Expression Of LL-37 In Renal Tubular Epithelial Cells HK2 And Initial Establishment Of Related Gene Knockout Cell Lines

[Objective]Detecting the expression of antimicrobial peptide LL-3 7 in renal tubular epithelial cells HK2 under infection,to explore the relationship between LL-37 and renal infection.Establishing the HK2 cell lines of LL-37 gene knockout simultaneously.[Methods]In vitro,different concentrations of LPS(0,0.1,1,lOug/ml)were added to renal tubular epithelial cells HK2,among them,Oug/ml LPS group was named control group,and 0.1,1,10ug/ml LPS groups were set as experimental group.After 12h of the LPS stimulation,We detected the expression of LL-37 mRNA and protein in HK2 cells with RT-PCR and western blot respectively,and measured the corresponding concentration of LL-37 protein in the culture supernatant by ELISA.At the same time,knocking out the LL-37 gene of HK2 cell by CRISPR/Cas9.Then we selected stable strains with the method of flow cytometry,puromycin and monoclonal selection.At last,use western blot to preliminary identify the gene knockout cell lines.[Results]The result of RT-PCR showed that the expression of LL-37 mRNA in experimental group were higher than control group except Oug/ml LPS group;western blot indicated that the expression of LL-37 protein were increased compared to control group;and the ELISA results demonstrated that 1,10ug/ml LPS can stimulate the secretion LL-37 in HK2 cell;The results of three method used above all revealed that LPS promoted the LL-37 expression in a dose-dependent.At the same time,The transfected efficiency of LL37-KO and LL37-HDR in HK2 cell was higher,and compared to normal HK2 cell,the western blot results of gene knock out cell showed no LL-37 expression.[Conclusion]The LL-37 expression in renal tubular epithelial cells HK2 without infection was very low.However,suitable concentration of LPS can stimulate the LL-37 expression in HK2 cell,and exhibited a dose-dependent,indicated that LL-37 may play an important role in the infection of renal tubular epithelial cells and participate in the immune response of kidney infections.At the same time,the result of WB illustrated an satisfied LL-37 gene knockout effect of HK2 cells in our experiment.