Experimental Study On Bioartificial Liver Support System Using Human Liver Cell Line

Liver failure caused by various reasons occurs frequently,but the curative effect of internal medicine combined therapies is not satisfactory.Bioartificial liver support system (BALSS) based on hepatocytes is an important measure to treat the patients,it can remove noxious substance and add to biotic activator.Ideal cell material for BALSS should include all the functions of normal human hepatocytes.At present,the cells used in BALSS mainly include porcine hepatocytes and C3A cell which derived from HepG2 cell line.It turns out that theⅢphase clinical trial of ELAD BAL with C3A cell has achieved certain effect on those patients who suffered from liver failure.But there were also many adverse effects happened during the treatment,such as haemorrhagia,hypersensitiveness,blood clotting and so on:In order to improve the function of liver cells,we used human augmenter of liver regeneration(hALR) in cell material of the bioatificial liver,and constructed extracorporeal BALSS to improve the therapeutic effect of it.1.Construction of recombinant plasmid of pcDNA 3.1(-) hALR Human ALR is a kind of thermostable cell factor which can stimulate hepatocytes to proliferate,inhibit NK cell,and protect the damaged hepatocytes.In this experiment,we extracted RNA from liver tissue,amplified the fragment of hALR,and constructed recombinant plasmid of pcDNA 3.1(-) hALR.After being appraised with sequencing,it was found to meet the requirements of application.2.Establishment and functional evaluation of hALR liver cell line Adopting measures of limiting dilution assay and translation of lidocaine,we chose an eugenic strain of cell which has metabolic capability of lidocaine from HepG2 cell line,transferred it with expression vector,pcDNA3.1(-)hALR,then screened it by G418.After cultured it for 50 generations,we evaluated its function of synthesis,metabolism and detoxification.The result shows that it grows well.The transformation efficiency of lidocaine is 65%,and diazapam is 79.33%.Human ALR were found in endochylema through indirect immunofluorescence. The cell infected by pcDNA 3.1(-) hALR can synthesize human albumin better than the uninfected one,and the level of AFP in culture fluid elevated from 1 to 118.0±56ng/ml.So the cell could be used in the BALSS.3.Bioreactor culture of hALR liver cell line On the basis of the liver cell line expressed hALR,we put 100 million of this kind of cells into the hollow fiber reactor,and construct the bioartificial liver culture system by peristaltic pumps,and cultured fluid with connection tube.After that the bioreactor were cultured for 72～96 hours,the culture fluid changed every day,and the cell,LDH,AFP were tested.When the cells were cultured for 72 hours or 96 hours,the cell number is increased by 7.60×10~8 and 9.02×10~8.The transformation efficiency of lidocaine is 88.4%at 24 hours.While the bioreactor was opened,the cells were found adhered on hollow fiber by inverted microscope and scanning electron microscope.No bacteria and mycoplasma were tested positive in fluid in the course of cell culture period.4.Construction of off-line BALSS and Study its effect on plasma discarded from patient who suffered from liver failure after plasma exchange(PE) We explored the measure of continuous blood purification on discard plasma after PE by bilirubin adsorbed and bioreactor;soon we used the plasma to PE for the patient again.The initial result showed that the cell had the functions of synthetic albumin and urea.The level of total bilirubin(TBil) is 332.22±22.54μmol/L before treatment,and it is 295.6±128.7μmol/L after bilirubin absorded(P＜0.01),but there are no difference in the item of albumin,urea nitrogen and blood sugar.Before the treatment of off-line BALSS,the level of TBil,AFP and albumin is 295.6±128.7μmol/L,21.2±17.5ng/ml and 26.44±2.186g/L.After the therapy,the level of them is 239.4±103.9μmol/L(P＜0.05),102±62.8ng/ml(P＜0.01),and 32.44±2.29g/L (P＜0.05) respectively.So the result shows that this BLASS can reduce the TBil and increase the synthesis of albumin,urea nitrogen and AFP.In a word,we have successfully constructed the recombinant plasmid of pcDNA 3.1(-) hALR,screened the liver cell line which can express hALR and transform lidocaton,and used the cell to construct bioreactor and off-line bioaritificial liver support system for liver failure patients,which can simplify the difficulty and complicacy of BAL,meanwhile reduce the need of normal plasma,raise the quality control and safety of the bioaritificial liver,and provide a new and reliable therapy to patients with hepatic failure.