| Objective:Based on the established HBV transgenic mouse model with pine mushroom extract (AMH), and a-interferon were applicated for the preventional chemotherapy using HBV transgenic mice in different groups, which aimed at evaluating the effects of AMH andα-interferon for the prevention of HBV transgenic mice oncogenesis in liver tissues. The influence of histology, biochemical, immunological changes, and other implications were explored in the HBV transgenic mice with oral AMH anda-interferon. Further explanation of the possible mechanism in liver oncogenesis with those drugs was investigated by inhibiting the HBV transgenic mice models with HCC. Method:Forty-six HBV transgenic C57BL/6J-TgN mice were randomly divided into four groups as follows; 1) AMHgroup; 2) a-interferon group; 3) a-interferon combined with AMH group, and; 4) control groups. There were 14 mice for each group, according to the different methods for preventive purpose. The blood was taken from the angular vein of an eye in the 11th month. The serum samples were measured for aspartate aminotransferase (AST). At the end of 12 months, each mouse model was weighed, and blood collected for further analysis. After then, all the models were sacrefied, and the liver tissues were stored in -80℃cryopreservation. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was tested for those of serum tumor necrosis factor (TNF-a), interleukin -2 (IL-2), Interleukin -10 (IL-10). The liver tissue from each mouse was taken and weighted. And then, the morphological changed from abnormal liver nodules, lung, spleen, kidney and lymph node tumors and metastases were observed under the. Part of the liver tissue was preserved in 10% formalin, and the remained part of the liver tissue immediately kept into the freezing tube in liquid nitrogen storage. Hepatic tissue Structural changes of liver tissues were microscopily observed in the above four groups, including non-cancerous tissue and oncogenesis tissue. It was necessary to identify the each occurrence of liver cancer in those mouse models in a specific amount and calculate in different groups. Several important immunohistochemistry tests were made in those liver tissues for analysis such as:1) Hepatitis B surface antigen (HbsAg); 2) transforming growth factorβ1 (TGF-(31); 3) transforming growth factorβ1 receptorⅡ(TβRⅡ); 4) phosphorylated Smad2 (P-Smad2); 5) Smad4, and; 6) Smad7. The result were observed and diagnosed by the microscope. Selected liver tissue were analyzed by the real-time fluorescence quantitative PCR detection (RT-PCR) such as:1) TGF-β1mRNA; 2) TβRⅡmRNA; 3) Smad2 mRNA. Results:1) Morphological changes in liver tissue:increased general form of the liver, swelling, liver tumor nodules as the white single or multiple nodules. Light microscope:diffuse swelling of liver cells, both cytoplasmic eosin staining (ground glass-like change), the nuclear bodies seen in eosinophils, liver cell necrosis, with infiltration of lymphocytes, scattered in the cytoplasm of a single liver stained with pyknotic deeply stained nuclear condensation. Obvious abnormalities were seen in the tumor mitotic cancer cells infiltrating to the surrounding liver tissues. After a little light on the performance level AMH plus a-interferon group had slightly lower percentage of cancer occurrence. However, it may not completely prevented from hepatic inflammatory changes and the incidence of cancer; 2) serum analysis. AST values in a-interferon group plus AMH group (215±64), a-interferon group (231±59) and AMH (294±51) group were lower than that in negative control group (322±45) (P< 0.05). AST values inα-interferon group, a-interferon plus AMH group AST values were lower than that in AMH group (P< 0.05); Serum IL-2 levels in a-interferon plus AMH group (58.43±14.42), AMH alone group (25.37±8.36), a-interferon group (35.84±12.36) were higher than that in negative control group (15.75±8.45) (P<0.05), in addition, IL-2 levels in a-interferon plus AMH group, a-interferon was significantly higher than the AMH group (P<0.05); compared with a-interferon group, IL-2 levels in a-interferon was increased (P<0.05); TNF-a level in a-interferon plus AMH group (12.03±6.54), AMH group (21.58±11.20), and a-interferon group (18.74±9.36) was significantly decreased than that in negative control group (45.97±14.03) (P<0.01); Serum IL-10 levels in a-interferon plus AMH group (160.25±70.03), AMH group (289.58±110.20), a-interferon group (240.69±95.36) compared with the negative control group (375.03±120.48) had decreased (P<0.05); serum IL-10 levels in a-interferon plus AMH group was lower than that in AMH group, a-interferon group (P<0.05); 3) Immunohistochemistry and RT-PCR analysis. AMH group (4.92±1.20), a-interferon group (4.57±1.09) and a-interferon plus AMH group (2.86±0.94) of liver tissue expression of TGF-β1 were lower than that in the negative control group (6.53±1.78) (P<0.05). In AMH, AMH group plus a-interferon group, and a-interferon group, the liver tissue expression of TGF-β1 showed significant changes (P<0.05); AMH group (3.51±1.78), a-interferon group (4.63±1.44) and AMH plus a-interferon group (5.72±1.55) in combination with Smad4 expression in liver tissues were higher than that in the negative control group (3.06±0.87) (P<0.05). AMH plus a-interferon group in combination with Smad4 expression in liver tissue was significantly higher than that in the AMH group (P< 0.05); AMH group (4.10±0.75), a-interferon group (4.57±0.93) and a-interferon plus AMH (5.38±1.25) in liver tissue P-Smad 2/3 expression was higher than that in the negative control group (3.36±0.62) (P<0.05). AMH plus a-interferon group in combination with P-Smad2/3 expression in liver tissues were higher than that in the AMH group, a-interferon group was statistically significant (P<0.05); AMH group (5.20±1.68), a-interferon group (5.49±1.85) and the a-interference plus AMH group (8.13±2.46) liver tissue expression of TβRⅡwere significantly higher than that in the negative control group (4.97±1.52) (P<0.01); AMH group, a-interferon group and a-interferon plus AMH group of liver tissue P-Smad2/3 were significantly lower than that in the negative control group (P<0.05). AMH group (5.19±1.91), a-interferon group (4.86±1.82) and negative control group (5.38±2.21) of liver tissue expression of Smad7 were higher than that in the a-interferon plus AMH group (2.17±1.64) (P< 0.05). The expression of TGF-β1mRNA in a-interferon plus AMH group was lower than that in the negative control group (P<0.05). AMH group plus a-interferon group with TβRⅡmRNA expression was higher than that in the negative control group (P< 0.05). AMH group, a-interferon group and a-interferon plus AMH group Smad2 expression were higher than that in the negative control group (P<0.05). Conclusion: 1) The process of HBV transgenic mice developing from chronic infection status to hepatocellular carcinoma formation can be in part to simulate that in humans; 2)AMH group in treatment of hepatitis B preventive C57BL/6J-TgNtransgenic mice could improve liver function and reduce levels of serum AST. It may inhibit the HBV virus detention in the liver cells, reduce the degeneration and necrosis of liver cells and slow the occurrence of oncogenesis; 3) AMH may increase the Thl-type cytokines levels and decreas the Th2 type cytokine levels in order to inhibit tumor formation; 4) AMH may reduce the incidence of hepatic cancer through influencing the TGF-β/Smad signal pathway transmission; 5) AMH plus a-interferon group showed better effective for the prevention of liver cancer in those animal models.
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