摘要
试验旨在研究牛支原体P48蛋白对胎牛肺(embryonic bovine lung,EBL)细胞增殖和凋亡的影响。收集不同时间段、不同浓度P48蛋白与EBL细胞共孵育的样品,通过MTT法检测细胞增殖率;DAPI染核法观察EBL细胞核形态变化;流式细胞技术检测该蛋白诱导EBL细胞的凋亡率;实时荧光定量PCR检测凋亡标志物的mRNA相对表达变化;Western blotting方法检测Bax和Beclin-1蛋白表达水平。结果显示:当作用时间为72 h,P48蛋白浓度在10μg/mL时,对EBL细胞增殖有极显著的抑制作用(P<0.01),在0.1和0.5μg/mL时对EBL细胞的增殖的抑制作用不显著(P>0.05);经蛋白诱导12 h细胞核形态未有明显变化,24 h细胞核形态发生皱缩和凝聚,48和72 h细胞核发生碎裂;凋亡标志基因mRNA表达在2和12 h没有明显提高,在24、48、72 h有显著提高,与作用时间呈正相关,同时凋亡相关蛋白Bax和Beclin-1的表达随之显著提高。流式细胞技术结果显示P48蛋白诱导EBL细胞凋亡率为48.44%。综上表明,牛支原体P48重组蛋白能够抑制EBL细胞的增殖,促进细胞凋亡,为进一步揭示牛支原体的致病机制提供参考依据。
The aim of this study was to investigate the effect of P48 protein on proliferation and apoptosis of embryonic bovine lung(EBL)cells.In this study,samples which co-incubated with P48 protein and EBL cells in different concentrations at different time points were collected,the proliferation rate of the cells was detected by MTT method,and the changes in the nuclear morphology of EBL cells were observed by DAPI staining method.Meanwhile,flow cytometry was used to detect the apoptosis rate of EBL cells induced by P48 protein.Real-time quantitative PCR was used to detect the changes in mRNA level of apoptotic marker genes,and Western blotting tested the Bax and Beclin-1 protein expressions.The results showed that under the condition of 72 h and 10μg/mL of protein concentration treatment,P48 extremely significantly inhibited EBL cells proliferation(P<0.01),while 0.1 and 0.5μg/mL protein concentration had no inhibitory effect(P>0.05).The nuclear morphology showed no significant change after protein induction for 12 h,but wrinkled and condensed at 24 h.The nucleus was fragmented,and a sprouted apoptotic body was appeared at 48 and 72 h.Apoptosis related genes expression showed no obvious increase at 2 and 12 h at mRNA level,but gradually increased at 24,48 and 72 h,and it showed a time-dependent manner.Accordingly,the expression of apoptosis marker proteins Bax and Beclin-1 significantly increased.Flow cytometry analysis showed that the apoptosis rate of EBL cells induced by P48 protein was 48.44%.In conclusion,P48 recombinant protein of Mycoplasmas bovis inhibited the proliferation of EBL cells and promoted their apoptosis,which provided reference for revealing the pathogenic mechanism of Mycoplasmas bovis.
作者
张生英
武小椿
邢小勇
刘佳
岳亚辉
张阳阳
贺健
温峰琴
包世俊
ZHANG Shengying;WU Xiaochun;XING Xiaoyong;LIU Jia;YUE Yahui;ZHANG Yangyang;HE Jian;WEN Fengqin;BAO Shijun(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)
出处
《中国畜牧兽医》
CAS
北大核心
2020年第10期3149-3157,共9页
China Animal Husbandry & Veterinary Medicine
基金
甘肃农业大学人才专项(GAU-KYQD-2018-04)。
