| Part ?Objective:To identify the subtype of hepatitis B virus(HBV)in HepG2.2.15 cells and evaluate the cell activity in order to evaluate whether the cells can be used as a good cell model in this experiment.Methods:HepG2.2.15 cells of logarithmic growth phase were collected and cultured in 6-well plates with 0.25,0.5,1.0,2.0 and 4.0×10~5cells/ml inoculation density.The supernatant of cell culture was collected every 48h and stored in sterile EP tubes for 5times.The expression of HBV-DNA and HBs Ag was measured by fluorescence quantitative PCR and chemiluminescence immunoassay to identify the subtypes of the cells The expression of HBs Ag and HBe Ag in HepG2.2.15 cells was detected by immunohistochemistry after the preparation of cell climbing tablets,and the activity of HepG2.2.15 cells was identified.Finally,the rule of HBs Ag and HBV-DNA secretion of HepG2.2.15 cells was analyzed by activity and subtype identification results.Results:The cells with a culture concentration of 1.0×10~5cells/ml had the best condition and the fastest proliferation.The results showed that the DNA sequence of the virus was more than 99%in accordance with the s and C sequence of ayw serotype.The results of virus activity showed that HBs Ag and HBe Ag were positive in HepG2.2.15 cells.Conclusion:HepG2.2.15 cells can be used as a good model for HBV research in vitro.Part ?Objective: To design an antigene locked nucleic acid(LNA)fragment for the coding chain of HBV preS1/preS2 region,and to screen and identify effective therapeutic target fragments that can specifically block the replication and expression of HBV in HepG2.2.15 cells.Methods: Based on HBV preS1/preS2 coding strand,RNA structure 6.0 software was used to design and synthesize 4 anti-gene LNA fragments at 3023~3037nt,3094~3109nt,3201~3215nt,3305~3320nt loci(represented by SQ1-4).HepG2.2.15 cells were transfected with cationic liposome lipofectamine 3000(lipo3000).Culture supernatants were collected on the 3rd,6th and 9th days after transfection.Real-time fluorescence quantitative polymerase chain reaction(FQ-PCR)The HBV-DNA concentration was measured,and the HBs Ag level was detected by chemiluminescence immunoassay experiments.Finally,the optimal ratio of LNA drug/lipo3000 was screened based on the experimental results,and whether the combined drug concentration was different from the individual drug concentration.Results: The anti-gene LNA fragments targeting the 3023 ~ 3037 nt site(SQ1)of the HBV preS1 coding chain and the 3305 ~ 3320 nt site(SQ4)of the preS2 coding chain have the most obvious inhibitory effects on HBs Ag expression and HBV-DNA replication.,6,9d,the average inhibition rate of SQ1 on HBs Ag was 36.17%,49.62% and61.94%,HBV-DNA was 26.96%,38.31%,56.08%,and the average inhibition rate of SQ4 on HBs Ag was 50.38% and 57.42,respectively.%,72.93%,HBV-DNA were 48.68%,52.96%,61.79%.Among them,the inhibitory effect of SQ1 according to the ratio of LNA drug/lipo3000 is 1?g/1.5?L is most obvious,and on the 3rd,6th and 9th days after administration,SQ4 according to the ratio of LNA drug/lipo3000 is 2?g/3?L.Through the comparison of combination drug experiments,the combination of HBV preS1 coding chain 3023 ~ 3037 nt site(SQ1)and preS2 coding chain 3305 ~ 3320 nt site(SQ4)can further enhance the inhibitory effect.Conclusion: Anti-gene LNA fragments targeting the 3023-3037 nt site(SQ1)of the preS1 coding strand of HBV and the 3305-3320 nt site(SQ4)of the preS2 coding strand have the most obvious inhibitory effect on HBs Ag expression and HBV-DNA replication.The optimal ratio of 3023~3037nt site(SQ1)anti-gene LNA drug to lipo3000 is 1?g /1.5?L,and the 3305 ~ 3320 nt site(SQ4)is 2?g/3?L.Co-administration of 3023 ~3037nt sites(SQ1)of the preS1 coding chain of HBV and 3305~3320nt sites(SQ4)of the preS2 coding chain can further improve the effect of medication.Part ?Objective: To explore the anti-HBV effect of HBV preS1 SQ1/preS2 SQ4 coding strand antigen LNA in vitro.Methods:RNA structure 6.0 software was used to design and synthesize anti-gene LNA fragments for HBV preS1 coding chain 3023-3037nt(SQ1)and preS2 coding chain3305-3320 nt sites(SQ4).The experiment was set up as blank control group,irrelevant sequence control group(USQ),entecavir(ETV)control group,SQ1 group,SQ4 group experimental group and SQ1/SQ4 experimental group,lipo3000 liposome mediated HepG2.2.15 cell transfection,collecting transfection The culture supernatants on the 2nd,4th,6th,8th,and 10 th days after staining were tested for the content of HBs Ag and HBV-DNA using chemical immunoluminescence technology and real-time fluorescence quantitative polymerase chain reaction(FQ-PCR),reverse transcription polymerase RT-PCR was used to detect HBV preS1 SQ1/preS2 SQ4 m RNA expression in cells.Finally,the cck8 method was used to detect the effect of anti-gene LNA on basic metabolism and apoptosis of HepG2.2.15 cells.Results: Anti-gene LNA fragments have strong resistance to nuclease degradation;SQ1 group and SQ4 group targeting HBV pre1/pre2 coding chain can effectively inhibit HBV replication,and the average inhibition on the second,fourth,sixth,eighth,and tenth days after SQ1 group The rates were 34.53%,33.06%,35.12%,49.71%,51.42%,the average inhibition rates after SQ4 group were 36.37%,38.05%,48.34%,55.12%,58.29%,and the average inhibition after SQ1/SQ4 group The rates were 47.08%,60.60%,49.50%,61.36%,61.56%,SQ1 and SQ4 and SQ1/SQ4 compared with ETV group,P <0.001.The experimental results showed that the relative expression levels of m RNA in blank control group,irrelevant sequence group,ETV group,SQ1 group,SQ4 group,and SQ1/SQ4 group were 0.996 ± 0.013,0.983 ± 0.019,0.732 ± 0.021,0.621 ± 0.021,0.596 ±0.018,0.463 ± 0.019,therefore,the SQ1 group,SQ4 group,SQ1/SQ4 group had a strong inhibitory effect on HBV preS1,preS2 gene m RNA expression,and the most significant SQ1/SQ4 group inhibition effect(P <0.001).The results of cck8 experiment confirmed that the drug had no significant effect on cell proliferation(P> 0.05).Conclusion: Anti-gene LNA against HBV pre1 coding chain 3023-3037 nt site(SQ1)and pre2 coding chain 3305 ~ 3320 nt site(SQ4)has better anti-HBV activity,the combination of the two can further improve anti-HBV activity,and Basically has no effect on cell metabolism and proliferation. |
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