| OBJECTIVE: To verify the genes showing differential expression in aflatoxin B! (AFBO-induced tree shrews' hepatocellular carcinoma (HCC) which were screened by cDNA microarray and discuss the role of these genes in hepatocarcinogenesis, through investigating the expression of SOD1, GSTA1, SCF, TPO, etc. in tree shrews' tissues of HCC, para-carcinoma and pre-HCC*as well as human samples of HCC and para-carcinoma, by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical methods.MATERIAL AND METHOD: Part 1, experiment on tree shrews: (1) Tree shrews were divided into two groups. The experimental group was treated with AFB1 and the control group was not. (D The mRNA expression of SOD1, GSTA1, SCF, TPO, IAP1, TMSB10, NOP120 in the tissues of HCC, para-carcinoma and pre-HCC of 11 animals that developed HCC were assayed the method of semi-quantitative RT-PCR. (D The DNA sequences of SOD1, GSTA1, SCF fragments which were amplified by RT-PCR were analyzed. @ The expression of SOD1 and GSTA1 protein in the tissues of HCC, para-carcinoma and pre-HCC from the HCC-developed animals, 120th-week biopsies from the animals no HCC developed in the experimental group, and 45th- and 120th-week biopsies from the animals in control group were examined with immunohistochemical S-P method. Part 2, experiment on human samples: (D The mRNA expression of SOD1, GSTA1 in 35 human HCC and para-carcinoma liver tissues were examined with the method of RT-PCR. (D The protein expression of SOD1 and GSTA1 in human HCC and para-carcinoma tissues were examined with immunohistochemical S-P method. (D The relationship between SOD1, GSTA1 protein expression and clinical-pathological parameters such as age, size, number and differentiation of* pre-HCC: 45* -week of the experiment, before HCC developed (the first HCC developed at 88th-week of the experiment).the tumor, serum AFP level, degree of cirrhosis, HBV infection, portal vein cancer-embolus, tumor capsule invasion, recurrence and metastasis were analyzed. RESULTS: Part 1, experiment on tree shrew: (1) HCC occurred in 64.5% (35/54) of tree shrews in experimental group;(1) The result of RT-PCT showed that the mRNA of SOD1, GSTA1, SCF, TPO, IAP1, TMSB10 and NOP120 were detected in all HCC, para-carcinoma and pre-HCC from the 11 HCC-developed animals. The expression level of SOD1 mRNA in HCC tissues was significantly lower than that in para-carcinoma and pre-HCC (1.200±0.926, 3.143 ± 1.134, 2.945 ± 1.432 respectively, all p < 0.01). The expression level of GSTA1 mRNA in HCC tissues was also significantly lower than that in para-carcinoma and pre-HCC (1.149 ± 0.320, 2.247± 1.201, 2.660±1.324 respectively, all/? < 0.05). The levels of SCF in HCC, para-carcinoma and pre-HCC increased by turns (0.201±0.123, 0.279 ± 0.147 and 0.390±0.149 respectively), of which the level in HCC was significantly lower than that in pre-HCC (p=0.003). The expression level of TPO mRNA in HCC, para-carcinoma and pre-HCC also increased by turns (0.161±0.096, 0.196 ± 0.110 and 0.231±0.046 respectively), of which the level in HCC were significantly lower than that in pre-HCC (p=0.045). There was no significant difference in mRNA expression levels of HIAP1, TMSB10 and NOP120 among HCC, para-carcinoma and pre-HCC (all p > 0.05). (3) DNA sequence of SOD1, GSTA1 and SCF, which were amplified from tree shrew's liver tissue, were similar to that of human correspondent genes with 89%, 89% and 90% homology respectively. (1) Immunohistochemical examination showed that the general scores of expression of SOD1 protein in HCC, para-carcinoma, pre-HCC and non-HCC 120th-week liver tissues of experimental group were 1.514 ± 1.084, 3.075 ±1.559, 2.800 ± 2.015 and 2.640±1.188 respectively. The general scores of SOD1 protein expression in 45th- and 120th-week liver tissues of control group were 3.075 ± 1.482 and 2.517±1.677 respectively. The general score of SOD1 protein expression in HCC was significantly lower than that in para-carcinoma, pre-HCC liver tissues, non-HCC 120th-week o... |
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