布鲁氏菌强毒株16M和疫苗株M5-90诱导巨噬细胞凋亡线粒体途径相关因子的表达比较  

Comparison of expression of factors related to mitochondrial pathways of apoptosis in macrophages induced with virulent Brucella strain 16M and vaccine strain M5-90

作　　者：陈鹏博[1] 任晓莉 张亚丽[3] 陈创夫[1] 王远志[3] 

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]莎车县农业局,新疆喀什844700 [3]石河子大学医学院病原生物学与免疫学教研室,新疆石河子832002

出　　处：《中国病原生物学杂志》2014年第3期193-197,共5页Journal of Pathogen Biology

基　　金：国家科技支撑计划项目(No.2013BAI05B05);国际科技合作项目(No.2013DFA32380);国家自然科学基金项目(No.31260596)

摘　　要：目的比较布鲁氏菌强毒株16M和疫苗株M5-90侵染小鼠巨噬细胞RAW264.7后其凋亡相关因子的转录和表达。方法建立16M、M5-90侵染RAW264.7模型,采用CFU计数法比较16M、M5-90侵染RAW264.7 4、8、12、24h后的胞内生存情况;采用qRT-PCR检测AIF、Bcl-2、Bax、Apaf-1、Bcl-xl基因转录水平的变化;采用ELISA检测TNF-α和Cyt C在细胞内的表达;采用激光共聚焦检测Cyt C在细胞内共定位表达情况。结果布鲁氏菌16M在侵染后4h^24h胞内CFU呈增多趋势,而M5-90CFU先增多后减少。qRT-PCR显示AIF基因的转录水平在侵染后4h^24h内呈上升趋势,且M5-90侵染组与16M侵染组比较差异无统计学意义(P>0.05);由M5-90侵染诱导的Apaf-1基因转录水平与16M侵染组比较有统计学意义(P<0.05);Bcl-xl的转录量随着侵染时间增长而不断增加,且16M诱导组与M5-90组比较差异无统计学意义(P>0.05);M5-90侵染组Bax和Bcl-2水平与16M组比较差异均有统计学意义(P均<0.05);M5-90侵染组Bax/Bcl-2比值与16M侵染组比较差异无统计学意义(P>0.05)。ELISA分析显示TNF-α、Cyt C分泌量随着时间增长而增加,且M5-90诱导组与16M组比较差异无统计学意义(P均>0.05)。激光共聚焦显示Cyt C定位在RAW264.7内,属于胞浆表达,且M5-90诱导的表达量与16M组比较差异无统计学意义(P>0.05),并随感染时间的延长不断增加。结论布鲁氏菌疫苗株M5-90侵染RAW264.7 4h^24h内,凋亡相关因子Cyt C、AIF、Bax/Bcl-2、Apaf-1的表达量高于强毒株16M侵染组,而Bcl-xl则相反,表明在此侵染阶段M5-90具有更强的促细胞凋亡作用。Objective To compare the transcription and expression of factors related to apoptosis in RAW264.7 murine macrophages after infection with wild Brucella strain 16M and vaccine strain M5-90. Methods RAW264.7 were infected with 16M and M5-90, and CFU counting was used to count the number of intracellular bacteria after 4 b, 8 h, 12 h, and 24 h. qRT-PCR was used to quantify the transcription of AIF, Bcl-2, Bax, Apaf-1, and Bcl-xl 4 h, 8 h, 12 h, and 24 h after RAW264.7 was infected with 16M and M5-90. ELISA was used to detect the expression of TNF-a and Cyt C, and laser confocal microscopy was used to clarify the intracellular location expression of Cyt C at the same time points described above. Results CFU results indicated that the survival of 16M in RAW264.7 tended to increase 4-24 h after infection and that the survival of M5-90 increased slightly at first and then decreased slowly, qRT-PCR results indicated that the level of AIF transcription tended to increase 4-24 h after RAW264.7 was infected with Brucella and that the level of AIF transcription was higher with M5-90 than with 16M （P=0. 0851）. The level of Apaf-1 transcription induced by M5-90 was significantly higher than that induced by 16M （P：0. 0160）, and so were the levels of Bax and Bcl-2 transcription （p values of 0. 0386 and 0. 0444, respectively）. The level of Bax/Bcl-2 transcription was higher in maerophages infected with M5-90 than in those infected with 16M, but the difference is not significant （P=0. 2288）. In addition, the level of Bcl-xl transcription increased over time, and 16M induced a higher level of transcription than did M5-90 （P=0. 0848）. ELISA results indicated that the amount of secreted TNF-a and Cyt C increased over time, and M5-90 induced a higher level of expression than did wild stains （p values of 0. 1373 and 0. 0981, respectively）. Cyt C was located in the cytoplasm of RAW264.7 according to eonfocal laser microscopy, and the strain M5-90 induced significantly greater expression of Cyt C than did 16M.

关 键 词：布鲁氏菌 巨噬细胞 凋亡相关因子 ELISA qRT—PCR 激光共聚焦 

分 类 号：R378.5[医药卫生—病原生物学]