| Hepatitis B is an infectious diseases caused by the hepatitis B virus and is seriously harmful to human health, whose main clinical manifestations is the acute and chronic damage to liver. The development of chronic hepatitis B is related with the host immune response, gene variation of the virus, as well as environmental, genetic and other factors. If long-term lack of effective control, the chronic hepatitis B can easily develop into the liver cirrhosis and liver cancer.Drugs for the treatment of chronic Hepatitis B at home and abroad is mainly interferon and nucleoside. These two kinds of drugs can produce certain inhibition effects on HBV in different mechanisms. Its shortcomings are also easy to ignore. For example, Nucleoside drugs can cause drug resistance, interferon is expensive and both of them can lead to inevitable drug side effects. Traditional Chinese medicine in our country has the resources advantage and the theoretical system of TCM treatment is also relatively mature. In alleviating clinical symptoms in patients with chronic Hepatitis B, Chinese medicine treatment effect is remarkable. However,the key of Hepatitis B prevention and control is still anti-virus. Chinese traditional medicine is one of the feasible way to explore effective anti HBV,which are potential research value.Hepatitis B virus(HBV) genome is divided into two parts: structural genes and regulatory gene, the structural gene area including S, C, P and X four open reading frames, regulate gene area includes four promoter(SP I, SPII, CP, XP) and two enhancer(Enh I, Enh II). Under the control of Enh II and CP, C gene codes two important viral proteins, HBeAg and HBcAg. HBeAg can be secreted to the outside of the liver cells after modified at amino and carboxyl end, HBcAg is capsid components involving assemble of virus particles. ObjectiveThe recombinant plasmid expressing preC + C gene under control of Enhancer II + BCP is constructed and transfect the HepG2 cells. The transfected cells are treated with Chinese herbal medicines to screen the traditional Chinese medicine with activity angainst HBV e antigen expression. Methods1. Extract HBV DNA from serum of a child patient with positive HBV infection.2. Amply HBV EnhancerII /BCP + preC + C area with PCR method and purify the amplified HBV DNA product.3. Insert the purified HBV-C target gene into the plasmid vector pBudCE4.1to constructe the recombinant plasmid HBV-C/pBudCE4.1.4. 45 kinds of single herb are screened by looking at the literature to prepare a decoction and dilute to 4 doses gradient for spare.5. Transfect the recombinant plasmid HBV-C/pBudCE4.1 into HepG2 cells by using the liposome transient transfection method.6. Use the prepared traditional Chinese medicine to treat the HepG2 cells; detect the level of HBeAg in Cell culture supernatant and lysates by ELISA; record and analysis the OD data by using the SPSS19.0 software.7. The data are expressed in x±s. Comparison between group is conducted by t test. Difference was statistically significant when P < 0. 05 is found. ResultsThe DNA sequencing showed that the recombinant plasmid HBV-C/ pBudCE4.1 is constructed successfully.Compared to the cell in the control group without the processing of the traditional Chinese medicines, in 45 kinds of traditional Chinese medicines, the content of HBeAg in the cell culture supernatant, which was processed through 4 doses(4 g/L?2 g/L?1 g/L?0.5g/ L)of serissa serissoides, radix scutellariae, thorowax and abrus mollis hance, all decreased significantly(P<0.01). Moreover, the control effect of the medicine appeared the dose-response relationship. 4 g/L of semen arecae, 4 g/L of ligustrum lucidum ait, 2 g/L of ferula and 4 g/L of polygonum cuspidatum also decreased the HBeAg content in the cell culture supernatant significantly(P<0.01).The content of HBeAg in the cell culture supernatant, which is processed through 1 g/L of ligustrum lucidum ait, 4 g/L of rhubarb and 4 g/L of radix astragali, decreased to some extent(P<0.05).Compared with the cell in the control group, the content of HBeAg in cell lysates, processed through 4 doses(4 g/L?2 g/L?1 g/L?0.5g/ L)of serissa serissoides and thorowax, decreased significantly(P<0.01) and the control effect of the medicine appeared the dose-response relationship. 4 g/L, 2 g/L and 1 g/L of radix scutellariae and abrus mollis hance decreased the HBeAg content in cell lysates significantly and they appeared the dose-response relationship. 4 g/L of ferula decreased the HBeAg content in cell lysates significantly(P<0.01). ConclusionIn the traditional Chinese medicines serissa serissoides, radix scutellariae, thorowax and abrus mollis hance, some small molecules ingredients may be contained to inhibit the HBeAg expression by regulating HBV enhancer II and core promoter activities, then reduce the secretion of the hepatitis b virus e antigen. |
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