【摘要】：豬肺炎支原體(Mycoplasma hyopneumoniae, Mhp)是引起豬支原體肺炎的一種病原,它以慢性、接觸性、高發(fā)病率為特點(diǎn)在豬群存在,與其他病原共同威脅著豬群的健康。豬肺炎支原體致病機(jī)理研究不清是當(dāng)前影響該病有效控制的核心因素。目的：本試驗(yàn)利用Mhp 168強(qiáng)、弱毒株具有來(lái)源于同一親本,而毒力不同的特點(diǎn),感染靶細(xì)胞——豬氣管上皮細(xì)胞上,探討其致病機(jī)理。方法與結(jié)果：本試驗(yàn)以5×103CCU/cell濃度的豬肺炎支原體強(qiáng)毒株(Mhp 168)、弱毒株(Mhp 168L)分別感染豬氣管上皮細(xì)胞(Porcine tracheal epithelial cells, PTEC),分別在感染后12h,18h,24h,30h用AO/EB雙染法檢測(cè)細(xì)胞凋亡率,發(fā)現(xiàn)在感染24h時(shí)細(xì)胞凋亡率達(dá)到最大值,強(qiáng)毒感染組顯著高于弱毒感染組(P0.01)；TUNEL(TdT-mediated dUTP Nick-End Labeling)檢測(cè)結(jié)果顯示,Mhp 168強(qiáng)毒感染PTEC后出現(xiàn)的陽(yáng)性細(xì)胞高于弱毒組。同時(shí),感染24h,采用Annexin V-FITC和PI雙染,通過流式細(xì)胞儀分析,強(qiáng)毒感染組和弱毒感染組細(xì)胞凋亡率分別為38.69%和61.26%。以上結(jié)果表明Mhp 168和168L均可引起細(xì)胞凋亡,且168株引起細(xì)胞凋亡作用強(qiáng)于168L株,以5×103CCU/cell濃度感染24h最為顯著。Mhp強(qiáng)、弱毒株感染PTEC細(xì)胞后,細(xì)胞內(nèi)caspase-8、caspase-12檢測(cè)結(jié)果顯示,與對(duì)照相比,Mhp強(qiáng)、弱毒株性均不引起caspase-8、12升高。用免疫熒光法檢測(cè)發(fā)現(xiàn),細(xì)胞色素c釋放至胞漿增加,Mhp 168感染組熒光強(qiáng)度高于Mhp 168L感染組；JC-1染色法檢測(cè)線粒體膜電位發(fā)現(xiàn),Mhp 168感染組綠色熒光強(qiáng)度高于Mhp 168L感染組。用N-乙酰半胱氨酸(NAC)預(yù)處理細(xì)胞后,出現(xiàn)強(qiáng)、弱毒感染組細(xì)胞的細(xì)胞色素c產(chǎn)生熒光強(qiáng)度降低,線粒體膜電位損失減少等關(guān)鍵的凋亡特征。結(jié)論：以上結(jié)果提示,Mhp強(qiáng)毒株感染PTEC細(xì)胞后,在線粒體途徑的凋亡過程中存在差異。
[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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28

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