Establishment Of HepG2 Cell Heterotopic Tumor Model In Mice And Study Of Gene Nanog And Oct4 Expression

Objective:The heterotopic tumor model of HCC in mice was established for comparative analysis.The tumor-forming ability and the invasiveness of liver cancer cells were observed.The histopathological characteristics of ectopic tumor formation and the expression of Nanog and Oct4 in different tissues and cells were preliminarily discussed.To provide reference for further study of hepatocellular carcinoma stem cells and establishment of animal models.Methods:1.Twelve male healthy BALB/c-nu mice were purchased and randomly divided into experimental groups(n=6)and control groups(n=6).The experimental group:The HepG2 cells in logarithmic growth phase were taken to adjust the cell concentration to2×10~7/m L,and then the HepG2 cells of 0.2m Lwere injected into the armpits of BALB/c-nu mice.The control group:The same amount of normal saline was injected into the armpit of BALB/c-nu mice.After modeling,the two groups of mice were routinely raised in an SPF-class aseptic laminar flow animal breeding room.The growth of the tumor was observed daily.The hair,mental status and diet of the mice was observed daily.The weight of the mice was weighed and recorded regularly.The mice of the experimental group were dissected to observe the tumor metastasis.2.Taking fresh tumor tissue for hematoxylin-eosinstaining and immunohistochemistry.Observing the cytological characteristics of the liver cancer tissue and the expression of AFP,Arginase-1,Glypican-3 and Hep Par1.3.Then RT-PCR was used to detect Nanog and Oct4 m RNA’s relative expression in heterotopic tumor tissues,HepG2 cells,and LO2 cells.4.The data obtained from the detection is processed by statistical software SPSS18.0.Results:1.The heterotopic tumor model of HepG2 cell in mice was successfully established.The BALB/c-nu mice of 6 in the experimental group all formed tumors,and the rate of tumor formation was 100%.Before modeling,the weight of mice in the experimental group was(18.14±1.06)g and the weight of mice in the control group was(18.27±1.30)g.There was no significant difference in body weight(P>0.05).After modeling,the weight of the mice in the experimental group gradually increased,reaching the maximum value of(25.24±1.24)g on the 35th day,and then showed a downward trend,with the weight value of(24.27±1.21)g on the 40th day.The weight of the mice in the control group also gradually increased,but there was no significant increase in the control group.The weight value was(22.17±1.32)g on the 35th day,and there was no decrease thereafter,and the weight value was(22.34±1.15)g on the 40th day.After modeling,the difference in body weight between the two groups was statistically significant(P<0.05).There were significant statistical differences in the body weight of mice in the experimental group among the three time points(P<0.05).However,there was no statistically significant difference in the body weight of the mice on the 35th and 40th days in the control group(P=0.874>0.05).The maximum diameter of the tumor nodule is(18.26±2.79)mm,the volume of the tumor nodule is(1723.78±896.90)mm~3,and the weight of the tumor nodule is(1.26±0.52)g.In the later period,the mice in the experimental group had dark skin,and their mental status,activity and diet were significantly worse than those in the saline control group.None of the mice in the experimental group had metastasis in vivo.In the process of modeling,all the mice in the experimental group survived with a survival rate of 100%.2.The hematoxylin-eosinstaining of HepG2 tumor tissues showed that cancer cells are of different sizes and morphologies,with obvious nuclear atypia,obvious nucleoli,more frequent abnormal mitotic images,rich cytoplasm,and cancer cells arranged in nests,with many blood vessels and few interstitial cells.There are many lymphocytes infiltrating in the interstitial space.Immunohistochemistry showed that AFP was positive in cytoplasm with a positive rate of 50%,Arginase-1 was positive in cytoplasm and nucleus with a positive rate of 90%,Glypican-3 was positive in cytoplasm with a positive rate of 80%and Hep Par1 was positive in cytoplasm with a positive rate of 90%.3.The expression level of gene Nanog and Oct4 m RNA in heterotopic tumor tissues and HepG2 cells were higher than the expression level of gene Nanog m RNA in LO2 cells,and the difference was statistically significant(P<0.05).Conclusion:1.A HepG2 cell heterotopic tumor model in mice was successfully established,that was confirmed by HE staining and immunohistochemical detection of characteristic indicators of hepatocellular carcinoma.2.The transcription levels of stem cell genes Nanog and Oct4 were significantly increased in the HepG2 cell heterotopic tumor model in mice.It is suggested that this model can be used for the related research of hepatocellular carcinoma tumor stem cells.