| Part Ⅰ Design,Screening and Identification of Antigene Locked Nucleic Acid in HBV S Coding ChainObjective: We designed and synthesized LNA fragments for hepatitis B virus S coding chain rich purine area,and selected HBV transgenic mice as the research objects,screened the best dosage,route of administration and drug delivery.Methods:(1)The experiment was designed to the dose group(0 g/g,0.1 g/g,0.2 g/g,0.3 g/g and 0.5 g/g),and the route of delivery group(intravenous,subcutaneous and intraperitoneal)and the way of Administration(one-time administration,1 /2 days,and 1 /1 days).(2)A LNA fragment complementing the 366-380 NT rich in purine region of the HBV S coding chain was designed and synthesized by RNA structure software,and HBV transgenic mice were transfected with cationic liposome.(3)Identification of LNA fragment stability by single strand specific endonuclease,the content of HBsAg in serum of mice was detected by ELISA method;the content of HBV DNA in serum was detected by fluorescence quantitative PCR.Results:(1)The results of enzyme digestion experiment showed that the ability of LNA anti nuclease degradation was stronger than other oligonucleotides.(2)Dose screening experiments showed that the inhibition of LNA on the expression of HBsAg and HBV DNA 0.2 g/g dose rate,the average inhibitory rates of HBsAg for third,fifth and seventh days were 30.47%,43.67% and 58.37%,respectively.The average inhibitory rates of HBV DNA were 31.82%,48.69% and 63.34%,respectively.(3)It was found that the inhibition rate of LNA on HBsAg and HBV DNA expression was the highest at the time of intravenous injection.The average inhibitory rates of HBsAg on third,fifth,seventh days were 30.92%,45.78% and 55.02% respectively,and the average inhibition rates of HBV DNA were 33.63%,50.79% and 58.99%,respectively.(4)The inhibition rate of LNA on HBsAg and HBV DNA was the highest at 1 /2 days,and the average inhibitory rates of third,fifth,seventh day HBsAg were 31.95%,41.08% and 50.62% respectively.The average inhibition rates of HBV DNA were 33.84%,51.89% and 56.12%,respectively.Conclusion:(1)The S coding chain of rich in purine domain 366-380 nt site,which is an effective target for hepatitis B gene therapy.(2)The stability of LNA is stronger than that of other oligonucleotides.(3)The medication 1 times(1 days /2),at a dose of 0.2 g/g body weight,suppressing the expression of LNA HBsAg and HBV DNA was the highest.Part Ⅱ Study on the Antiviral Effect of Locked Nucleic Acid in Transgenic MiceObjective: To investigate the antiviral effect of anti gene LNA on HBV S coding chain in transgenic mice.Methods:(1)30 HBV transgenic mice were randomly divided into blank control group,unrelated sequence group,lamivudine group,antisense LNA group and antigene LNA group.(2)Lamivudine group was set by gavage method.Blank group,unrelated sequence group,antisense LNA group,antigene LNA group by intravenous injection method.(3)After administration via orbital venous blood and serum.The ELISA method was used to detect the content of serum HBs Ag in mice;serum HBV DNA was detected by fluorescence quantitative PCR;liver m RNA of HBV was detected by RT-PCR;The content of HBs Ag in liver cells was detected by immunohistochemistry;HE staining was used to observe the changes in the structure of liver and kidney cells by anti gene LNA.Results:(1)After administration of first,third,fifth,seventh and fourteenth days,HBs Ag average inhibition rates of antisense LNA group were 11.06%,26.55%,35.40%,42.48% and 14.16%,the average inhibition rate of HBV DNA were 16.76%,35.31%,47.47%,45.29% and 21.37%,respectively.And the control group(blank group,unrelated sequence group,lamivudine group)compared with statistically significant difference(P < 0.05);HBs Ag average inhibition rates of antigene LNA group were 17.24%,30.17%,43.53%,57.76% and 21.12%,the average inhibition rates of HBV DNA replication were 16.52%,37.18%,50.27%,61.46% and 28.79%,respectively.Compared with the antisense LNA group,there was statistically significant difference(P < 0.05).The relative expression of HBV m RNA was 0.33,the rate of HBs Ag positive cells in hepatocytes was 31%,which was significantly different from that in the control group(P < 0.05).No abnormal changes were found in liver and kidney biochemistry and HE staining.Conclusion: According to the HBV S coding chain of the antigene LNA containing purine region 366-380 nt site has a good antiviral effect in transgenic mice and has no toxic side effects.It provides the theoretical basis and experimental basis for the hepatitis B gene therapy. |
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