Cytology Of Biliary Exfoliative Cells And Expression Of Genes Associated With Carcinogenesis Of Cholangiocarcinoma In Biliary Cells In Bile Drainage

Background and objectives:Cholangiocarcinoma is a malignant biliary tumour arising from cholangiocytes,the epithelial cells lining in the bile duct.Owing to the lack of sensitive and specific methods of diagnosis,cholangiocarcinoma usually is advanced disease which could not been resected and has a poor prognosis when it is diagnosed.How to diagnose the early cholangiocarcinoma and to improve the patient survival rate are the study focus currently.We studied the biliary cells morphology in the bile drainage,the cell growth in culture and the expression of genes associated with carcinogenesis of cholangiocarcinoma(CDX2,MUC1,MUC2,MUC3,MUC5AC,TFF1),for understanding the biological behaviour of cholangiocarcinoma.Our study provides evidences of the mechanism of cholangiocarcinogenesis at cellular and molecular levels. This may benefit to the early diagnosis of cholangiocarcinoma.Methods:1.Collecting the biliary exfoliative cells from the bile drainage in patients with obstructive jaundice using different centrifugalization methods.The cell morphology was observed by cell smear and by Wright-Giemsa's and Hoechst33258 staining.2.Establishing a method for culturing biliary cells in vitro.3.Extracting total RNA from the biliary exfoliative cells in the bile drainage.Comparing the expression of genes associated with carcinogenesis of cholangiocarcinoma(MUC1,MUC2,MUC3,MUC5AC,TFF1,CDX2) in cholangiocarcinoma and in cholangiolithiasis by semiquantitative RT-PCR to research the mechanism of cholangiocarcinogenesis at cellular and molecular levels.Results:1.There were considerable a mount of biliary exfoliative cells collected from a six-hour bile drainage by using filtration or gradient centrifugalization methods ex vivo.The biliary cells from the bile drainage maintained the original morphology of the cells.The morphology of cells from benign or malignant biliary diseases was distinguishable.2.The biliary exfoliative cell in the cholangiocarcinoma bile drainage could be grown from several days to one month in regular culture system in vitro.The cells in long term cultured maintained the characteristics of cholangiocarcinoma.3.The biliary exfoliative cells from the cholangiocarcinoma bile drainage expressed high level of MUC1,MUC2,CDX2 mRNA, compared with these in the biliary cells from the cholangiolithiasis bile drainage(p＜0.01).They all expressed MUC5AC and TFF1 mRNA,but the level in the biliary cells from the cholangiocarcinoma bile drainage was higher than that in the biliary cells from the cholangiolithiasis bile drainage(p＜0.01).MUC3 mRNA expression could be only detected in cells from the cholangiolithiasis bile drainage.Conclusion:1.We increased the number of the biliary exfoliative cells collecting from the bile drainage by improving the method of collecting and staining. The sensibility and specificity were enhanced in our cytopathological method.The cells in the bile drainage of both benign and malignant biliary diseases were in a considerable large amount with characteristic morphology.They could be used as a tool in differential diagnosis in clinic and as an important pathway of understanding the mechanism of cholangiocarcinogenesis at cellular and molecular levels.2.The biliary exfoliative cell in the cholangiocarcinoma bile drainage could grow in regular culture system in vitro.Part of the cells in long term cultures maintained malignant tumour cell characteristics. Cholangiocarcinoma cells culture in vitro provided a new approach of researching the mechanism of cholangiocarcinogenesis.3.MUC1,MUC2,MUC3,MUC5AC,TFF1,CDX2 genes,which primary express in gastrointestinal tract mucosa,aberrantly expressd in the biliary cells from the cholangiocarcinoma bile drainage.The levels of these genes expressed higher in the biliary exfoliative cells from the cholangiocarcinoma bile drainage than in the biliary exfoliative cells from the cholangiolithiasis bile drainage.Aberrant expression of these genes may play a rule in the mechanism of cholangiocarcinogenesis.