| BACKGROUNDFirst, hepatitis B is an infectious disease, which can seriously threaten to human health and lead to liver fibrosis and cirrhosis. Although an effective inoculation of hepatitis B vaccine has greatly reduced the incidence of hepatitis B in recent years, there are still 35 million people with chronic HBV infection all over the world. Therefore, study of the pathogenesis, diagnosis, prevention and treatment of hepatitis B is still in great need.At present, the main cell models are cancer cell lines during study the HBV in vitro, such as HepG2.2.15 cells. They can secret HBsAg, HBeAg and Dane particles stablely, and replicate high level of HBV-DNA in the cells. However, cancer cell lines can not show the physiological activity of non-cancerous cells. After the long-term subcultured, the expression of the important drug-metabolizing enzymes(cytochromes P450) decreases significantly, so they can not be an ideal target cell for selecting some anti-virus drugs. Primary hepatocytes play an important role in examination of the drug biotransformation and searching for drug metabolites. Practices shows that the primary hepatocytes have become the important target cells during studying something about CYP450 enzymes. Second, there is increasing attention both at home and abroad on the reason of HBV persistent infection and how to change this status. Some studies have shown that the reason of HBV persisitent infection is the immunological tolerance, which is caused by some HBV antigens escape the immune onslaught engendered by specific cells. How to eliminate the immunological tolerance has becomes a key problem for CHB patients. Therapeutic hepatitis B vaccines can induce the production of the specific immune response in order to suppress HBV virus. However, due to the differences of histocompatibility antigens among different species, there will be insufficient number of suitable animal models for research before clinical trial of therapeutic hepatitis B vaccines. Therefore, our laboratory prepared HBV/HLA-A2.1 double transgenic mice, in order to solve the problem of therapeutic hepatitis B vaccines'pre-clinical screening.OBJECTIVEFirst, to find a method in isolating and culturing the HBV-Tg mice primary hepatocytes in vitro, and observe the effects of alcohol on the primary hepatocytes' damage sensitivity and secretion of HBsAg.Second, to observe the induction of specific immune response to HBV/HLA-A2.1 dTg mice and the effects of virological indicators (HBsAg/ HBV-DNA) of specific immunity peptides.METHODSFirst, the primary hepatocytes was separated from HBV-Tg mice and wild Balb/c mice with the modified two-steps collagenase perfusion. Cells growth was observed within 7 days, then the levels of albumin, urea, lactate dehydrogenase, HBsAg and HBV-DNA were examined, which were in the supernatant of the primary hepatocytes separated from HBV-Tg mice. Alcohol was added to the primary hepatocytes in order to induce the oxidative stress. Observe the differences of damage sensitivity between the cells from the HBV-Tg mice and wild Balb/c mice, and the effects of alcohol on HBsAg levels.Second, HBV-specific epitopes was used for subcutaneously immunized three transgenic mice (HBV-Tg, HLA-A2.1, andHBV/HLA-A2.1 dTg). Some time afterwards, observing the changes of liver injury on the histology, the secretion of IFN-y by spleen cells and the ratio of CD3+, CD4+, CD8+in the spleen cells. Observe the expression of HBsAg, HBV-DNA dynamically.RESULTSFirst, the laboratory using the modified two-step collagenase perfusion separate and culture the primary hepatocytes from HBV transgenic mice. We can not only successfully isolated and cultured the primary hepatocytes, but also high output, each mice can obtain a total of 107 cells, the ratio of living cells can reach 90% after purified. The cells were cultured in the 24-well plate or 96-well plate coating with gelation, the morphological changes were observed daily. At the first day, the living cells spreaded over the bottom of the culture plates. Meanwhile some round and non-adherent dead cells drift on the supernatant. Living cells adhered absolutely,and become larger, polygonal, more cytoplasmic vacuoles and lipid dropletsTwo or more nucleus in cells can be seen. At the second day, some adjacent cells contact witn each other by pseudopodia, the cell surface became larger and stretched more thoroughly. There were no morphological changes until the 7th day, the cells gradually retracted, became smaller and lower transparency, not translucent after 7 days. More and more dead cells appeared in the culture supernatant. Cell culture supernatants were collected every day, the expressions of albumin, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and virological indicators of HBsAg and HBV-DNA were detected. Albumin levels were found in the highest level in the 2 to 5 days, the levels of alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase kept a high level in the beginning, after the first two days fall to a stable level; virological indicators of HBsAg and HBV-DNA maintain a high level in 5 days; there were difference sensitivities observed between HBV-Tg and wild-type Balb/c mouse when alcohol acted as an oxidative stress factors. At the same time, alcohol can also influence the expression of HBsAg of HBV-Tg mouse hepatocytes. Primary hepatocytes cultured for 24 hours, being treated with different concentrations of alcohol (0.13%,0.5%,2%,8%) for 16 hours, and then detecting the AST levels in the supernatant. As the concentration of alcohol increased, the levels of AST released into the medium increased gradually. Calculating the rates of specific cytotoxicity between two groups of cells. The results showed that the inhibition ratio of HBV-Tg mouse hepatocytes was significantly higher than wild Balb/c mice in the 0.5% and 0.13% group. There was no significant difference between the 2% and 8% group in cell toxicity. Observing the relationship between alcohol and the HBsAg levels, we found that in the concentration of alcohol 0.13%, the expressions of HBsAg was significantly higher than the negative control group. With increasing the concentration of alcohol, the HBsAg levels decreased. Although there was no significant difference between the experimental and negative group in the concentrations of higher than 0.5%, the expression of HBsAg had a trend of lower than negative control.Second, HBV/HLA-A2.1 double transgenic mice we prepared can stably expressed HBsAg, HBV-DNA, and lymphocytes had a higher expression of human-derived MHC-â… (HLA-A2.1). We use CTL-specific epitope peptides HBc18-28 plus Th-specific epitope peptides HBc128-140 emulsified with incomplete Freund's adjuvant. HBV-Tg, HLA-A2.1, HBV/HLA-A2.1 dTg three kinds of transgenic mice were immunized subcutaneous. After a period of time after immunization, we found that CD3 + /CD8 + T cells and IFN-y secretion of HBV/HLA-A2.1 dTg and HLA-A2.1 experiment groups was significantly higher than that other control groups; HBV/HLA-A2.1 dTg mice liver tissue revealed an acute inflammatory response, while other groups of mice liver tissue reactions were relatively mild; There were no significant differences in the levels of HBsAg and HBV-DNA between pre and post immunization in the same strains.CONCLUSIONSFirst, more hepatocytes can be isolated by the method of modified two steps collagenase perfusion. The stability of cell membrane restored to normal after 2 days cultivation. ALB can be secreted stabily for 5 days. Cells began to lose normal function slowly and fall off after cultured 7 days. HBV-Tg mice hepatocytes was more sensitive and vulnerable than Balb/c mice hepatocytes when damage was caused by alcohol. Certain concentrations of alcohol also can promote the secretion and expression of HBsAg. These datas are coincide with several related articles internal and abroad. So the primary hepatocytes separated from our HBV-Tg mice can be used for studying the injury mechanisms of alcohol combined with HBV, and the effects of alcohol on hepatitis B virus replication and other aspects.Second, HBV/HLA-A2.1 dTg mice were subcutaneously immuned by specific epitopes of hepatitis B. The results confirmed that the specific epitopes activate specific immune cells of HBV/HLA-A2.1 dTg mice successfully, and may creat inflammation against liver cells which have HBV replication. Unfortunately, though specific immune responses in the body had increased, HBsAg and HBV-DNA had not found decreaseing significantly. May be due to the large individual differences and low expression levels in the expression of virological indicators between mice, or slight changes are not easy to find. On the other hand may be our specific immune peptide combination were also not reasonable. In a word, the characteristics of the HBV/HLA-A2.1 double-transgenic mice and whether suitability for the hepatitis B vaccine preclinical studies and experiments need to be further confirmed. |
PDF Full Text Request