Study On Molecular Pathology Of Hepatocelluar Carcinoma In P21～(HBsAg/HBsAg) Transgenic Mice

Hepatocelluar Carcinoma(HCC) is one of the leading malignancies worldwide. More than 1 million of people in the world suffered from HCC every year and 5 year survival rate is lower than 5%. A number of etiological factors, particularly hepatitis B virus (HBV) infection, are involved in the development and progress of HCC. HBV is a highly specific virus, generally infects neither cultured cells nor ordinary experimental animals. This has impeded the research on HBV to some extent. Therefore it is necessary to establish an appropriate animal model for further studies on the pathogenesis and molecular mechanisms of HBV-induced HCC.Yang Xiao's team established a HBV gene knock-in transgenic mouse containing the complete sequence of HBsAg gene. HBsAg was introduced into p21 locus of the mouse by homologous recombination and ES cell culture. The transgenic mice that can express HBsAg steadily were obtained by microinjection and embryonic transplantation. The integration of HBsAg gene was confirmed by Southern Blot and in situ hybridization. HBsAg mRNA could be detected in the liver, kidney and testis of the transgenic mice. Both heterozygous and homozygous transgenic mice could survive normally after birth. The mice were denominated as p21^HBsAg/HbsAg transgenic mice.Here the molecular pathology of the liver and hepatic tumors in p21^HBsAg/HbsAg transgenic mice was investigated.The pathological changes in the liver of p21^HBsAg/HbsAg transgenic mice were aggravated by month. The liver turned to be dark and sclerotic with scatteredformation of nodules and tumors. The tumors were nodular, scattered and massive. Lymph nodes surrounding hepatic portal and mesentery became swollen.The biochemical parameters were assistance to estimate hepatic injuries. The activities of AFP, ALT and AST were significantly higher than those in wild control mice while CHO, TG, TP were lower. These changes were similar to those observed in human chronic hepatitis, indicating that in the early stage metabolic changes in liver had developed and hepatic injuries might have occurred in the liver of HBV transgenic mice.Light microscope examination showed that liver cells with HE staining were swollen and infiltrated with inflammatory cells. Fat degeneration arose with punctuate, focal and crumblike necrosis, atypical hyperplasia and finally HCC developed. Hepatpocarcinoma cells were highly or moderate differentiated, forming cord-like and acinar-like structure. The nuclei of cancer cells were dark stained with karyokinetic figures. HCC was found in 31 cases of 18 month old p2iHBsA8/HBsAg transgenic mice (31/60), and remote lymph node metastasis developed in 2(2/31). 44 cases of HCC in 24 month old mice (44/60), of which, 4 lymph node metastasis (4/44). Metastasis of HCC in elderly transgenic mice was here first reported in the world.The ultrastructure examination on the hepatocarcinoma cells of 18 month old p2jHBsAg/HBsAg transgenic mice showed that the proportion of nuclei in plasma did not increase and the nuclei were irregular. There were two or more nucleoli and inside the nuclei were pseudoinclusions, with invaginated karyotheca. Mitochondria became enlarged and round with thinner matrix, shorter and fewer ridges. The hepatocytes looked smooth and were adjacent closely but not connected. In 24 month old p2|HBsAg/HBsAg tfausggjug mjc^ me nuclei of hepatocarcinoma cells were irregular, lobular or multiple. The proportion of nuclei in plasma increased and the karyotheca were invaginated and heterochromatin increased and condensed under the karyotheca.Mitochondria degenerated, containing more glycogen granules. Laminar inclusions were seen in some of the cancer cells, which were similar to the ultrastructure of hepatocarcinoma cells in human.Under transmission electron microscope(TEM) nucleo-chromosome agglutinated to the margin with irregular shape and the nuclear membrane was accidented in early stage of apoptosis. In middle stage, the chromatin in the nuclei condensed to the margin in the shape of crescent. The nuclear pores disappeared and the nuclear membrane crimpled as ripples. In late stage of apoptosis the nuclei became pycnositic and the cell membrane sprouted and formed vacuoles and apoptosis bodies. All these changes in morphology of cells were the characteristics of apoptotic cells.Flow cytometry detection showed that apoptosis rate of the hepatocytes in p2jHBsAg/HBsAg transgenic jujgg was iower than that of wild mice of the same months. Apoptosis rate of the hepatic carcinoma cells was higher than that of hepatocytes in p2jHBsAg/HBsAg transgenic mjce of the same months. The apoptosis rate of hepatic carcinoma cells in p2iHBsAg/HBsAg transgenic mice was high, as confirmed by the two methods.Immunohistochemistry study revealed that p53 positive signal appeared in the nuclei of hepatocytes and hepatocarcinoma cells of p21 HBsAe/HBsAs transgenic mice as brown or yellowish brown. The positive cells were locally or extensively distributed. The expression of p53 tended to be enhanced in the development of hepatocarcinoma in p21 HBsAg/HBsAg transgenic mice. The expression of p53 in cancerous cells was stronger than that in para-tumor tissues and liver tissue, indicating that p53 was involved in the development of HCC. MMP-2 were largely located in the plasma of hepatocarcinoma cells of p2iHBsAg/HBsAg transgenic mice, but not expressed in the liver of p2iHBsA8/HB!|Ag transggnjc mjce ^^ w^ mjce jhe expression of MMP-2 was stronger in liver cancer cells with lymph node metastasis than that without lymph nodemetastasis, implying that the high expression of MMP-2 was associated with HCC metastasis of p21HBsA8/HBsA8 transgenic mice. The result was consistent to the study on the role of MMP-2 in human HCC.The results here showed that p2iHBsAg/HBaA? transgenic mice manifested pathologic pictures, physical and biochemical indexes and molecular changes similar to those observed in human HCC patients. p2iHBsAg/HBsA8 transgenic mice were ideal animal model for investigate the role of HBsAg in HBV-related HCC, especially theearly molecular events.