| Part Ⅰ Culturing the HepG2.2.15 Cell and Identificating the Activity and Subtype of HBVObjective:To identify the activity and subtype of HBV in HepG2.2.15 cell.Methods:HepG2.2.15 cell was routinely cultured,culture supernatant and and cells were collected in logarithmic growth phase.The level of HBsAg、HBeAg and HBcAb in supernatant were detected by enzyme-linked immunosorbent assay(ELISA);Content of HBV-DNA in supernatant was detected by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR);HBV subtype was identified by high throughput gene sequencing;HBV genotype and drug resistance mutation were detected by molecular hybridization;the expression of HBsAg and HBeAg intracellular were detected by immunohistochemistry.HepG2.2.15 cell was cultured at 5 concentrations(0.25,0.5,1.0,2.0 and 4.0×10~5 cells/mL),supernatants were collected every 48 hours,collected 5 times in succession and detected the content of HBeAg and HBV-DNA.Results:HBV activity of HepG2.2.15 cell was identified as follows:HBsAg(OD)was0.36±0.11;HBeAg(OD)was 2.37±0.08;HBcAb(OD)was 1.73±0.17;and HBV-DNA was(4.75±0.06)×10~5IU/mL.Sequence alignment of viral S and C region showed that they were consistent with HBV ayw subtype with the coincidence rate of more than 99%.HBV genotype is B+D,there is no drug-resistant mutation site.Immunohistochemical result showed that both HBsAg and HBeAg intracellular were positive.The best culture concentration was 1.0×10~5 cells/mL with faster and more stable proliferation and perfect state.Conclusion:With good expression of HBV activity and no drug-resistant mutation site,HepG2.2.15 can be used as a good model for HBV-related studies in vitro.Part Ⅱ Designing,Screening and Identification the Anti-gene Locked Nucleic Acid targeting HBV C Encoding ChainObjective: Designing,screening and identification the anti-gene locked nucleic acid(LNA)fragment targeting HBV C encoding chain which can specific blockade the HBV replication and expression and become effective therapeutic targets in Hep G2.2.15 cell.Methods: Seven anti-gene LNA fragments that targeting 1914-1928 nt,1969-1983 nt,2002-2016 nt,2053-2067 nt,2069-2073 nt,2162-2176 nt and 2404-2418 nt site respectively(expressed as SQ1-7)of HBV C encoding chain were designed and synthesized by RNAstructure5.0 and transfected Hep G2.2.15 cell mediated by cationic liposome lipofectamine 3000(lipo3000).The level of HBe Ag in supernatant were detected by enzyme-linked immunosorbent assay(ELISA);the content of HBV-DNA in supernatant was detected by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR).And screening the optimum proportion of LNA drug / lipo3000.Results: Anti-gene LNA fragments targeting 2162-2176 nt and 2404-2418 nt site(SQ6 and SQ7)of HBV C encoding chain had the most obvious inhibition effect on HBV-DNA replication and HBe Ag expression.On the 3rd,6th and 9th day after administration,the average inhibition rates of HBe Ag of SQ6 were 43.91%,59.95% and 59.07% respectively,and the HBV-DNA were 42.91%,50.11% and 51.14%respectively;the average inhibition rates of HBe Ag of SQ7 were 44.18%,60.81% and 60.02% respectively,and the HBV-DNA were 47.26%,54.16% and 52.22% respectively.The inhibition effect is the most significant when the ratio of LNA drug/lipo3000 was 4μg/6μL,on the 3rd,6th and 9th day after administration,the average inhibition rates of HBe Ag of SQ6 were 44.16%,60.14% and 60.27% respectively,and the HBV-DNA were 47.71%,56.66% and 53.26% respectively.Conclusion: Anti-gene LNA fragments targeting 2162-2176 nt and 2404-2418 nt site of HBV C encoding chain showed the best anti-HBV effect,and the optimal ratio of anti-gene LNA drug to lipo3000 was 6μL/4μg.Part Ⅲ Anti-HBV Effect of Anti-gene Locked Nucleic Acid targeting HBV C Encoding Chain in VitroObjective: To investigate the anti-HBV effect of anti-gene LNA targeting HBV C encoding chain in vitro.Methods: Anti-gene LNA fragments(SQ6,SQ7)targeting 2162-2176 nt and 2404-2418 nt site of HBV C encoding chain were designed and synthesized by RNA structure 5.0 software.The experiment consisted blank control group,lipo3000 control group,unrelated sequence control group(USQ),entecavir control group(ETV),SQ6 and SQ7 experimental group.Transfected the Hep G2.2.15 cell by lipo3000,each group was given corresponding drug every other day for 5 consecutive times.Cell culture supernatants were collected on the 2nd,4th,6th,8th and 10 th day respectively,cells were collected on the 10 th day to determine the expression of HBV C m RNA intracellular.The level of HBe Ag in supernatant were detected by enzyme-linked immunosorbent assay(ELISA);the content of HBV-DNA in supernatant was detected by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR);and the expression of HBV C m RNA intracellular was detected by reverse transcription-polymerase chain reaction(RT-PCR).The effect of anti-gene LNA on cell proliferation and apoptosis was detected by CCK8 test and flow cytometry.Results: On the 2nd,4th,6th,8th and 10 th day after administration,the average inhibition rates of HBe Ag of SQ6 group were 34.86%,47.99%,61.62%,61.51% and 59.49% respectively,and the HBV-DNA were 33.38%,54.97%,59.99%,53.94% and 50.59% respectively,compared with other groups,P<0.001;the average inhibition rates of HBe Ag of SQ7 group were 36.33%,50.93%,64.67%,62.45% and 61.36% respectively,and the HBV-DNA were 33.71%,55.82%,61.17%,54.95% and 51.82% respectively,compared with other groups,P< 0.001.The electrophoretic gray scale ratios of amplified product of HBV C mRNA in blank control group,lipo3000 control group,USQ,ETV,SQ6 and SQ7 to amplified product of internal reference gene(set to 1)were 0.973±0.017、0.967±0.011、0.969±0.019、0.602±0.013、0.376±0.009 and 0.371±0.007,respectively.CCK8 and flow cytometry showed that anti-gene LNA had no significant effect on cell proliferation and apoptosis.Conclusion: Anti-gene LNA targeting 2162-2176 nt and 2404-2418 nt site of HBV C encoding chain can effectively inhibit the HBV replication and expression and no significant effect on cell proliferation and apoptosis in Hep G2.2.15 cell. |
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