猪肺炎支原体强、弱毒株诱导猪气管上皮细胞线粒体通路凋亡差异的研究

发布时间：2018-08-19 14:14

【摘要】：猪肺炎支原体(Mycoplasma hyopneumoniae, Mhp)是引起猪支原体肺炎的一种病原,它以慢性、接触性、高发病率为特点在猪群存在,与其他病原共同威胁着猪群的健康。猪肺炎支原体致病机理研究不清是当前影响该病有效控制的核心因素。目的：本试验利用Mhp 168强、弱毒株具有来源于同一亲本,而毒力不同的特点,感染靶细胞——猪气管上皮细胞上,探讨其致病机理。方法与结果：本试验以5×103CCU/cell浓度的猪肺炎支原体强毒株(Mhp 168)、弱毒株(Mhp 168L)分别感染猪气管上皮细胞(Porcine tracheal epithelial cells, PTEC),分别在感染后12h,18h,24h,30h用AO/EB双染法检测细胞凋亡率,发现在感染24h时细胞凋亡率达到最大值,强毒感染组显著高于弱毒感染组(P0.01)；TUNEL(TdT-mediated dUTP Nick-End Labeling)检测结果显示,Mhp 168强毒感染PTEC后出现的阳性细胞高于弱毒组。同时,感染24h,采用Annexin V-FITC和PI双染,通过流式细胞仪分析,强毒感染组和弱毒感染组细胞凋亡率分别为38.69%和61.26%。以上结果表明Mhp 168和168L均可引起细胞凋亡,且168株引起细胞凋亡作用强于168L株,以5×103CCU/cell浓度感染24h最为显著。Mhp强、弱毒株感染PTEC细胞后,细胞内caspase-8、caspase-12检测结果显示,与对照相比,Mhp强、弱毒株性均不引起caspase-8、12升高。用免疫荧光法检测发现,细胞色素c释放至胞浆增加,Mhp 168感染组荧光强度高于Mhp 168L感染组；JC-1染色法检测线粒体膜电位发现,Mhp 168感染组绿色荧光强度高于Mhp 168L感染组。用N-乙酰半胱氨酸(NAC)预处理细胞后,出现强、弱毒感染组细胞的细胞色素c产生荧光强度降低,线粒体膜电位损失减少等关键的凋亡特征。结论：以上结果提示,Mhp强毒株感染PTEC细胞后,在线粒体途径的凋亡过程中存在差异。
[Abstract]:Mycoplasma suis (Mycoplasma hyopneumoniae, Mhp) is a kind of pathogen that causes mycoplasma pneumoniae pneumonia. It is characterized by chronic, contact and high incidence in pigs, and it threatens the health of pigs together with other pathogens. Unclear studies on the pathogenic mechanism of mycoplasma pneumoniae are the core factors affecting the effective control of the disease. Objective: to investigate the pathogenicity of Mhp 168-strong attenuated strain from the same parent and different virulence on the target cell-pig trachea epithelium. Methods and results: in this experiment, 5 脳 103CCU/cell concentration of mycoplasma pneumoniae virulent strain (Mhp 168) and attenuated strain (Mhp 168L) were used to detect the apoptosis rate of porcine trachea epithelial cells (Porcine tracheal epithelial cells, PTEC),) at 12h, 18h, 24h, 24h and 30h, respectively, using AO/EB double staining method. The results of Tunel (TdT-mediated dUTP Nick-End Labeling) analysis showed that the positive cells in PTEC infected with MHP 168 were higher than those in the attenuated group. At the same time, the rate of apoptosis was 38.69% and 61.26%, respectively, after 24 hours of infection with Annexin V-FITC and Pi. The results of flow cytometry showed that the apoptosis rate was 38.69% in the high toxic infection group and 61.26% in the attenuated infection group. The results showed that both Mhp 168 and 168L could induce apoptosis, and the apoptotic effect of 168L strain was stronger than that of 168L strain, the concentration of 5 脳 103CCU/cell was the most significant. After infecting PTEC cells with attenuated strain, caspase-8 caspase-12 was detected. Compared with the control, Mhp was stronger, and no increase of caspase-8 was induced by the attenuated strain. The fluorescence intensity of Mhp168 infected group was higher than that of Mhp 168L infected group by immunofluorescence assay. The green fluorescence intensity of Mhp168 infected group was higher than that of Mhp 168L infection group. After pretreatment with N-acetylcysteine (NAC), the cytochrome c and mitochondrial membrane potential loss in the cells infected with Nacetylcysteine were decreased. Conclusion: these results suggest that there is a difference in apoptosis of mitochondrial pathway in PTEC cells infected with Mhp virulent strain.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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