| Background The improvement of early diagnostic technique is one of the critical factors to elevate cholangiocarcinoma therapeutic effect. We have confirmed that human FXYD6 is related with cholangiocarcinoma differentiation in transcription level. By gradually understanding the relation between human FXYD6 and cholangio- carcinoma, a new tumor marker for early cholangiocarcinoma diagnosis can be provided, meanwhile we can know more about the carcinogenesis and develop-ment of cholangiocarcinoma.Objective 1. To prepare the rabbit anti-human FXYD6 polyclonal antibody. 2. To validate the differential expression of hFXYD6 mRNA and protein among normal bile duct, high grade cholangiocarcinoma and low grade cholangiocarcinoma. 3. To investigate the effects of human FXYD6 antisense on the proliferation and invasion of human cholangiocarcinoma cells.Methods 1. B cell etitope analysis, polypeptide design, prokaryotic expression, protein purification, animal immunization and antibody purification were done to prepare rabbit anti-human FXYD6 polyclonal antibody. 2. SYBR?Green I fluorescence quantitation Real Time PCR and immunohistochemistry were used to validate the differential expression of hFXYD6 mRNA and protein among different differentiated bile duct carcinoma. 3. Antisensenueleic acids technique was used to investigate the relation between hFXYD6 and cholangiocarcinoma cells proliferation and invasion. Human cholangiocarcinoma cell line QBC939 was transfected with the plasmid expressing human FXYD6 antisense. Meanwhile, the empty vector and non-transfection group were designed. The mRNA transcription level of hFXYD6 was assayed by real-time reverse transcriptase polymerase chain reaction with SYBR Green I, and the hFXYD6 protein expression was detected by immunohistochemistry. MTT and the colony-forming assay was used to measure the ability of cell growth. The cell cycle distribution was analysed by flow cytometry and Transwell chamber model was employed to test the ability of cellinvasion in vitro.Results 1. High titer rabbit anti-human FXYD6 polyclonal antibody was successfully prepared with ELISA and immunohistochemistry verification. 2. Real Time PCR showed that hFXYD6 was expressed in normal bile duct, high grade cholangiocarcinoma and low grade cholangiocarcinoma; the level of high grade was higher than normal group and low group than high group. The immunohistochemistry result was the same. 3. In comparison with the cells transfected with empty vector or without transfection, QBC939 cells transfected with hFXYD6 antisense had a significant decrease in mRNA transcription and protein expression. The cell doubling time was augmented (46.8 h vs 34.5 h, 35.3 h), whereas the colony formation was decreased (24.3%±5.3% vs 61.0%±8.5%, 58.0%±5.6%; both P values < 0.001). The G1-phase cell population was increased (66.4%±2.9% vs 33.5%±2.3%, 39.4%±3.7%; both P values < 0. 001) and S-phase cell population was decreased (18.6%±1.6% vs 36.2%±2.1%, 34.1%±1.6%; both P values < 0. 001). The cells moved from the upper chamber into the lower one in Transwell chamber assay had no marked difference (32.8± 6.2 vs 34.4±5.3, 29.4±5.2; respectively P= 0.659, P= 0.355).Between the cells without transfection and the cells transfected with empty vector, there were no significant differences in cell doubling time, colony forming ability, cell cycle distribution and the ability of cell invasion in vitro.Conclusion 1. Human FXYD6 expression correlates with bile duct carcinoma differentiation. From normal bile duct, high grade cholangiocarcinoma to low grade cholangiocarcinoma, the hFXYD6 mRNA and protein level increase progressively. 2. Transfection of hFXYD6 antisense can inhibit the ability of cell proliferation, but it has no effect on the ability of cell invasion in human cholangio-carcinoma cell line QBC939 in vitro.
|
PDF Full Text Request