| Objective:Establish an experimental animal model for studying the invasion and metastasis of hepatocellular carcinoma,and detect the expression of HCC tumor markers,epithelial mesenchymal transformation markers and lymphocytes around the cancer nest in the metastasis model,so as to further explore the changes of tumor microenvironment of metastatic HCC.Methods:(1)Construction of animal models on HCC metastasis:Hep G2 cells were cultured and the concentration of Hep G2 cells was adjusted to 2×10~7 cells/m L.Thepurchased 20 BALB/c-nu mice were randomly divided into the experimental group and the control group,with 10 mice in each group.The experimental group was injected withHep G2 cell suspension 0.2m L(2×10~7 cells/m L)through the tail vein,and the control group was injected with the same amount of normal saline through the tail vein.The mice were weighed regularly,and the active state,mental state,diet,skin color changes and so on were observed regularly.(2)HE stained:the mice were killed,the common metastatic organs and tumor forming tissues of HCC were stained with HE,the metastasis of HCC was observed,and the growth and distribution characteristics,invasion and migration ability of Hep G2cells were analyzed.(3)Immunohistochemistry:detection of alpha-fetoprotein(Alpha-fetoprotein,AFP),arginase-1(Arginase-1,Arg-1),phospholipid proteoglycan-3(Glypican-3,GPC-3),hepatocyte paraffin antigen-1(Hepatocyteparaffin-1,Hep Par-1),Epithelial cadherin(Epithelial cadherin,E-cadherin)and Neural cadherin(Neural cadherin,N-cadherin)in tumor tissues and metastatic organs.The expressions of CD4~+T lymphocytes,CD8~+T lymphocytes,CD20~+B lymphocytes and interleukin-2(IL-2)around the metastatic tumor nest were detected.(4)Observation under microscope:the infiltration oflymphocytes around the cancer nest.(5)Statistical analysis:SPSS 18.0 statistical software was used to analyze the obtained data.Results:(1)An animal model of hematogenous metastasis of HCC was successfully established:during the injection of tumor cells into the mice in the experimental group,one mouse died due to the rapid injection.The remaining mice had a model survival rate of 90%and a tumor formation rate of 89%.Hep G2 cells can metastasize to the liver,lung,spleen,kidney,adrenal gland and invade the bone and abdominal wall through the tail vein,the liver metastasis rate was 44%,the lung metastasis rate was 78%,the kidney metastasis rate was 11%,the adrenal metastasis rate was 33%,and the spleen metastasis rate was 22%.(2)HE staining results:There are a large number of cancer nests in the liver,the cancer cells are of different sizes and shapes,the cell atypia was extremely obvious,and the pathological mitotic figures are more common.The cancer cells were arranged in nests,and the interstitium contained a large number of lymphocytes infiltrated.(3)Immunohistochemical results:AFP,Arg-1,GPC-3,and Hep Par-1 are all expressed in the metastasis tissue of HCC metastasis model,but the positive rates vary.The strong positive rate of AFP was 33%,the strong positive rate of Arg-1 was 89%,the strong positive rate of GPC-3 was 78%,and the strong positive rate of Hep Par-1 was 89%.Weakly positive expression of N-cadherin and negative expression of E-cadherin in HCC metastatic tissues;CD20~+B lymphocytes and IL-2 were positively expressed in the lymphocytes around the cancer nest of HCC metastatic tissue,CD4~+T lymphocytes were weakly positive,and CD8~+T lymphocytes were negatively expressed.Conclusion:(1)The expressions of AFP,Arg-1,GPC-3 and Hep Par-1 in HCC metastases suggest that the combination of multiple indicators has a certain clinical reference value for the diagnosis of HCC.(2)It was confirmed that the change of tumor microenvironment is an important factor affecting the invasion and metastasis of HCC. |
