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We sought to determine the expression of molecular markers in an animal model of cholangiocarcinoma compared with those in human cholangiocarcinoma.Cholangiocarcinoma is a rare disease characterized by early intrahepatic and extrahepatic spread, which seriously limits the efficacy of surgery. Establishing an experimental model to study the cholangiocarcinogenesis is desirable.Sprague-Dawley rats weighing 300 ± 50 g were used for the study group. The animals were given 0.3% thioacetamide in tap water continuously. Thirty mass-forming peripheral cholangiocarcinoma patients also were studied. Expression of epidermal growth factor receptor (EGFR), MUC1, MUC2, MUC5AC, MMP-2, MMP-9, and p53 in both human and experimental rat cholangiocarcinoma was examined using immunohistochemistry.Using thioacetamide 0.3% as a hepatoxin to induce cholangiocarcinoma in rats, microfoci of cancerous cells were detected from 12 weeks, and all experimental rats displayed diffuse mass-forming cholangiocarcinoma after 24 weeks. EGFR was strongly expressed in 14 (47%) of 30 human cholangiocarcinoms and 24 (100%) of 24 rat cholangiocarcinomas, respectively. MUC1 was strongly expressed in all human and rat cholangiocarcinomas, whereas MUC2 and MUC5AC were focally and weakly expressed. MMP-2 and MMP-9 were strongly expressed in 22 (73%) of 30 human cholangiocarcinomas and 24 (100%) of 24 rat cholangiocarcinomas, respectively. p53 overexpression was detected in 9 (30%) of 30 human cholangiocarcinoma and none of the rat cholangiocarcinoma, respectively.The expression of EGFR, apomucins, MMPs, and p53 in rat cholangiocarcinoma was strongly homologous to human cholangiocarcinoma. Thioacetamide-induced cholangiocarcinoma in rats provides an excellent model for investigating cholangiocarcinogenesis in vivo.