Characteristics And Functional Evaluation Of An Immortalized Human Hepatoyte Cell Line

At present,the morbidity of acute liver failure(ALF) caused by various reasons step up year by year.Because of its urgent onset and quick progression,the mortality is very high.So the treatment for ALF is always an urgent problem in medicine.Bioartificial liver(BAL) technique based on cultural hepatocytes procured considerable advancement recently.It is considered as an effective method to treat ALF or to bridge the patient to orthotropic liver transplantation(OLT).The choice of ideal hepatocyte for BAL systems is critical in this approach.Since the porcine hepatocyte and human hepatoma cells are different from the normal human hepatocyte in functional characteristics as well as they could cause the immunologic rejection, xenogenic virus infection,and tumorgenesis when they applied in BAL.Therefore, establishment of BAL systems based on humanized hepatocyte cell line is the direction for the research and developed of BAL systems.Objective To establish an immortalized human hepatocyte cell line(IHCL);to analyze its biological and functional characteristics;and to evaluate the possibility of its application in bioartificial liver as cell material.Materials and Methods The primary human hepatocytes were respectively transfected with simian virus 40 large T antigen(SV40 Tag) and human telomerase reverse transcriptase(hTERT),and then the immortalized hepatocyte cell line(IHCL) was established from the proliferated cell clone with alpha 1 proteinase inhibitor(API) positive staining.The mRNA expression of API,CK18,UGT1,GS,GST,HBCF and ALB in IHCL were detected by RT-PCR and protein expression ofAPI,CK18,UGT1, GS,GST were demonstrated by immunofluorescence staining and Western blot respectively.The IHCL were cultured with microcarriers and the cell density was analyzed by MTT.The IHCL were co-cultured with the serum or plasma from patients with acute liver failure for different time,and then the concentration of TB, DB,LDH and BUN in serum or plasma was measured by automatic biochemistry analyzer. Results The established IHCL has similar appearance compared with the primary human hepatocytes and has been passaged more than 80 times,mRNA expressions of SV40 Tag,hTERT,API,CK18,UGT1,GS,GST and HBCF were shown by RT-PCR and those related proteins were demonstrated by immunofluorescence and Western blot in the IHCL.IHCL growth was well in microcarriers and reached to high density. Functional analysis indicated that TB and DB levels in serum were decreased and BUN was increased gradually when IHCL cultural with serum derived from patients with severe liver failure,the differences before and after co-culture have statistical significance.Conclusion The immortalized human hepatocyte cell line maintains the major biological and functional characteristics of primary human hepatocytes and was suitable for application in bioartificial liver as cell material.