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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 apoptosis-related factors after macrophages RAW264.7 infected with brucella virulent strain 16M and vaccine strain M5-90. Methods RAW264.7 cells infected with 16M and M5-90 cells were established. The intracellular survival of RAW264.7 cells infected with 16M and M5-90 cells was evaluated by CFU counting at 4, 8, 12 and 24 hours. The expression of AIF, The changes of transcription level of Bcl-2, Bax, Apaf-1 and Bcl-xl were detected by ELISA. The expression of TNF-α and Cyt C were detected by ELISA. The co-localization of Cyt C in cells was detected by laser scanning confocal microscope. Results The intracellular CFU of Brucella 16M increased at 4h ~ 24h after infection, but M5-90CFU first increased and then decreased. qRT-PCR showed that the transcriptional level of AIF increased from 4 h to 24 h after infection, and there was no significant difference between M5-90 infected group and 16M infected group (P> 0.05) The transcriptional level of Apaf-1 gene was significantly higher than that of 16M group (P <0.05). The transcript level of Bcl-xl increased with the increase of infection time, (P> 0.05). The levels of Bax and Bcl-2 in M5-90 infection group were significantly different from those in 16M group (all P <0.05) -2 ratio and 16M infection group showed no significant difference (P> 0.05). ELISA analysis showed that the secretion of TNF-α and Cyt C increased with time, and there was no significant difference between M5-90 induction group and 16M group (all P> 0.05). Confocal laser scanning showed that Cyt C was localized in RAW264.7 cells and expressed in cytoplasm. The expression of M5-90 in M5-90 cells was not significantly different from that in 16M group (P> 0.05), and increased with the prolongation of infection time. Conclusion The expression levels of Cyt C, AIF, Bax / Bcl-2 and Apaf-1 were significantly higher in Brucella vaccine strain M5-90 infected with RAW264.7 within 4 h to 24 h than that of virulent 16 M , Whereas Bcl-xl was the opposite, indicating that M5-90 has a stronger pro-apoptotic effect during this stage of infection.