Study Of The Effect Of RNA Interference With HIF-1α Gene On Pulmonary Artery Endothelial Cells In Broiler Chickens

The aim of this study was to investigate the effects of RNA interference HIF-1αgene on the growth and proliferation of Pulmonary artery endothelial cell(PAECs)in broilers.The PAECs of AA broilers aged 10 to 18 days were cultured in vitro to establish the cell hypoxia model and construct the RNA interference lentivirus vector recombinant plasmid.At different time(12h,24h,48h),different groups(normal group(NC group),no load group(UP group),HIF-1αgene knockdown effect was realized by transfection of PAECs in HIF-1αgroup(HRNAi group),hypoxia group(NC-A group),no-load hypoxia group(UP-A group)and HIF-1αhypoxia group(HRNAi group).Cell morphology observation,apoptosis,immunofluorescence identification,gene and protein expression levels of HIF-1αand VEGF,and RNA-seq detection were used to verify the RNA interference effect of HIF-1αgene.The test results are as follows:1.HIF-1αinterfering lentivirus LV416＿PDS019-PL-sh RNA-GFP-gas-HIF-1α-535(HIF-1α-535)was successfully constructed,and the proto-liquid titer of lentivirus was 3×10~8 TU/m L,which proved to have good interference effect.2.Under the condition of hypoxia,PAECs cell proliferation and apoptosis were promoted,and the gene expression levels of HIF-1αand VEGF were significantly increased;HIF-1αinhibited proliferation and promoted apoptosis,and the expression levels of HIF-1αand VEGF were significantly down-regulated.3.Compared with NC group,there were 1070 differentially expressed genes in HRNAi group,of which 235 were up-regulated and 835 were down-regulated;In NC-A group,131and 215 were up-regulated and down-regulated,respectively.There were 371 up-regulated genes and 642 down-regulated genes in the HRNAi group compared with the NC-A group.Compared with the HRNAi group,there were 107 up-regulated genes and 237 down-regulated genes.Enrichment analysis combined with GO database and KEGG pathway database showed that CAMs,ECM,MAPK and other pathways were significantly regulated,and these pathways were closely related to cell proliferation and apoptosis.Conclusion:Hypoxia can induce the proliferation and apoptosis of PAECs in broilers,while HIF-1αcan reduce the proliferation and apoptosis of PAECs in broilers,and induce the down-regulation of COL4A and up-regulation of ITGα8 and MKP5,and regulate the changes of CAMs,ECM and MAPK pathways.These results provided a basis for the mechanism of pulmonary artery remodeling,and also laid a foundation for the gene drug screening of ascites syndrome in broilers.