Dissertation > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis B

Culture of Primary Hepatocytes from HBV-Tg Mice and the Fumdamental Use of HBV/HLA-A2.1 Mice

Author TaoZuo
Tutor KongXiangPing;YiXueRui
School Southern Medical University,
Course Infection within the scientific
Keywords Primary hepatocytes Alcohol Oxidative Stress Therapeutic vaccine
CLC R512.62
Type Master's thesis
Year 2011
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Background of a hepatitis B infection is a serious threat to human health, infectious diseases, and is the main lead to liver fibrosis and cirrhosis. Although in recent years, effective vaccination of hepatitis B vaccine greatly reduces the incidence of hepatitis B, but around the world there are still about 35 million chronically infected. Therefore, the study of hepatitis B pathogenesis, diagnosis, prevention and treatment still we need to vigorously carry out. Vitro model of hepatitis B research cancer line cells, such as HepG2.2.15 cells. Can stabilize the secretion of HBsAg, HBeAg, Dane particles, and can be detected by intracellular HBV-DNA, etc., but, after all, is a cancer-line cells, can not exhibit the physiological activity of the non-cancerous liver cells after long-term subculture its surface is a very important drug-metabolizing enzyme CYP450 expression was significantly reduced, the screening of antiviral drugs can not be good. The primary hepatocytes training for drug biotransformation pathways of research looking for metabolites of the drug may provide a simple and effective model. Practice has proved that the primary hepatocytes has become the CYP450 enzyme closely related to the experimental model. B, hepatitis B why the the chronic development is easy to chronic HBV and how to change the hotspot of hepatitis B research at home and abroad, is difficult. Some studies, hepatitis B The reason is easy to chronic hepatitis B virus antigen substance-specific cellular immune attack can evade the body's immune tolerance. How can break hepatitis B patients become key blocking hepatitis B chronic hepatitis B virus immune tolerance. Treatment of hepatitis B vaccines is the principle of the clearance of HBV virus-specific immune response induced body become a hot topic. Histocompatibility antigen differences between species, the treatment of hepatitis B vaccine because of the lack of a suitable animal model, and the impact of its evaluation. Prepared HBV/HLA-A2.1 double transgenic mice in the laboratory, want to be able to solve the problem of the treatment of hepatitis B vaccine preclinical studies. Objective To establish the separation of primary hepatocytes of HBV transgenic mice (HBV-Tg mice), culture methods, and observed alcohol damage sensitivity of HBV-Tg mouse primary hepatocytes and HBsAg secretion. Second, the observed induction of specific immune peptides HBV/HLA-A2.1 double transgenic mice (HBV/HLA-A2.1dTg mice) specific immune responses and virological indicators HBsAg, HBV-DNA. Method using the optimization step perfusion improved separation cultured primary hepatocyte HBV-Tg mice and wild-type Balb / c mice; observe the growth state of the cells were plated in 7 days, and the detection of HBV-Tg mouse primary liver cell culture supernatant albumin, urea, lactic dehydrogenase, HBsAg, HBV-DNA secretion and expression; using alcohol as oxidative stress injury substance, observation of HBV-Tg mice and wild-type of Balb / c mice primary hepatocytes damage sensitivity differences, and to observe the impact of alcohol on the HBV-Tg mice HBsAg. Second, with the HBV specific epitope peptide immunized subcutaneously HBV-Tg, HLA-A2.1, HBV/HLA-A2.1 dtg three transgenic mice, to observe the change in mouse liver tissue damage after a period of time, the secretion of spleen cells secretion of IFN-γ and spleen cells, CD3, CD4, CD8 cell ratio. And dynamic observation of the expression of the mouse serum HBsAg, HBV-DNA. Results of one of the laboratory using a modified two-step perfusion method, hepatitis B transgenic mouse primary hepatocytes were cultured, not only can we successfully isolated and cultured primary hepatocytes of HBV-Tg mice, and the yield is high, each the mice share of a total of 107 cells, live cells after purification ratio can be up to 90%; 24-well plates coated with gelatin advance or cultured in 96-well plates to its daily observation of morphological change one day live cells on the basic covered with plates at the end the upper drift some rounded, not translucent, non-adherent dead cells. Activity adherent cells SHOP down completely, cells were ranging from large, polygonal, the volume becomes large, vacuolization of cytoplasm multi and lipid droplets, cells can be seen that two or more than two nuclei, 2 days and some neighboring cells rely extensor long pseudopodia are linked to each other, greater cell surface area, stretching more fully, a few days after the morphological changes until 7 days 7d cells gradually retracted, smaller size, reduce transparency, the culture supernatant More the emergence of the round, not translucent, the shedding of dead cells. The cells were collected daily culture supernatant detect albumin, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and virological indicators HBsAg and HBV-DNA expression. Found albumin level is the highest level of 2d ~ 5d; alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase beginning of a high level, is reduced to a stable level after two days; virological indicators HBsAg and HBV-DNA in decking 5d within basically be able to maintain a higher level; alcohol as oxidative stress injury factors observed alcohol HBV-Tg and wild-type Balb / c mouse primary hepatocyte damage sensitivity differences, as well as alcohol HBV-Tg expression in mouse primary hepatocytes of HBsAg, we found that the damage 16h after decking 24h primary hepatocytes treated with different concentrations of alcohol (0.13%, 0.5%, 2%, 8%), to detect the cell culture supernatant AST, two kinds mice AST levels overall trend with increasing alcohol concentration, the cells released into the medium AST increased gradually. Calculate the specific cell toxicity rate of the two groups of cells. The results are shown HBV-Tg mouse primary hepatocytes at 0.13%, 0.5% inhibition rate was significantly lower than the wild-type of Balb / c mice. Group 2%, 8% two cell toxicity was no significant difference. The observed alcohol HBsAg of HBV-Tg mouse primary hepatocytes expression of alcohol concentration of 0.13%, HBsAg expression level was significantly higher than the negative control group, a statistically significant difference. HBsAg expression decreases with increasing alcohol concentration. Alcohol concentration higher than 0.5%, no significant difference between the experimental group and negative group, but the expression of HBsAg levels lower than the negative control group. Prepared HBV/HLA-A2.1 dTg mice can stably expressing HBsAg, HBV-DNA and other virological indicators lymphocyte surface also have high levels of the human-derived MHC-I (HLA-A2.1) expression. We use recognized CTL specific epitope peptide HBc18-27 plus Th specific epitope peptide HBc128-140 as the experimental group, OVA257-264 as the OVA the control group, blank control group does not impose any experimental factors. The polypeptides of the different groups, respectively, with a non-complete Freund adjuvant, subcutaneous immunization of HBV-Tg, HLA-A2.1, HBV/HLA-A2.1 dtg three transgenic mice. After 5 weeks after immunization detected HLA-A2.1 HBV/HLA-A2.1 dTg of IFN-γ secretion of spleen cells of the mouse experimental group was significantly higher than that of HBV-Tg mice and HBV/HLA-A2.1 of the the blank of dTg mice, OVA control group; HBV/HLA-A2.1 dTg experiments with mice spleen cells CD3 / CD8 positive cells ratio was significantly higher than the OVA control group; doing the liver pathology HE staining found HBV/HLA-A2. 1 dTg mice experimental liver tissue similar to mild acute inflammatory response, while the other group of mice liver tissue reactions are relatively mild; observed with germline mice after immunization serological HBsAg and HBV-DNA changes found no significant difference. Conclusion First, we isolated hepatocytes step perfusion improved, the number of multi-effect decking, after two days, the liver cells adherent basic recovery due to collagenase digestion caused part of the damage of the cell membrane. And 5d, the liver cells maintained a good synthetic albumin ability decking 7d, the cells began to fall off, slowly lose normal function of the liver cells. Influencing factors with alcohol as a damage HBV-Tg mouse primary hepatocytes more sensitive to alcohol, and are more vulnerable to injury, and a certain concentration of alcohol will also promote the secretion and expression of HBsAg. These data with many articles related reports are consistent, thus confirming our culture HBV-Tg mouse primary hepatocytes for the alcohol combined hepatitis B infection liver damage and alcohol influence of hepatitis B virus replication . Second, we use the HBV specific epitope peptide immunized subcutaneously HBV/HLA-A2.1 dTg mice, the results confirm the immunological polypeptide activates the double transgenic mice specific immune cells, and may be generated on HBV replication the inflammatory response of the liver cells. Unfortunately, while the increased organism-specific immune response, did not find mice HBsAg and HBV-DNA decreased. The reason may be the expression of individual differences in mouse virological indicators, low expression levels, a slight change is not easy to be found, the other reason may be not enough to optimize the specific immune polypeptide composition. Characteristics of the double transgenic mice, and whether it is suitable for the treatment of hepatitis B vaccine preclinical study also requires further experiments and confirmed.

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