Immortalization Of Human Proximal Tubule Cells By Transfection Of Human Adenovirus 5 E1A Gene

Objective Immortalization of human proximal tubule cells was performed by transfection of human adenovirus E1A gene (ad5E1A). The function and growth characteristic of transfected cells were determined for turther research. Methods Ad5E1A gene was amplified by RT-PCR from 293 cell line. Then the production of RT-PCR was cut by the restriction enzyme KpnI and EhoI. The enzymed vector pShuttle CMV and insert were ligated with T4-ligase. Recombinated pShuttle CMV -ad5E1A was transfected the primary cultured human proximal tubule cells (PTC). The transfected cells were selected in the kanamycin positive LB plate. The positive clone was identifyied by RT-PCR and immunofluorescence for ad5E1A mRNA and protein expression. The growth property of transfected cells was determined by MTT and FACS method. The CKP, LDH, NAG and Na~+-K~+-ATPase level of transfected cells was detached for the functional dertermination as compared with HK2 cell line. Results Ad5E1A gene was amplified from 293 cell line by RT-PCR correctly. After the ad5E1A gene was cut by double restrictionenzyme Kpnl and Ehol and ligated with pShuttle CMV vecter, the recombination of the plasmid was proved that the gene sequence and opening reading frame (ORF) was correct by sequencing. Then the recombinated plasmid was transfected the PTC with lipofectamine. The positive clone could be selected out and cultured more than 30 passages stably in vitro. It had been documented that ad5ElA gene and protein expressed in the transfected cells by RT-PCR and immunofluorence. The result of MTT indicated that the growth speed of transfected cells was much faster than control group (PO.05). The result of FACS revealed that the proportion of cells in GO/Gl phase significantly decreased as compared with control group (P<0.05). The titration of PKC, NAG and NaT-IC-ATPase was higher than HK2 cell line (PO.05). Conclusion The immortalization of proximal tubule cells can be obtained by the transfection of ad5ElA gene. The transfected cells have characteristics of immortalization and some of functions are superior to HK2 cell line.