Establishment Of Animal Hepatocellular Carcinoma Model And The Study Of P53，p21, STAT3and CyclinD1Genes Expression

Objective:To Establish DEN-induced Hepatocarcinoma animal model and to observe thehistomorphology changes in rats during carcinogenic process；to detect the P53、p21、CyclinD1and STAT3genes transcription and protein expression level inDEN-induced liver cancer tissues of rats, and discuss their role and the possiblemechanism in the occurrence and development of hepatocellular carcinoma.Methods:1.The60rats were randomly divided into hepatocellular carcinoma model groupand control group, hepatocellular carcinoma model group has36rats and controlgroup has24rats, hepatocellular carcinoma model group drink0.1mg/mL DENfreely which configured with the sterile distilled water, Control group drinkingsterilized distilled water, drinking for20weeks; Each group were sacrificed6rats atthe11weeks and15weeks, at the20weeks all rats were killed.2.Using optical microscopy to observe the liver tissue morphology changesatdifferent points in time(at the end of11,15and20weeks);3.Reverse transcription-polymerase chain reaction (RT-PCR) was used to detectthe mRNA expression levels of p53, P21, CyclinD1and STAT3genes in normalgroup and DEN-induced Hepatocellular Carcinoma animal model group.4.Using Western blotting to detect the normal group and model group mice p53,P21, CyclinD1and STAT3protein expression levels；5. Using Immunohistochemical SP method to detect the protein expression levelsand location of p53, P21, CyclinD1and STAT3in normal group and DEN-inducedHepatocellular Carcinoma model group；6. Statistic analysis was performed with SPSS17.0to processing Experimentaldata and independent sample t test was used for Statistic analysis; to compare thecorrelation Between the various used Spearman rank correlation analysis (Spearmancorrelation coefficients test).The mean difference is statistically significant at thep-value＜0.05and p-value＜0.01level. Results：1. The general observation of liver tissue shows that with the extension of DENinduced time, the tissue surface begins to rough and darken, the texture hardens, theendge gets blunt, at the end of the20weeks liver Tissue surface appears the varioussizes of white lesions. The morphological change results show that as time goes on,the liver tissue damage, atypical hyperplasia and liver cirrhosis, there are3cases ofliver cirrhosis and8cases of liver cancer at the end of the20weeks.2. The analysis of Agarose gel electrophoresis of RT-PCR product showed thatthe average band intensity ratio of P53product was0.87±0.03in the Model group,when Compared with the control group (0.39±0.03), the difference was statisticallySignificant(p<0.01). mRNA transcription level of P21gene was0.51±0.05in modelgroup， when Compared with the control group(0.69±0.02), the difference wasstatistically Significant (p<0.05); mRNA transcription level of STAT3gene was0.31±0.02in model group, when Compared with the control group(0.05±0.008),thedifference was statistically Significant(p<0.01); mRNA transcription level ofCyclinD1gene was0.79±0.02in model group， when Compared with the controlgroup(0.66±0.03), the difference was statistically Significant (p <0.05).3. The western blot results showed that the expression of P53was0.45±0.16inmodel group, when compared with control group(0.2±0.09)the difference wasstatistically significant (p <0.01)；The P21protein expression was0.91±0.19, in modelgroup, when compared with control group (0.51±0.14), the difference was statisticallysignificant(p <0.01); The STAT3protein expression was0.58±0.08in model group,when compared with control group(0.37±0.09), the difference was statisticallysignificant(p <0.05); CyclinD1protein expression0.65±0.1in model group, whencompared with control group (0.42±0.11), the difference was statistically significant(p <0.05).4. The Immunohistochemistry results showed that P53, P21, STAT3andCyclinD1proteins positive expression rates in the liver cancer tissue animal modelwas73%、64%、73%and55%. The protein IOD sum value of P53was404.22±32.98In the model group, and73.01±6.2in control group, the protein IOD sum value of P21was279±22.51and6.81±2.03, respectively. the protein IOD sum value ofSTAT3was4878±55.08and2373±58.8, respectively. the protein IOD sum value ofCyclinD1was668.4±23.09and138.86±31.49, respectively; When compare theprotein expression level of each index in model group with the level of control group,the difference was statistically significant (p <0.01).5. Correlation analysis of positive expression rates showed that, P53and P21protein expression has no correlation (p=0.773r=0.297), positively correlated withCyclinD1(p=0.022r=0.297), negatively correlated with STAT3(p=0.032, r=-0.324); P21has negative correlation with CyclinD (p=0.018r=-0.354) and STAT3(p=0.031r=-0.494); STAT3has positive correlation with CyclinD1(p=0.034r=0.221).Conclusion:We have used DEN to induce hepatocellular carcinoma in rats for20weeks, andestablished hepatocellular carcinoma animal model successfully; The expression ofP53, P21, STAT3and CyclinD1genes in model group tissue was up regulated, thechange of P53, P21, STAT3and CyclinD1gene expression level may play importantrole in the DEN-induced liver cancer model.