| Carcinogenesis and development of hepatocellular carcinoma(HCC) is a complicated process of multiple gene participated and multiple phase developed, analysis of gene expression will help the diagnosis of HCC.However,the single tumor marker has limitations on diagnosis because of unsatisfaction of sensitivity, specificity and practicability on apply of clinic.So the improvement of technology is demanded to aggregate analyze the mutiple genes in the process of carcinogenesis, and apply the markers scientifically and reasonable to diagnose HCC.AIM:To choose genes from literature and cDNA library constructed by SSH.On the basis of bioinformatics to pick out oncogenes,such as ANGPT2,BIRC5,CLDN10, E2F1,GPC3,HGF,MMP2,PIN1,SPINT2,TFDP1 and anti-oncogeneand,such as APC,DLC1,MGMT,P16,PRDM2,PTEN.Apply RT-PCR to detect the express levels from normal liver,hepatitis,liver cirrhosis to HCC.In conclusion,molecular diagnostic index was established to detect hepatocellular carcinoma using genes whose express level differs markedly from the normal tissue.METHODS:To choose 81 tissue samples from surgical resection at random including 5 normal liver,4 hepatitis,12 liver cirrhosis,60 HCC and paracancer, extract RNA,apply RT-PCR to detect the express level of 16 genes controlled by G3PDH.2-△△CT method was used to calculate the genes express quantity.and then establish the molecular diagnostic index according to the gene numbers of abnormal expression.ROC curve was used to evaluate the diagnostic value of molecular diagnostic index.The method of immunohistochemistry was used to analyze the consistency of GPC3 and HGF expression in mRNA and protein level of tissue, ELISA to analyze the consistency of GPC3 and HGF expression in mRNA and protein level of blood serum.RESULTS:The quantitation value of GPC3,E2F1 and BIRC5 increased gradually from HV,LC to HCC.General average of paracancer and cancer also increased gradually from HV,LC to HCC.MGMT,DLC1 and PRDM2 decreased gradually. Statistics analysis of mean quantitation value of genes in HV group,LC group, HCCgroup showed BIRC5,CLDN10,GPC3,TFDP1,DLC1,HGF,MGMT,PRDM2 has statistic significance.According to chi square test and quantitation,we choose GPC3,E2F1,BIRC5,MGMT,DLC1 and PRDM2 to develop MDI,the result showed MDI was 0 in NL abd HV group,0.83±0.83 in LC group,1.55±1.14 in N2 group, 4.42±1.20 in HCC group.MDI increased gradually from paracancer to cancer.The result of ROC curve showed when the diagnosis is more than 2,the sensitivity and specificity were satisfactory.Survival curve of low index is higher than high index and low index group had better prognosis.Compare the cases of AFP positive with the cases which index value were more than 2,the result showed the sensitivity of MDI was higher than AFP.The GPC3 result of immunohistochemistry showed 45 HCC sample was high expression out of 60,ELISA showed the expression level of HCC was higjher than NL.The HGF result of immunohistochemistry showed paracancer was stained stronger than HCC,ELISA showed the expression level of HCC was higjher than NL.CONCLUSION:The result of RT-PCR showed 8 genes quantitation value out of 16 was significantly difference in NL,HV,LC,HCC.6 choosed genes as MDI to detect HCC showed MDI bears high specificity and sensibility.MDI can be potential used to identify and detect early HCC effectively by analyzing the combination of more screening genes.CPC3 express level is coincidental in protein and mRNA.On the basis of RT-PCR and immunohistochemistry showed HGF express level in blood serum maybe excreted by paracancer.
|
PDF Full Text Request