Basic Research Of Human Immortalized Hepatocytes And Clinical Trials Of Bioartificial Liver With Human Hepatoma Cell Line

Background and Objective Liver failure and deficiency is often life threatening. Bioartificial liver support system is an attractive approach for an alternative to orthotopic liver transplantation. Although the primary human hepatocytes would be ideal for bioartificial livers, the shortage of hepatocytes and the difficulty to increase the doubling time of human hepatocytes in vitro limit the clinical application. Telomere is a ribonucleoprotein that plays a pivotal role in maintaining telomere length, chromosome stability and expansion of primary cells. Human telomerase reverse transcriptase (hTERT) is the determinative factor for telomerase activation. The objective of this experiment is to construct eukaryotic expression vectors containing hTERT cDNA, and study the isolation and culture methods of human primary hepatocytes. Which provide an effective and fast method for evaluating stable transfection of hTERT and a groundwork for establishing immortalized hepatocytes. Moreover, we studied the efficiency and safety parameters of the bioartificial liver with human hepatoma cell line(C3A) in treating the patients suffering from liver function failure and deficiency.Methods hTERT cDNA and GFP cDNA were cloned by PCR. Based on the technique of genetic recombination, hTERT and GFP were constructed into the eukaryotic expression vectors VR1012 and pEGFP-C1, formed the recombinant vectors of VR1012-GFP-hTERT and pEGFP-C1-hTERT. We checked up the positive clones with enzymes digestion, PCR and sequencing. Then transfected the correct recombinant vectors into the HepG2 cells, studied the location and expression of hTERT and GFP in the HepG2. We accepted the trypsin and collagenase digestive methods to isolate human primary hepatocytes from surgical exairesis hepatic tissue. We choosed 17 hospitalized patients who suffering from chronic severe liver function failure. According to the rules of random and control, 12 cases accepted the plasma diafiltration and bioartificial livertreatment, 5 cases in the control group only accepted the plasma diafiltration therapy, the two groups had the same internal medicine treatment. The vital signs, liver panel, blood coagulation, α-fetal protein were tested before, during and after the treatment. Using the CHISS software to perform the statistical analysis.Results The first, the consequences of enzymes digestion and PCR were match to the anticipation. The second, the fusion protein of hTERT and GFP distributed mainly in the nuclei of HepG2. the cell viability, adherence and growth ability with collagenase digestion were better than that with trypsin digestive method. The third, the 84^th survival rate of the treatment group and control were 83% vs 40% (P<0.01 ). The life span of the two groups were 84.58±1.56 vs 54.60±4.18 days (P>0.05) .The COX's proportional harzard model showed the bioartificial liver therapy was the major factor to reduce the relative ratio and extend the life span. The bilirubin decreased gradually during the bioartificial liver treatment. The concentration of AST and AFP were higher than the baseline and the control group during the therapy. There were no tumorigenic evidence at the end of follow-up.Conclusion The successful construction of recombinant eukaryotic expression plasmids of VR1012-GFP-hTERT and pEGFP-C1-hTERT, which provide an effective and fast method for evaluating stable transfection of hTERT and a basis for studying immortalized hepatocytes. Collagenase digestive method has the advantages of simplify, large quantity of cells. The performance of the bioartificial liver is good, which can improve the patients' physicial condition, reduce the bilirubin, increse the survival rate. The safety has been proved well.