牛支原体感染牛巨噬细胞对线粒体途径介导凋亡的影响

Effect of Mycoplasma bovis Infection on Mitochondrial Pathway Mediated Apoptosis in Bovine Macrophages

[目的]探究牛支原体石河子分离株感染牛巨噬细胞(BoMac)能否通过线粒体凋亡途径影响细胞增殖活力和凋亡。[方法]通过培养基培养及测定颜色变化单位(CCU)绘制牛支原体菌株生长曲线,确定其最高活力的培养时间;使用处于活力最高时的牛支原体以不同感染复数(MOI)和不同感染时间感染牛巨噬细胞,分别利用实时荧光定量PCR和Western blotting试验检测线粒体凋亡通路核心分子Bax和Bcl-2的表达情况,通过CCK8试验检测细胞增殖活力水平,流式细胞术检测细胞凋亡率。[结果]生长曲线分析结果显示,牛支原体石河子分离株12~54 h处在对数生长期,54~84 h处在平台期(滴度为109 CCU/mL),因此,选用培养54 h的牛支原体石河子分离株感染细胞。实时荧光定量PCR与Western blotting检测结果一致,即随感染时间和MOI的增加,促凋亡蛋白Bax基因及其蛋白表达量呈现上升趋势,抑凋亡蛋白Bcl-2基因及其蛋白表达量呈现下降趋势,其中牛支原体石河子分离株感染24 h时,Bax基因表达量极显著增加(P<0.01),Bcl-2基因表达量显著降低(P<0.05),Bcl-2/Bax显著下降(P<0.05);当MOI为1000时Bax蛋白表达量极显著增加(P<0.01),Bcl-2蛋白表达量显著降低(P<0.05),Bcl-2/Bax极显著下降(P<0.01)。CCK8试验结果显示,随着感染时间和MOI的增加,牛巨噬细胞活力极显著降低(P<0.01),增殖抑制率为35%~45%。流式细胞术检测结果显示,感染后,牛巨噬细胞凋亡率为25%~45%。[结论]随着感染时间和MOI的增加,牛支原体石河子分离株可通过线粒体凋亡途径标志因子Bax和Bcl-2的表达诱导牛巨噬细胞凋亡,为进一步解析牛支原体的致病机制提供理论基础。

[Objective]The aim of this experiment was to investigate whether infection of bovine macrophages(BoMac)with Mycoplasma bovis isolate from Shihezi could affect cell proliferation activity and apoptosis through the mitochondrial apoptosis pathway.[Method]The growth curve of Mycoplasma bovis isolated from Shihezi was drawn by medium culture and color change unit(CCU)to determine the culture time.Using Mycoplasma bovis,which was at its highest level of vitality,to infect BoMac with different infection multiples(MOI)and infection time,Real-time quantitative PCR and Western blotting tests were used to detect the expression of core molecules Bax and Bcl-2 in the mitochondrial apoptosis pathway.The cell proliferation activity level was detected by CCK8 assay,and the cell apoptosis rate was measured by flow cytometry.[Result]The analysis of the growth curve showed that Mycoplasma bovis isolated from Shihezi was in the logarithmic growth phase from 12 to 54 h,and in the plateau phase from 54 to 84 h(with a titer of 109 CCU/mL).Therefore,the Mycoplasma bovis isolated from Shihezi cultured for 54 h was selected to infect the cells.The results of Real-time quantitative PCR and Western blotting were consistent,indicating that with the increase of infection time and MOI,the pro-apoptotic protein Bax gene and its protein showed an upward trend,while the expression of the anti-apoptotic protein Bcl-2 gene and its protein showed a downward trend.Among them,after 24 h of infection with Mycoplasma bovis isolated from Shihezi,the expression of the Bax gene was extremely significantly increased(P<0.01),the expression of the Bcl-2 gene and Bcl-2/Bax were significantly decreased(P<0.05).When the MOI was 1000,Bax protein expression was extremely significantly increased(P<0.01),Bcl-2 protein expression was significantly decreased(P<0.05),and Bcl-2/Bax was extremely significantly decreased(P<0.01).The CCK8 test results showed that with the increase of infection time and MOI,the activity of BoMac was extremely significantly decreased(P<0.01),and the proliferation inhibition rate was 35%to 45%.The results of flow cytometry showed that the apoptosis rate of BoMac after infection was 25%to 45%.[Conclusion]The results of this experiment indicated that as the infection time and MOI increasing,the Mycoplasma bovis isolate from Shihezi could induce apoptosis of BoMac through the expression of mitochondrial apoptosis pathway markers Bax and Bcl-2,providing a theoretical basis for further elucidating the pathogenic mechanism of Mycoplasma bovis.