Establishment Of Ascites Model Of Human Hepatocellular Carcinoma Hepg2 In Nude Mice And Study On Its Effect On Hepg2

Background and objective: Hepatocellular carcinoma(HCC)is one of the most common and refractory malignant tumors in human body,and corresponding five-year survival rate is relatively low,especially regarding advanced HCC with ascites.Meanwhile,tumor microenvironment can promote malignant progression of tumor cells and shorten the survival time of patients.For that reason,following the establishment of a transplanted tumor model of HCC in nude mice with the presence of ascites and using fluorescent to display tumor microenvironment,this paper attempted to investigate the mechanism of tumor microenvironment that might promote malignant progression of tumor cells.Materials and methods: HCC cell line Hepg2 in logarithmic growth phase purchased from the cell bank of Chinese Academy of Sciences was injected into the micro-injection needle at a concentration of 1×106 cells/50?L PBS;green fluorescent nude mice bred at our laboratory were anesthetized with chloral hydrate,small incision was then made in the right upper quadrant of the mouse,followed by direct injection of a needle into the left lobe of the liver under direct vision;subsequently,the above prepared Hepg2 was injected using micro-injection pump within 15 min to ensure that Hepg2 was localized in the hepatic tissue completely.Results and analysis: A total of 11 rats were inoculated,among which 1 mouse was treated in the early stage of Hepg2 inoculation,and was found to have Hepg2 localized in the liver without the formation of ascites;remaining 10 mice were treated in the late stage of malignant tumor with massive hemorrhagic ascites formation,ascites routine test and biochemical examination showed malignant ascites.Cells of ascites were obtained and cultured in vitro,early Hepg2 coexisted with host green cells,afterwards,the number of green cells declined with passage increased.All intraperitoneal organs(including peritoneum and mesenterium)except ascites were confirmed by pathological examination,malignant cells with dense arrangement,dark stained nucleus and evident mitotic activity were filled within tumorous nodules;tumor necrosis and bleeding were a bit more common,accompanied by host cell infiltration with green fluorescent protein mostly.In addition,2mice were observed to have Hepg2 metastases in the lung.Otherwise,in Hepg2-1 cells which were monoclonal from ascites microenvironment,the expression rate of Oct4,Nanog and CCN was significantly higher than that of Hepg2 cells,and the cell proliferation rate,invasion and migration ability of Hep G2-1 cells were higher than that of Hepg2 cells.Only the expression of AFP was not significant.Conclusion and prospect: In situ inoculation of Hepg2 in the liver of nude mice with green fluorescent protein in all its cells can be used to replicate a highly invasive and widely spread tumor model,and can also be associated with a 100%(10/10)cancerous ascites formation.Meanwhile,whether in solid tumor tissue or ascites,Tumor cells and tumor microenvironment cells that grow along with tumor cells and provided by the host can be differentiated according to whether there is green fluorescence or not.Under the influence of tumor microenvironment cells,Hepg2 cells which don't emit green fluorescence with high expression of Oct4,Nanog and CCN,become more malignant than the control.These results lay the foundation of animal and cell experiments for further study on the cellular and molecular biological mechanisms of tumor microenvironment promoting tumor malignant progression and target molecular interference.