关键词: 腹膜间皮细胞, 原代培养, 猪, 猪鼻支原体

Abstract: 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% type Ⅰ collagenase 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.

Key words: peritoneal mesothelial cell, primary culture, swine, Mycoplasma hyorhinis