Construction And Preliminary Application Of A Vector For Hepatocyte Immortalization Based On The PiggyBac Transposon

Background and Objection:Bioartificial liver(bioartificial liver,BAL)support system as an effective treatment of acute liver failure and an alternative to liver transplantation is being considered worldwide.Its support role is mainly derived from good biological metabolic function of liver cells,Therefore,the ideal liver cell source is the core component of the bioartificial liver system.Now commonly used in biological artificial liver cell material is porcine hepatocytes and tumor-derived cell lines.However,these two kinds of cells not only in function differ from human liver cells,but also carry the potential of zoonosis,anaphylactic reactions or tumorigenic potential safety hazard.Human-derived cell lines as materials of BAL is becoming a hot spot of current research.Immortalized hepatocytes applied to BAL with good prospects for well-differentiated hepatocellular functions,proliferative superiority and low cost of cell culture.Immortalized cells induced by human telomerase reverse transcriptase(hTERT)can maintain good function of differentiation and fewer chromosomal damage.Reversible immortalization strategies provide new unique means and ideas for cellular materials problems.The rationale is to introduce an immortalizing gene into primary cells,expanding the cells in culture,and finally,efficiently removing the immortalizing agent.The resulting cell population will be essentially identical to the starting primary cells,but greatly increased in number and without probability of tumorigenisis and viral infection.Design of carrier will be the research hotspot recently from the present situation and tendency of the development of the technology.Based on the previous researches,we will make a preliminary exploration of constructing a vector for virus-free hepatocyte reversible immortalization based on piggyBac carrier system,aiming at the safety of the human-derived cell lines.First,we transfected the hTERT(human telomerase reverse transcriptase)into adult hepatocytes(HL-7702)to establish a new immortalized adult hepatocyte line(hTERT-L02),then we conducted a pilot study and contrast on the morphous,proliferation,fuction between two types of the hepatocyte lines,So as to contribute to providing ideal biomaterials,this is crucial for the development of BAL treatment.Methods and materials1.Constructing the eukaryotic expressing vector of hTERT.We obtained attB1-Kozak-eGFP/F2A/neo-attB2,attB1-Kozak-hTERT-attB2 and attBl-hTERT/E2A/eGFP/F2A/neo-attB2 through PCR amplification,then we recovery the product through gel extraction,and then we through Gateway,a recombinant clone technology including BR reaction and LR reaction,and endonuclease digestion and conjunction to construct the vector PB-hTERT/E2A/eGFP/F2A/neo.The same principle to build empty plasmid vector PB-eGFP/F2A/neo and auxiliary plasmid vector pCAGG-PBase.2.Transfecting of the recombinant eukaryotic expressing vector and detecting the integration of hTERT.Gene expression analysis by real-time PCR for HL-7702 cells co-transfected by PB-hTERT/E2A/eGFP/F2A/neo and pCAGG-PBase were further conducted.Positive cells clones were selected and cultured singly,G418 maked it clear that the ectopic hTERT gene had been stably integrated in hTERT-L02.Then the expression of hTERT gene and protein were detected respectively by qRT-PCR and western-bolt.3.Contrasting on the morphous,proliferation,fuction between two types of the hepatocyte lines.Morphous of cells was observed by light microscope and HE staining.Proliferation of the cells was examined by CCK8 detection method;the expression of some functional genes was detected by qRT-PCR.The functionally related protein ALB and CYP2E1 were detected by immunofluorescence,meanwhile ALB and BUN from culture supernatants were evaluated by automatic biochemical analyzer.4.Transfecting the pCAGG-PBase again into hTERT-L02 and detecting the expression of hTERT gene by qRT-PCR.5.Statistical Analysis.Differences between two groups were analyzed using the Student’s t test;and differences between groups were analyzed using analysis of variance for repeated measures followed by a Newman-Keuls test.The level of significance was defined as p<0.05.All statistical tests were performed using SPSS software version 13.0.Results:1.Construction of the eukaryotic expressing vector PB-hTERT/E2A/eGFP/F2A/neo.Successful construction of immortalized hepatocellular vector PB-hTE RT/E2A/eGFP/F2A/neo,whose target sequence should be the same as GenBank’s,was confirmed by PCR,restriction enzvme digestion and gene sequencing.2.Gene expression in liver cells and associated protein can be detected by qRT-PCR and western-bolt.And the mRNA and protein levels of cell lines was significantly higher than before transfected cell lines.3.Morphous of cells was observed by light microscope and there was no change after we transfected hTERT into HL-7702,4.Proliferation experiments suggested that the capacity of proliferation was significantly enhanced after transfecting the hTERT into HL-7702.5.Expression of functional gene could be detected by qRT-PCR.Show that it did not affect the function of synthesis,metabolism and transformation after transfecting the hTERT into HL-7702.6.Cytochrome P450 and albumin was detected by using immunocytochemical method.The examination of hepatocytes supernatants showed that the general biochemical functions of cells were not infulunced by transfection.7.Successfully removal of purpose gene fragment after transfecting the pCAGG-PBase again into hTERT-L02.ConclusionIn this research we successfully constructed an eukaryotic expressing vector of hTERT(PB-hTERT/E2A/eGFP/F2A/neo)and established a new immortalized hepatocytes line hTERT-L02.Its proliferative capacity in vitro was improved than hepatocytes line HL-7702.There is no difference in the morphology and biological function between hTERT-L02 and HL-7702.But proliferation experiments suggested that the capacity of proliferation was significantly enhanced in hTERT-L02.These indicated that the cell could resolve the problem of exiquity of the liver cells for the BAL,which may be used to improve the clinical security of immortalization hepatocyte.