猪腹膜间皮细胞的分离培养及初步应用

Isolation and Culture of Swine Peritoneal Mesothelial Cells and Its Preliminary Application

本研究旨在建立猪原代腹膜间皮细胞(peritoneal mesothelial cell, PMC)的分离和体外培养方法,并将其初步应用于猪鼻支原体(Mycoplasma hyorhinis,Mhr)体外感染细胞模型。采用0.1%Ⅰ型胶原酶消化猪大网膜组织分离猪PMC细胞,通过形态学、免疫学方法鉴定细胞。使用Mhr感染PMC细胞,观察细胞形态变化,并利用乳酸脱氢酶(lactate dehydrogenase, LDH)法检测细胞活性损伤情况。结果显示,倒置相差显微镜观察显示,分离培养的细胞早期呈拉网状,5~8 d后生长可达融合状态,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微绒毛。结果证实分离培养的细胞为猪PMC细胞,纯度达95%以上。Mhr感染后,光镜下可观察到细胞发生明显皱缩,感染组细胞的LDH释放量较对照组细胞显著升高。本研究成功建立了猪原代PMC细胞的分离培养方法,并初步用于Mhr体外感染,为研究Mhr等引起浆膜炎的病原与宿主的互作机制提供了体外细胞模型。

The aim of this study was to establish a method for the isolation and in vitro culture of porcine primary peritoneal mesothelial cells(PMC)and preliminarily apply it to an in vitro infection model of Mycoplasma hyorhinis(Mhr).Porcine omentum was digested with 0.1%typeⅠcollagenase to isolate primary porcine PMC.The cells were identified by morphology and immunological assays.The PMC cells were infected with Mhr.Thereafter,the morphological changes were observed and the cell activity damage was detected using the lactate dehydrogenase(LDH)method.Inverted phase contrast microscope observation showed that the isolated cells were pulled reticular at the early stage,and could grow up to fusion state 5-8 days later,with uniform size and polygonal slab stone-like appearance.Immunofluorescence analysis showed that the cells were positive for vimentin and cytokeratin-18,but negative for factor VIII-related antigen and CD45.Microvilli on the cell surface were observed under scanning electron microscope.It was confirmed that the isolated and cultured cells were PMC,and the purity was more than 95%.After Mhr infection,significant shrinking of the cells could be observed under light microscopy,and LDH release was significantly higher in the infected group of cells compared with the control group of cells.This study successfully established the method to isolate and culture the primary porcine PMC cells,and was initially used for Mhr infection in vitro,which could provide an in vitro cell model for studying the interaction mechanism between pathogens polyserositis-causing such as Mhr and the host.