Construction Of Immortalized Human Hepatocyte Cell Lines Derived From Human Telomerase Reverse Transcriptase And Caspase-3siRNA

BackgroundLiver failure is a serious threat to human health due to various reasons. Clinical application of orthotopic liver transplantation was limited because of expensive fee and donor shortage. Bioartificial liver support system is an attractive approach for an alternative to liver transplantation. Bioartificial liver support system temporarily substitude liver function with the in vitro device until the autologous liver function recovery or proceeding liver transplantation. In the perspective of bioengineering, hepatocyte is a core part of the bioartificial liver support system. the ideal hepatocyte for the bioartificial liver support which origined from human, with normal phenotype and easy to access, cultured with rapid growth and proliferation in vitro and maintain the differentiation state in the highest density with all biological metabolic function of mature hepatocytes. However, human primary hepatocytes cultured in vitro under harsh conditions and limited survival time, proliferation, had to culture and shortages of sources. Therefore, it is essential to construct human liver cell line with good differentiation and proliferation ability for development of BAL.An important step in cell immortalization is activation of the human telomerase reverse transcriptase （hTERT）. Transferred exogenous genes of hTERT into telomerase-negative cells can be restored in the purpose of to repairing and maintaining telomerase activity and length of telomere cells.thereby extending the life of the cells under in vitro culture conditions, even immortalization will be developed. Caspase-3is a key protease in mammalian apoptosis and in the heart of the apoptotic cascade response pathway is the only way of the cascade of apoptosis protein. Human primary hepatocyte was transfected by lentiviral vectors with siRNA-mediated Caspase-3gene silencing. The constructed immortalized human liver cells in the genome with fidelity and stability and the synthesis of cell metabolic function have great advantage.This study was to construct immortalized human liver cell lines which origin from human primary hepatocyte stably transfected with human telomerase reverse transcriptase gene and inhibition expression of caspase-3siRNA by cellular immortalization and resistance apoptosis principle with application of gene cloning, RNAi interference and lentiviral expression vectors and other technology. The establishment of immortalized human liver cell lines HepGL was build by two lentiviral vectors with hTERT gene and of Caspase-3RNAi. Biological characteristics and functions of immortalized human liver cell lines HepGL was to be appraised and the feasibility of seed cells for bioartificial liver also to be evaluated. The dissertation is divided into five chapters:Chapter I Construction of lentiviral vector pLENT6.3/V5-hTERT-IRES-RFPObjective:To construct lentiviral vector carrying target gene of telomerase reverse transcriptase （hTERT） pLENT6.3/V5-htert-IRES-RFP and marker gene of red fluorescent protein （RFP） eukaryotic expression vector for gene transfection primary hepatocytes experimental basis.Methods:（1） Construction and identification of lentiviral phTERT-IRES-RFP:PCR amplification of the hTERT gene and cloned into pIRES-the RFP vector by BgⅢ and EcoRI digestion and cloned into the pIRES-the RFP vector between the BgⅢ and EcoRI sites. PCR products were purified, digested with plastic recycling, plasmid pIRES-the RFP as a template, vector digested, T4-DNA ligase reaction. Will connect the products were transformed into E.coli DH5a kanamycin picked positive clones line PCR, restriction enzyme digestion and sequencing phTERT-IRES-RFP plasmid.②Construction and identification of recombinant vector pLENT6.3/V5-htert-IRES-RFP:PCR amplification of hTERT-IRES-the RFP and cloned into the MCS-V5, pLenti6.3-lentiviral vector digested with BgⅢ and NheI, cloned into the the pLenti6.3-MCS-V5vector between. BamHI and NheI sites. PCR products were purified, digested with plastic recycling, pLenti6.3-MCS-V5lentiviral vector as a template, vector digested with T4-DNA ligase reaction. The ligation product was transformed into E. coli STB13competent, painted on LB plates containing ampicillin neomycin, pick a single colony were cultured, and plasmid was extracted and sequenced to confirm the pLENT6.3/V5-htert-IRES-RFP carrier.③Packaging and collection of virus containing particles supernatant of hTERT-the RFP:the use of liposomal transfection of lentiviral the carrier pLENT6.3/V5-htert-IRES-RFP packaging plasmid （containing pLP1, pLP2and pLP/VSVG three plasmid） were cotransfected into293FT cells, packaging collecting of virus, concentration and titer determination. Results:（1） Construction and characterization of results in lentiviral phTERT-the IRES-RFP: This experiment was successfully amplified lasted about3.4Kbp of hTERT fragment, restriction analysis and sequencing confirmed that hTERT has been cloned into the pIRES-RFP vector, and successful obtain phTERT-IRES-RFP product.②Construction and identification of recombinant vector results in pLENT6.3/V5-htert-IRES-RFP. hTERT-IRES-RFP fragment was amplified by PCR and cloned into pLenti6.3-MCS-V5lentiviral vector construction was slow virus recombinant plasmid pLENT6.3/V5-htert-IRES-RFP. The recombinant plasmids were sequenced and confirmed the recombinant plasmid pLENT6.3/V5-htert-IRES-RFP build success.③Preparation of results containing the supernatant of hTERT-RFP virus particles: The pLENT6.3/V5-htert-IRES-RFP and packaging plasmid transfected293FT cells, and termination of the supernatants were collected72h after transfection, ie containing supernatant of hTERT-the RFP virus particles. Titer formula to calculate the titer of the lentiviral5.88×106TU/mL.Conclusion:The pLENT6.3/V5-htert-IRES-RFP was constructed successfully using lentiviral vector carrying hTERT eukaryotic expression vector.Chapter ⅡConstruction Ccaspase-3siRNA lentiviral vectorObjective:Construction of RNAi DNA plasmid vector for the expression of target gene expression of Caspase-3sequence, and filter out the interference target, and the lentiviral vector packaging.Methods: ①Construction of siRNA interference vector:the target gene, caspase-3gene sequence was be collected through application of public Web site in accordance with the principles of RNA interference sequence design. The design four pairs of siRNA interference target sequence were SR₁, SR₂, SR₃, SR₄SR_neg （C-） negative control, four pairs of double-stranded siRNA to an oligo were inserted into the siRNA expression vector pcDNA TM6.2-GW/EmGFPmiR build four siRNA expression competent cells.aplasmid was transformed into DH5. Colony PCR screening of positive clones were sequenced using vector universal primers and verified the consistency of the fragment inserted in the recombinant clones and design of the oligo sequence, amplified plasmid spare.②Transfection and screening of siRNA interference vector:The application of interference plasmids were transiently transfected HEK293cells in the logarithmic phase. Fluorescence was observed after24hours. The qPCR and Weatern Blot interference vector in gene and protein level on the inhibition of the target protein leve was to be used. According to the results of qPCR in pcDNA TM6.2-GW/EmGFP, si-SR₂was the best after transient transfection, the inhibitory effect of the target gene is the most obvious.③Construction and packaging lentiviral expression vector:Application Gateway recombination technology using Gateway recombination technology to build the lentiviral expression vector pLENT6.3/V5-SR₂. After matching, the reorganization of the lentiviral vector oligo sequence completely correct. Lentiviral vector was constructed successfully amplified plasmid spare; lentiviral packaging293FT cells and testing of the titer.Data processing:Results were expressed as mean values±standard deviation （x±SD）, A value of a=0.05was considered the standard of inspectability significant. We used the SPSS13.0software for statistics analysis. The results of Quantitative real-time RT-PCR were analysed by one-way ANOVA. LSD method was used for multiple comparisons between groups for homogeneity of variance; otherwise Tamhane’s T2method was used.Results:①To design4interference target, respectively, were SR₁, SR₂, SR₃, SR₄, SR_neg （C-） as negative control, and siRNA expression vector pcDNATM6.2-GW/EmGFPmiR success connection, build the four siRNA expression plasmid plasmid successfully connected.②Construction four paris of caspase-3RNAi plasmid vector after sequencing, contrast, amplification and extraction.After qPCR detection of single-factor analysis of variance showed, four siRNA transfection significantly affect the HEK293cells, expression of Caspase-3expression （Welch=27965.102, P<0.001）, between the two groups of multiple comparison （Tamhane’s the T2law） Display:siRNA2transfer dye in HEK293cells, Caspase-3mRNA relative expression amount is9.84±0.461（P<0.001）, on inhibitory effect of the target gene is the most obvious.③using the Gateway recombination technology to build the lentivirus expression vector pLENT6.3/V5-SR₂detection sequencing sequence is entirely correct, the lentiviral vector was constructed successfully. The titer determination of packaged lentiviral was3.6×105TU/ml.Conclusion:The siRNA lentiviral vector of Caspase-3gene was successfully constructed.Chapter III Isolation and culturation of primary hepatocytesObjective:To establish separate and culture methods for human primary hepatocytes in vitro.Methods:Human primary hepatocytes was obtained by using multi-point puncture perfusion technique with pre-perfusate solution at38℃and Ⅱcollagenase solution perfusate filling prior to digestion and centrifugation of human liver tissue. Hepatocyte morphology was observed by inverted microscope and electron microscope and functional identification were identified.Results:The amount of human primary hepatocytes was5x105and the viability was90%in the average size of3cm×2cm×1cm of liver tissue after multi-point puncture perfusion, digestion and centrifugation. Electron microscopy showed hepatocytes were cobblestone-like growth with non-proliferation, a typical large nucleus and little cytoplasm after cultured24hours. Glycogen PAS staining and ALB expression of cultured hepatocytes were positive.Conclusions:The multi-point puncture perfusion hepatocytes technique was simple and easy, with advantages of high yield, good viability and high purity and specific biological functions, so it would be applied widely in ordinary laboratory.Chapter IV Establishment and characterization of immortalized human hepatocytesObjective:Lentiviral mediated human telomerase reverse transcriptase （hTERT） gene and Caspase-3of siRNA successively transfected human primary hepatocytes to build immortalized human liver cell lines （HepGL） and the morphological and functional identification were evaluated..Methods:To obtain HepGL-H liver cell line through human primary hepatocytes infected by expression of recombinant lentivirus infection of the h-TERT gene with blasticidin screening positive clones. To obtain immortalized human liver cell line （HepGL） through HepGL-H cells infected by caspase-3-siRNA lentivirus fluid. The morphology growth and proliferation of transfected hepatocytes were observed by phase-contrast microscopy respectively. PAS （the peri odicacid-Schiff, the PAS） staining of glycogen content of the cells, ALB expression of HepGL cells of immunocytochemical identification and chromosome karyotype were observed, by The functional genes and surface markers of gene expression were evaluated by RT-PCR. ALT, AST and urea of HepGL cell culture supernatant concentration were detected by automatic biochemical analyzer and to draw the growth curve.Results:The establishment of an immortalized human hepatocytes HepGL typical morphological characteristics of primary hepatocytes, but also has better proliferative capacity, karyotype analysis showed that the karyotype was normal; RT-PCR detection HepGL of expression to expression of hTERT, P4503A, albumin, ASGPR, GS, GST-π, HBCF-X, HNF4, CK18mRNA of liver cell biological characteristics, automatic biochemical analyzer the determination HepGL cell culture supernatant of ALT, AST, concentration showed a decreasing trend. Urea concentration showed a gradually increasing trend.Conclusion:The immortalized human liver cell lines HepGL was established in this study. HepGL is similar to primary hepatocytes in the morphological features and biological functions which can become the ideal seed cells for bioartificial liver and liver cell transplantation materials.Chapter V Security of immortalized human hepatocytesObjective: To explore the oncogenicity of immortalized human liver cells HepGLMethods:The experiment was divided into HepGL group and C3A group completely randomly assigned to two groups. According to a random number table method, each group of nude mice10. The two groups were injected HepGL and C3A cells with subcutaneous0.2mland a number of2.0×106in the neck anf back of nude mice respectively. To observe the skin cells tumorigenic and daily measurement of tumor size, and organization of the injection site tumors and brain, liver, lung and kidney tissue samples were cut after five weeks.Results:No growing tumor in inoculation site was showed in experimental group. There was no significant difference between inoculation district organizational structure and the site of non-vaccinated. Liver, lung, brain, renal pathological section without metastasis were showed in histological observation. C3A group had a total of20inoculation site of tumor, and tumorigenicity was100%. Uniform shape and size of nude mice implanted tumor cells, the nuclei deeply stained mitotic figures were showed more cells a trabecular arrangement.Conclusion:Immortalized human liver cells HepGL hasn’t tumorigenicity with a good application security.