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

发布时间：2018-01-10 18:20

本文关键词：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　出处：《中国病原生物学杂志》2014年03期 　论文类型：期刊论文

【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(P0.05);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(P0.05);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(P0.05);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(P0.05)。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(P0.05),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。
[Abstract]:Objective to compare the transcription and expression of apoptosis-related factors in murine macrophages (RAW264.7) infected by brucella virulent strain 16M and vaccine strain M5-90. Methods 16M was established. The RAW264.7 model infected with M5-90 was used to compare the intracellular survival of 16MN M5-90 infected with RAW264.7 4 (812m) by CFU counting method. QRT-PCR was used to detect the changes of Bcl-xl gene transcription level in AIF-Bcl-2BaxAX. The expression of TNF- 伪 and Cyt C in cells was detected by ELISA. The expression of Cyt C in cells was detected by confocal laser. Results the intracellular CFU of Brucella 16M increased at 4 h and 24 h after infection. However, M5-90 CFU increased first, then decreased. QRT-PCR showed that the transcription level of AIF gene increased within 24 hours after infection. There was no significant difference between M5-90 infection group and 16m infection group (P 0.05). The transcriptional level of Apaf-1 gene induced by M5-90 infection was significantly higher than that of 16M infection group (P 0.05). The transcription amount of Bcl-xl increased with the time of infection, and there was no significant difference between 16M induction group and M5-90 group. The levels of Bax and Bcl-2 in M5-90 infected group were significantly higher than those in 16M group (P < 0.05). There was no significant difference in Bax/Bcl-2 ratio between M5-90 infection group and 16M infection group. Elisa analysis showed that TNF- 伪. The secretion of Cyt C increased with time. There was no significant difference between M5-90 induction group and 16M group (P < 0.05). Laser confocal focus showed that Cyt C was located in RAW264.7 and belonged to cytoplasmic expression. The expression of M5-90 was not significantly different from that of 16M group (P 0.05). Conclusion Brucella vaccine strain M5-90 infects RAW264.7 for 4 h within 24 hours, and the apoptosis-related factor Cyt Con AIF is increased with the prolongation of infection time. The expression of Bax-Bcl-2 Apaf-1 was higher than that of 16M virulent strain, but Bcl-xl was the opposite, which indicated that M5-90 had a stronger apoptotic effect at this infection stage.
【作者单位】：石河子大学动物科技学院;莎车县农业局;石河子大学医学院病原生物学与免疫学教研室;
【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)
【分类号】：R378.5
【正文快照】： 布鲁氏菌病是由布鲁氏菌引起的一种慢性传染病[1],可导致人发生波浪热、心内膜炎、关节炎和骨髓炎等症状[2],在动物可引起流产和不育[3]。布鲁氏菌能够长期在宿主巨噬细胞内生存和增殖,并逃避巨噬细胞的杀伤作用[4]。细胞的凋亡是胞内病原菌生存的关键机制[5],布鲁氏菌抑制巨

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