| Hepatitis B Virus(HBV) infection and series of diseases caused by HBV are great threats to human health around the world. Scientists from all over the world have conducted various researches and projects in different prospective in order to effectively prevent and treat HBV infection. The current measures in the prevention and treatment of HBV infection mainly includes Hepatitis B vaccines, interferon and nucleotide analogues. Though HBV vaccines can prevent the infection, they have no effect to already infected patients, immune tolerate patients and immune escape mutant virus. Moreover, the current anti-HBV drugs all have the disadvantages of huge side effect(interferon), drug resistance(nucleotide), low antigen seroconversion rate and other shortcomings.Building the cell model is one of the most convenient and efficient ways to research into the virus infection history and biological characteristics, evaluate the effectiveness of drug, optimize treatment pan and develop new vaccines. The existing cell model for virus research includes virus infection cell model, virus transient transfection cell model, virus stable transfection cell model, and so on. Regarding to the virus infection cell models, though HBV receptor molecules Sodium taurocholate co-transporting polypeptide(NTCP) is discovered in recent years, its efficiency is still low and infection operation is complex, and due to the strict demands of the species and tissue specificity for HBV infection, so the applications of virus infection cell model are constrained. Moreover, it is extremely hard for virus transient transfection cell model to maintain long term stable virus replication in hepatic carcinoma cell line, thus this makes it unsuitable for standardized research like drug sensitivity test. For the past 20 years, many HBV cell models have been well-established for stable for virus transient transfection cell model to maintain long term stable virus replication in hepatic carcinoma cell line, sucn as the international recognized Hep G2.2.15 and Hep AD38 cells. Some research institutes have developed drug resistant mutational HBV stable cell lines for their research needs. However, there is no Chinese epidemic strain genotype B and C HBV based stable virus replication cell lines and multiple drug-resistant cell model.Moreover, some human-induced mutant cells cannot reflect the virus natural hereditary properties. Therefore, establishing a stable virus replication cell model based on chinese epidemic strain genotype would bring significant advancement to the evaluation of anti-HBV drugs and localization of HBV prevention plan.We established Chinese epidemic strain genotype C HBV stably transfected cells using PB transposon combined with other technologies in the early stage. The objective of the research is to evaluate the parameters of the cell and to apply it on in vitro anti-HBV pharmacodynamic evaluation. The goal is that this cell model can provide ideal cell models for anti HBV drug evaluation.Objective: Analysis the pre-established cell lines, namely Hep G2.CW and Hep G2.LER, which were stably transfected with HBV Chinese epidemic strain genotype C HBV. Evaluate these cell lines with the typical HBV stably transfected cells mainly by relative replication indexes. Test the reliability of these cell lines using commonly-used clinical nucleoside drugs. Design the lentiviral vectors for new short hairpin RNA(sh RNA) expression and evaluate the feasibility of its application on anti HBV.Methods: 1、Property analysis of HBV stable transfection cell: 1) Quantitative chemiluminescence assay for expression of hepatitis B virus surface antigen(HBs Ag) and e antigen(HBe Ag) in Hep G2.LER and Hep G2.CW cell line supernatants; 2) Fluorescence quantitative PCR assay of HBV DNA in Hep G2.CW and Hep G2.LER cell line supernatants; 3) Qualitative analysis of intracelluar expression of HBs Ag and HBe Ag by ELISA; 4) Indirect immunofluorescence assay of intracelluar expression of HBs Ag; 5) Indirect immunofluorescence assay of intracelluar expression of HBc Ag; 6)Qalitative detection of the expression of hepatitis B virus pre-S1 antigen(HBV Pre S1) in Hep G2.CW and Hep G2.LER cell line supernatants.2 、 Nucleoside drugs commonly used in clinical were adopted to evaluate reliability of Hep G2.CW and Hep G2.LER: lamivudine(LAM), entecavir(ETV), adefovir dipivoxil(ADV) with concentration gradient of 0, 0.001, 0.01, 0.1, 1, 10, 100 μM respectively, were acting on Hep AD38(positive control), Hep G2.CW and Hep G2.LER cell lines. After cell culture for nine consecutive days, HBV DNA in cell supernatant was detected by fluorescence quantitative RCR method.3、 Construction of sh RNA lentiviral vector and evaluation of its anti-HBV application: Sap I sites in lentiviral vector backbone were mutated and structures for small hairpin RNA expression were subcloned into the lentiviral vector. sh RNA targeting HBV conserved sequences were designed and cloned into the lentiviral vector, the recombinant lentivirus containing sh RNA sequence were packaged and the virus titers were measured, evaluate the effects of single sh RNA and series of sh RNAs expression on the lentivirus titers; HBV liver cell line Hep G2.CW(MOI=3) was stably transfected through recombinant lentivirus infection. Stable clones were selected using blasticidin for one week, then cells were re-digested and inocuated in 24-well plates at a concentration of 105 cells/well. Expressions of HBs Ag、HBe Ag and HBV DNA in cell supernantes were detected 96 h after cell inocuation.Results: 1、Analysis on HBV stably transfected cells: 1) HBs Ag concentration in Hep G2.2.15, Hep G2.CW and Hep G2.LER cell supernatant are 79 ng/ml, 85 ng/ml and 8 ng/ml respectively under same condition; HBe Ag concentration in Hep G2.2.15, Hep G2.CW and Hep G2.LER cell supernatant are 63 NCU/ml, 78 NCU/ml and 70 NCU/ml respectively under same condition. 2) quantity of HBV DNA in Hep G2.CW and Hep G2.LER cells are both higher than in Hep G2.2.15. 3) Intracelluar HBs Ag in Hep G2.CW is higher than Hep AD38, while intracelluar HBe Ag in Hep G2.CW is lower than Hep AD38. Intracelluar HBs AG and HBe AG in Hep G2.LER are both lower than positive control cell Hep AD38. 4) Hep G2.CW and Hep G2.LER cell lines both have high expression of HBs Ag. 5) Hep G2.CW and Hep G2.LER cell lines both have high expression of HBc Ag. 6) Average OD values of HBV Pre S1 in Hep AD38, Hep G2.CW and Hep G2.LER cell supernatants are 2.5, 3.5, and 0.8 respectively.2、Commonly used nucleoside drugs for HBV infection therapy in clinical was adopted to evaluate reliability of Hep G2.CW and Hep G2.LER. In positive control cell Hep AD38 and Hep G2.CW, quantity of HBV DNA in cell supernatants decreases uniformly with the concentration of drugs increased. However for Hep G2.LER, quantity of HBV DNA in cell supernatants has a decreased sensitivity to LAM and ETV. ETV turns out to have an inhibition effect on HBV DNA only when concentration is above 1 μM, while the inhibitive effect of ADV on HBV DNA decreased when concentration is higher than 0.1 μM.3、Successfuly constructed a lentiviral vertor expressing new sh RNA. Highly effective inhibition of HBV replication was realized by sh RNA which targeted HBV DNA conservative sequences. sh RNA expression of two kinds or three kinds in series has a better effect than expressing single sh RNA.Conclusion: Two stably transfected cell lines Hep G2.CW and Hep G2.LER can support HBV replication and expression. Hep G2.CW is sensitive to all the three commomly used drugs while Hep G2.LER has a decreased sensitivity to LAM and ETV. Our new-type sh RNA expressing lentiviral vetor can be a potential effective method to prevent HBV infection. |
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