| OBJECTIVE: To explore the potential relationship between calculus and cholangiocarcinoma through observing the effects of bile from patients with biliary calculi (CB) and that from patients with tumor of bile duct (TB) on the growth of human cholangiocarcinoma cell line (FRH-0201) .METHODS: One primary hilar cholangiocarcinoma cell line ( FRH-0201) already established and proved stable by us was selected and was cultured to the 160th generation, Clinical specimen were collected, of which CB (including choledocholithiasis > calculus of intrahepatic duct and cholecystolithiasis) , TB( including hilar cholangiocarcinoma and intrahepatic cholangiocarcinoma) and NB(normal bile) specimen were used for this study. The specimen were centrifuged (800r/min), degerminged through double-deck filter membranes with 0.22um orifice; Concentration Gradient dilution of bile was undertaken and cytotoxicity was tested to get the final concentration so as to eliminate the toxic effects of different bile to the cell; The proliferative effect of bile was measured by methabenzthiazuron (MTT) assay including the effects of different biles to FRH-0201 ( transverse )and that of different concentrations of the same bile( longitudinal ); Cell growth curve and doubling generation time were observed; Cell cycle, proliferation index, the ratio of S phase, GO / G1 phase and apoptosis were analyzed by flow cytometry; Cell clone formation was made; Light microscope and transmission electronmicroscope( TEM ) were used to observe the cell form and the internal structureo With these methods, the cytoactivity and reproductive activity of the cell treated by CB and TB was analyzed ?RESULTS: Cytotoxicity diseappears when the final concentration of each bile was diluted to 1 % , The FRH-0201 cell can grow faster with 1 % CB and 1 % TB, the doubling generation time was 17.9h(CB) ? 17.9h(TB) compared with NB (24.Oh) and the control group (24.lh); CB and TB significantly promoted the proliferation of FRH-0201 cells, especially in 48-72h, and the proliferation is time- and concentration-dependent; the cell clone was obvious with CB and TB; the respective cloning efficiency with 1%CB^ 1%TB, 1 %NB and the control group is (51.2±3.%)% > (50.0±2.9)%. (34.4±4.2)%, (33.9±3.6)% -, The percentage of S phase with 1 % CB [(58.4±3.05)% ]and 1 % TB [(46.6±0.35)%] for 48h increased remarkably ( P<0.05) compared with the control group [(29.75±1.77)%], and the percentage of G0/G1 phase with 1% CB [(39.55±4.75)%] and with 1%TB [(35.33±3.O3)%] for 48h decreased remarkably ( P<0.05) compared with the control group [(69.75±1.06)%]o Proliferative index of FRH-0201 increased significantly after being treated with 1%CB [(59.88±3.98)%] and with 1%TB [(62.43±3.90)%] for 48 h ( PO.05) compared with the control group [(30.25±1.07)%]<, FRH-0201 was affected in the way of changing the cell cycle and cell apoptosis by TB and CB?CONCLUSION: CB and TB can promote the proliferation of human cholangiocarcinoma cells (FRH-0201)except NB, they function by altering the cell cycle and the cell apoptosis; CB and TB have the tumor promoter activity, which may account for the high incidence of cholangiocarcinoma among patients with biliary calculi and bile tract neoplasms- For patients with cholangiocarcinoma that is unresectable or that is incompletly resected, the tumor grows faster, thus, chole-entero by-pass should be made to avoid the direct contact of bile and tumor and to avoid the promotion of TB to theproliferation of the tumor ?...
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