【作者】 黄媛媛；

【导师】 熊祺琰； 王先炜；

【作者基本信息】 南京农业大学 ， 兽医硕士（专业学位）， 2023， 硕士

【摘要】 猪鼻支原体(Mycoplasma hyorhinis,Mhr)的临床感染率极高,可诱导多发性浆膜炎、关节炎为主的多种炎症,严重危害养猪业的健康发展。目前对Mhr的研究较少,其致病机制不明。合适的细胞模型的缺乏是深入开展Mhr致病机制研究的重要制约因素,也阻碍了相关防控技术产品的研究与评价。另一方面,Mhr感染猪中仅少部分出现临床疾病,提示Mhr菌株毒力可能存在较大差异。感染菌株毒力的判定对猪场Mhr的防控与治疗具有极其重要的意义,但目前Mhr的毒力判定仅能通过猪体内攻毒进行,耗时耗力且成本高昂。间皮细胞是浆膜的主要细胞成分,是Mhr感染致病的重要靶细胞。本研究旨在分离猪原代腹膜间皮细胞(peritoneal mesothelial cell,PMC),建立Mhr体外感染细胞模型,并利用该模型比较不同毒力Mhr菌株体外感染结果的差异,尝试建立Mhr菌株毒力的体外快速比较评价方法。具体研究内容和主要结果如下:第一,猪原代PMC细胞的分离、培养和鉴定。采集猪腹腔大网膜组织,通过Ⅰ型胶原酶或胰蛋白酶-EDTA消化分离猪原代PMC细胞,通过扫描电镜、表面特征性抗原免疫学检测方法鉴定细胞。结果显示,采用0.1%Ⅰ型胶原酶能够快速、高效地分离获得猪原代PMC细胞。分离的原代细胞在培养早期呈拉网状,5-8天后可达融合状态,细胞大小均一,呈多边形铺路石状。间接免疫荧光试验结果显示细胞波形蛋白、角蛋白18抗原呈阳性,第VIII因子相关抗原、白细胞CD45抗原呈阴性。扫描电镜下可见细胞表面有丰富的微绒毛。鉴定结果证实所分离获得的细胞为PMC细胞,纯度可达95%以上。第二,Mhr体外感染PMC细胞研究。将Mhr强毒株感染PMC细胞,检测Mhr黏附情况,观察细胞形态学变化,利用CCK-8法和乳酸脱氢酶法检测细胞活力损伤情况,通过q PCR检测感染后PMC细胞胞间连接分子、炎症因子、凝血-纤溶平衡相关调节因子等基因的mRNA水平变化。结果显示,感染后Mhr可大量黏附于PMC细胞上,细胞明显皱缩、活力明显受损。感染12 h后细胞E-钙黏蛋白(E-cadherin)、紧密连接蛋白-1(zonula occludens-1,ZO-1)基因转录水平显著下降,炎症因子IL-1β、TNF-α、IL-6、IL-8基因转录水平显著上升,组织因子(tissue factor,TF)和纤溶酶原激活物抑制剂-1(plasminogen activator inhibitor-1,PAI-1)基因转录水平显著上升,而组织型纤溶酶原激活物(tissue plasminogen activator,tPA)基因转录水平显著下降。研究结果提示Mhr感染PMC细胞后可诱导细胞损伤,上调炎症因子水平,并促进PMC细胞的凝血-纤溶平衡调节向纤维蛋白积累方向倾斜。第三,不同毒力Mhr菌株体外感染PMC细胞的比较评价。将Mhr强弱毒株感染PMC细胞,同上评价细胞损伤情况、炎症因子和凝血-纤溶平衡相关调节因子等基因的mRNA水平变化。结果发现强毒株感染组细胞形态学改变更为严重,细胞活力显著低于弱毒株感染组,细胞凋亡率显著高于弱毒株感染组。感染后PMC细胞IL-1β、IL-8、IL-6和TNF-α基因的转录水平均显著升高,但强弱毒株感染组间差异无明显规律性,TF、PAI-1基因转录水平显著上升,tPA基因转录水平显著下调,但强弱毒株感染组间无显著差异。进一步尝试将Mhr强弱毒株感染猪肺泡巨噬细胞,发现四种炎症因子基因转录水平均上调,且强毒株感染组显著高于弱毒株感染组,炎症因子TNF-α体外共孵育可显著提高PMC细胞的TF、PAI-1基因转录水平,降低tPA基因转录水平,提示Mhr强弱毒株可能通过感染巨噬细胞释放的炎症因子对PMC细胞凝血-纤溶平衡调节产生不同的间接影响。本研究成功建立了猪原代PMC细胞分离培养方法,并以此为模型开展了Mhr体外感染研究,细胞损伤、炎症、凝血-纤溶平衡调节相关指标的检测结果与临床多发性浆膜炎的特征性变化一致,为Mhr提供了良好的细胞感染模型。此外,不同毒力Mhr菌株感染PMC细胞的比较研究结果提示感染后PMC细胞活力损伤和细胞凋亡检测可用于Mhr菌株毒力的初步比较评价。更多还原

【Abstract】 Mycoplasma hyorhinis(Mhr)has a very high clinical infection rate.It can cause a variety of inflammatory conditions,typically polyserositis and arthritis,which pose a serious threat to the pig industry’s health.Currently,the studies on Mhr are limited,and the pathogenic mechanism of which is unclear.The lack of appropriate cellular models is a significant impediment to the deep research on the pathogenesis of Mhr,as well as to the development and evaluation of related prevention and control technology products.On ther other hand,only a small proportion of Mhr-infected pigs developed clinical disease,implying that the virulence of Mhr strains varies greatly.The determination of the virulence of infected strains is critical for the prevention,control,and treatment of Mhr in pig farms.However,it can only be evaluated by in vivo assault test on pigs,which is time consuming and costly.As the major cellular component of the serosa,mesothelial cells are the crucial target cells of Mhr.The aim of the current study is to isolate the porcine primary peritoneal mesothelial cells(PMC)and establish an in vitro infection cell model of Mhr.Furtherly,this model would be used to compare the results when infected with Mhr strains of different virulence,in order to establish a rapid in vitro evaluation method for the virulence of Mhr strains.The specific research contents and main results are as follows:First,the isolation,culture,and identification of porcine primary PMC cells.Porcine omentum tissue was collected and the primary PMC cells were isolated by type I collagenase or trypsin-EDTA digestion.The isolated cells were identified by scanning electron microscopy and immunological detection of surface characteristic antigens.The results showed that porcine primary PMC cells could be isolated rapidly and efficiently using 0.1%type I collagenase.The isolated primary cells show a network like morphology in the early stage of cultivation,and can reach a fusion state after 5-8 days.The cells are uniform in size and appear as polygonal slab stone-like appearance.The results of indirect immunofluorescence test showed that cells were positive for vimentin and cytokeratin 18 antigen,and negative for factor VIII-related antigen and CD45 antigen.Abundant microvilli on the cell surface were observed under scanning electron microscopy.The results confirmed that the cells obtained were PMC cells with a purity of over 95%.Second,research on in vitro infection of PMC cells by Mhr.PMC cells were infected by Mhr.After infection,the adhesion of Mhr was first detected,as well as the morphological changes of cells.The CCK-8 and lactate dehydrogenase methods were taken to detect the damage of cell viability,The mRNA transcriptional changes of genes involved in intercellular junctions,inflammation and coagulation-fibrinolysis balance regulation were determined by q PCR,respectively.The results demonstrated that Mhr could adhere to PMC cells in large quantities after infection,resulting in significant cell shrinkage and impaired vitality.The transcript levels of tight junction protein ZO-1 and E-cadherin genes decreased significantly after 12 hours of infection.The transcript levels of the inflammatory cytokines IL-1,TNF-,IL-6,and IL-8 genes increased significantly.Additionally,the transcript levels of tissue factor(TF)and plasminogen activator inhibitor 1(PAI-1)genes increased significantly,while those of tissue plasminogen activator(tPA)gene obviously decreased.According to the findings,Mhr infection of PMC cells caused cell damage,upregulates inflammatory cytokines,and encourages the regulation of coagulation-fibrinolysis balance of PMC cells toward the direction of fibrin accumulation.Third,a comparative evaluation on in vitro infection of PMC cells with Mhr strains of different virulent was performed.The virulent and nonvirulent strains of Mhr were used to infectPMC cells.The cell damage as well as the changes in the mRNA levels of genes such as,inflammatory cytokines and coagulation-fibrinolysis balance regulators were evaluated as above.The results showed that the morphological changes of cells were more severe in the group infected with the virulent strain,and the cell viability was significantly lower than that of the group infected with the nonvirulent strain.The cell apoptosis rate was significantly higher than that of the group infected with the nonvirulent strain.The transcript levels of IL-1β,IL-8,IL-6 and TNF-αgenes were significantly increased in PMC cells after infection,but there was no obvious regulating in the differences between the groups infected with virulent and nonvirulent strains.The transcription levels of TF and PAI-1 genes were significantly increased,while the transcription levels of tPA genes were significantly down regulated.However,there was no significant difference between groups infected with strong and weak strains.Furtherly,we tried to infect porcine alveolar macrophages with Mhr virulent and nonvirulent strains.It was found that all the four inflammatory cytokines upregulated in mRNA level,and the changes in the virulent strain-infected group was significantly higher than those of the nonvirulent strain-infected group.In vitro co-incubation with TNF-αsignificantly increased TF and PAI-1 gene transcripts and decreased tPA gene transcripts,suggesting that the virulent and nonvirulent Mhr strains may have different indirect effects on the regulation of coagulation-fibrinolysis balance in PMC cells through the release of inflammatory cytokines from infected macrophages.In the present study,we successfully established a method for isolating and culturing porcine primary PMC cells from pigs,and used it as a model to conduct in vitro infection research of Mhr.The results of cell injury,inflammation,and coagulation-fibrinolysis balance regulation-related indicators after Mhr infection were consistent with the characteristic changes of clinical polyserositis,which provided a good cell model for Mhr cell infection.In addition,the results of a comparative study of PMC cells infected with Mhr strains of different virulence suggested that the detection of PMC cell viability damage and apoptosis after infection can be used for preliminary evaluation of the virulence of Mhr strains.更多还原