The Study Of The Establishment Of Hepatoma Model In Green Flurescent Protein Transgenic Mouse And Its Change Of Fluorescence Protein

Background and ObjectiveHepatocellular carcinoma(HCC) is a malignant carcinoma with poor prognosis.Five years survival rate is under5%.The mobility of HCC in all human malignant tumor around the worldwide is about5.6%.The incidence in china is higher, about30.3/100000, especially in fusui of Guangxi province and Qidong of Jiangsu province. Etiology and pathogenesis of HCC is still unclear.The one of the reasons may be the lack of an ideal experimental animal model of HCC.Until now, HCC model has been established in the following animals:rat, rabbit, dog, tree shrews. The chemical induced HCC models established include spontaneous HCC model, chemical induced HCC model, transplanted HCC model and transgenic HCC model. Among them, the chemical induced and transplanted models are widely used. The chemical drugs which are used for inducing HCC are aromatic amines, nitrosamines and yellow aspergillin and so on. Among them the most commonly used is diethylnitrosamine. Recently, transgenic animals have been used for the animal model, especially GFP transgenic mice. However, HCC model in GFP transgenic mice has never been reported.We would try to establish a kind of HCC animal model, using GFP transgenic mice, And to explore whether existing or losing of GFP in the carcinogenesis of HCC model.MethodsSixty of6-9weeks old male GFP transgenic mice (weight27.71±1.88g), were randomly divided into2groups:50in experimental group,10in control group. GFP transgenic mice in DEN/CC14/ethanol/DEN experimental group (50mouse) were given i.p injection of DEN as an initiator of liver carcinogenesis at a dose of100mg/kg body weight in the first week. Experimental groups were given gastric lavage of5%ccl4at a dose of0.05mL/10g,2/W from3weeks to7weeks. At the same time, experimental groups were given9%ethanol in the drinke of water for20weeks. Experimental groups were given gastric lavage of10%ccl4at a dose of0.05mL/10g,2/W from8weeks to12weeks; Experimental groups were given gastric lavage of20%ccl4at a dose of0.05mL/10g,2/W from13weeks to15weeks; Experimental groups were given i.p injection of DEN as an initiator of liver carcinogenesis at a dose of50mg/kg body weight in the16week;There was no drug using from17-20weeks; Control group(10mice) was given a single i.p. injection of sodium chloride as the same volume as DEN solution in the frist week; Control group (10mice) was given gastric lavage of sodium chloride at dose of0.05mL/10g,2/W from3weeks to15weeks, and was given a single i.p injection of sodium chloride as the same volume as DEN solution in the16week. The weight change of the mice in two groups was monitored weekly; Experiment group GFP transgenic mice were sacrificed in4,12,16,20weeks and its liver tissue were made into frozen section in order to observe the fluorescence expression; Its tissue were dynamic observated pathologic routine in HE staining; The mice were sacrificed in the twentieth weeks in order to culture liver tumor in primary;The fluorescence was expressed and distributed in the tumor cells.The tumor in the GFP mice liver which was induced in twentieth weeks was transpanted in nude mice;The aim was to observe the change of the fluorescence in the nude mice.ResultsFifteen of GFP transgenic mice died in the experimental group,and the mortality rate was30%(15/50). None of GFP transgenic mice died in the control group. At twentieth weeks, liver cancer incidence rate of GFP transgenic mouse in DEN/CC14/ethanol/DEN experimental group were77.14%(27/35), no liver cancer was found in the control group. The morphology of hepatitis, cirrhosis, precancerous lesions, carcinogenesis in GFP transgenic mice were observed in experimental group in4,12,16,20weeks. Fluorescence was existed in liver frozen sections which were taken from GFP transgenic mice in4,12,16,20weeks. The cytoplasm and nuclei of primary cultured liver tumor cells which were taken from the liver cancer in experimental group expressed GFP. Fluorescence was observed in the tumor transplantated experiments until1.5months.ConclusionsLiver cancer models could be succefully established in GFP transgenic mouse by using DEN/CC14/ethanol/DEN. Green fluorescent protein could be remained in the liver cells or GFP transgenic mouse model of hepatocellular carcinoma.