【作者】 窦春青；

【导师】 周宁新；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2007， 博士

【摘要】 背景胆管癌早期诊断技术的改进是提高胆管癌疗效的关键因素之一。本课题组前期工作已在转录水平证实hFXYD6为胆管癌分化相关抗原。通过对FXYD6蛋白与胆管癌关系的逐步揭示，在为胆管癌早期诊断提供新的肿瘤标志物同时，也有望对胆管癌的发生演进发展有进一步认识。目的1．制备兔抗人FXYD6蛋白多克隆抗体。2．比较hFXYD6 mRNA、蛋白质在不同分化胆管癌组织的表达差异。3．探讨hFXYD6反义核酸对人胆管癌细胞体外增殖和侵袭能力的影响。方法1．通过B细胞表位分析，设计hFXYD6抗原多肽，经原核表达、蛋白纯化、动物免疫、抗体纯化等过程，制备兔抗人hFXYD6蛋白多克隆抗体。2．采用SYBR^? GreenⅠ荧光定量Real Time PCR及免疫组化方法检测不同分化胆管癌hFXYD6 mRNA及表达差异。3．通过反义核酸技术研究hFXYD6蛋白与人胆管癌细胞增殖侵袭关系。构建hFXYD6反义核酸真核表达载体pcDNA3.1（-）／hFXYD6（-）并转染人胆管癌QBC939细胞，同时设pcDNA3.1（-）空载对照组和空白对照组。Real Time PCR和荧光免疫组化鉴定转染结果；MTT、平板克隆形成实验检测细胞体外增殖活性；流式细胞仪检测细胞周期；Transwell侵袭小室模型检测细胞体外侵袭能力。结果1．经ELISA及免疫组化证实，成功制备高效价兔抗人FXYD6蛋白多克隆抗体。2．SYBR^? GreenⅠ荧光定量Real Time PCR结果表明：人FXYD6基因在正常胆管、高分化胆管癌及低分化胆管癌均存在表达；低分化组高于高分化组（P＜0.001），高分化组高于正常组（P＜0.001）。免疫组化支持这一结果。3．与空白组和空载组比较，反义组细胞hFXYD6 mRNA及蛋白表达量降低；细胞群体倍增时间增加（46.8h vs 34.5h，35.3h），细胞克隆形成率降低（24.3％±5.3％vs 61.0％±8.5％，58.0％±5.6％；P值均＜0.001）；细胞周期中G1期细胞比例明显升高（66.4％±2.9％vs 33.5％±2.3％，39.4％±3.7％；P值均＜0.001），S期比例明显减少（18.6％±1.6％vs 36.2％±2.1％，34.1％±1.6％；P值均＜0.001）；Transwell侵袭小室中24h穿膜细胞数无显著改变（32.8±6.2 vs 34.4±5.3，29.4±5.2；分别为P=0.659，P=0.355）。空白组和空载组细胞间均无明显差异。结论1．hFXYD6与胆管癌分化相关，从正常胆管、高分化胆管癌到低分化胆管癌，hFXYD6表达量依次递增。2．hFXYD6反义核酸抑制人胆管癌QBC939细胞体外增殖活性，但对其侵袭能力无明显影响。更多还原

【Abstract】 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.更多还原