LPS在50~100μg/mL浓度下可引起MAC-T细胞自噬,200μg/mL可抑制此过程,同时细胞凋亡随着LPS浓度上升而加剧。说明LPS可引起MAC-T细胞自噬同时诱导细胞凋亡,为大肠埃希氏菌诱导牛乳腺炎发生机制的研究提供参考。To explore the effect of lipopolysaccharide(LPS)on autophagy and apoptosis of bovine mammary epithelial cells(MAC-T),CCK-8 and lactate dehydrogenase(LDH)assays were used to detect different concentrations of LPS(100,150,200,250μg/mL)in MAC-T cells.LPS with concentrations of 0(control),50,100,and 200μg/mL were selected from the above groups to establish experimental groups.Hoechst33342 was used to detect the nuclear morphology.qPCR was used to detect the mRNA expressions of autophagy and apoptosis genes(LC3B,P62,BAX,Bcl-2,ATG5,ATG7,Beclin-1,and caspase-3)in MAC-T cells.The expressions of autophagy and apoptosis(LC3B,P62,BAX,Bcl-2,ATG5,ATG7,Beclin-1,caspase-3)related proteins in MAC-T cells were detected by Western blot.The results showed that the viability of MAC-T cells decreased with the increase in LPS concentration and time,and the LDH in the cell supernatant increased with the increase in LPS concentration.Hoechst33342 staining showed that nuclear morphology showed atypia,chromatin fragmentation,and psychosis.qPCR and Western blot results showed that LPS at the concentration of 50-100μg/mL could induce autophagy in MAC-T cells,and 200μg/mL could inhibit this process.At the same time,apoptosis was increased with the increase of LPS concentration.In conclusion,LPS can induce autophagy and apoptosis of MAC-T cells,which may provide a reference for the mechanism of Escherichia coli-induced bovine mastitis.

关 键 词：牛乳腺上皮细胞 脂多糖 自噬 凋亡 

分 类 号：S857.26[农业科学—临床兽医学]