Isolation And Purification Of PMVECs From Plateau Pika And Preliminary Study On Cx40 Signal Pathway

Ochotona cruzonia,a native species of the Qinghai-Tibet Plateau,has developed a unique adaptation strategy at phenotypes,physiological and biochemical levels,as well as cellular and molecular levels over a long period of evolution.Therefore,it has been regarded as one of the best model animals for the study of hypoxia adaptation.Blunted hypoxic pulmonary vasoconstriction(b HPV)is one of the typical characteristics of hypoxic adaptation in plateau pika,and its pathogenesis and regulatory mechanisms have long been of interest to researchers.Lung endothelial cells can regulate the contraction,relaxation,proliferation and apoptosis of vascular smooth muscle cells.Therefore,endothelial cells may play an important role in the b HPV of plateau pika,which is an important research point.Therefore,studies on the isolation,culture,purification and identification of pulmonary microvascular endothelial cells(PMVECs)of plateau pika can lay a foundation for subsequent studies on the molecular mechanism of b HPV in plateau pika.The main findings of this study are as follows:1.Taking plateau pika as the research object and rabbit and mouse as the reference,PMVECs were cultured by tissue block attachment method and enzyme digestion method,respectively.The results showed that the enzyme digestion method required short time and high cell purity for cell culture.2.Lung microvascular endothelial cells were purified by differential adhesion,immunomagnetic beads separation method and the combination of the two methods,respectively.The results showed that the purity of the cells obtained by the combination of the two methods was the highest.3.The pulmonary microvascular endothelial cells of plateau pika,rabbit and mouse were identified by cellular immunofluorescence staining,cellular immunochemical staining and other methods,and the identification results were all positive,which proved that the cells obtained from the cultured cells were PMVECs.4.The extracted genomic DNA of plateau pika was used to verify three pairs of primers CD31,v WF and VEGF,and the results showed that CD31 had the best effect.Then the extracted RNA of plateau pika was used to measure the expression of CD31 in cells by q RT-PCR,and the results showed that: the expression of CD31 in positive cells was significantly higher than that in negative cells,so the obtained cells were identified as PMVECs.5.The OD value was measured by enzyme plate analyzer,and the growth curve of PMVECs of plateau pika,mouse and rabbits was plotted.The results showed that the growth rate of plateau pika PMVECs was fast.6.Total RNA was extracted from the lung tissues of the hypoxic-treated and non-hypoxic-treated plateau pika,and the full-length transcriptome data of the lung tissues were measured.The expression level of Cx40 was measured by q RT-PCR,and the experimental results showed that the expression level of Cx40 in the hypoxic group was lower than that in the untreated group,and the expression was down regulated.Therefore,it was speculated that plateau pika inhibited vasoconstriction response by reducing the expression level of Cx40,resulting in the passivation of pulmonary vasoconstriction response,and then adapt to the hypoxic environment.