Establishment Of Immortalized Yak Renal Tubular Epithelial Cell Line Mediated By HPV-E6/E7 Gene

This research established a reliable yak tubular epithelial cell line for the increasing demand of yak-study in vitro cells.It is focused on original culture tubular epithelial cell and its characteristics;on transfection of E6/E7 gene;the study on preimmortalization,the results as following.1,The practical application level,the relative content of keratin was mensuratd to make the clear conclusion on the 3 method,by tissue explants adherent method,precipitation method and digestion method,for the original culture tubular epithelial cell method.The results as following: precipitation method and digestion method can get the considersible number of epithelioid cells,the tissue explants adherent method can get a small number of epithelioid cells and a amount of fibroblast cells;the relative content of keratin form 3 methods is with 1.0006% by digestion method,0.0021% by the tissue adherent and 0.0034% precipitation method;Immuno histochemistry of CK-18 protein on original cultured epithelioid cells was positive;western blot of E-cadherin was positive;Those menifestd the original cultured cell was the tubular epithelioids cells.2,The optimal retio with plasimd and liposome is above 1:3 on expression of HPV-16 E6/E7 gene by qPCR with steady signicicant difference between groups.The optimal ratio of plasimd and liposome is 1:3 for target gene which base on the different expression of the HPV-16 E6 and E7 gene.The expression of E6/E7 gene in two cells was screened with micro needle.3,The A cell with faster growth was seleted for research.The DNA gel electrophoresis for E6 and E7 gene was positive;the content of keratin between original cultured cells and immortalization cells was no difference(P>0.05);the gray value of each bar for E-cadherin has significant difference;the 42 gernerations of immortalization cells was significantly more than 9 generations of original cultured cell..The tubular epithelial cells are not tumorigenic for the S180 ascites tumor and solid tumor for bio-safety assessment.The cell line can be used in following and clinical experiment.The yak retular epithelial cell was successfully established by HPV-16 E6 and E7 gene with 42 generations than original cultured,with epithelial characteristics,with the properties of renal tubular epithelium and bio-safety.