【作者】 梁军才；

【导师】 缪林；

【作者基本信息】 南京医科大学 ， 内科学， 2011， 硕士

【摘要】 胆管癌是来源于肝内或肝外胆管上皮细胞的恶性肿瘤,在肝脏原发肿瘤中,胆管癌的发病率仅次于肝癌。其发病隐匿,临床症状及体征出现较晚,恶性程度很高。胆管癌占所有人类恶性肿瘤的2%以下,占消化道肿瘤的3%。1840年Durand Fardel首次提出胆管癌。近年来胆管癌的发病率和病死率呈上升的趋势,5年生存期低于5%。目前胆管癌多倾向于以手术为主的综合治疗,但手术治疗仅对部分的早期患者适用。然而,由于胆管癌所处的位置特殊,在胆管尚未被肿瘤完全堵塞之前患者的临床表现不明显,故早期诊断困难,临床上收治的大多是晚期患者。故大约80%的胆管癌患者由于发现时已处于进展期或转移,不能手术切除,而只能行姑息治疗。由于胆管癌对常规的放疗和化疗方法也不敏感,因此,探讨一种新的有效治疗途径,改善其预后,具有十分重要的意义。中药单体蟾蜍灵来源于中华大蟾蜍和黑框蟾蜍耳后腺及皮肤腺分泌的浆液,分子式为C24 H34 O4,相对分子质量为386.5,研究表明这种成分具有抗癌、强心、镇痛及麻醉等作用,有可能是通过干扰细胞周期而抑制白血病细胞的增殖,通过改变凋亡相关基因如bcl-2家族、抑制异常活化MAPK等多种途径诱导肿瘤细胞分化和凋亡。其对肝癌、胃癌、结肠癌细胞的相关作用已有报道,但是在对胆管癌细胞的作用及其机制方面研究鲜有报道。我们以蟾蜍灵单体作用于人胆管癌细胞QBC939,检测其对细胞增殖抑制以及cyclin E和p27的mRNA和蛋白表达影响,旨在探讨蟾蜍灵对胆管癌细胞的影响以及可能的分子机制,为临床治疗提供理论依据。目的通过观察蟾蜍灵对人胆管癌细胞QBC939的增殖及cyclin E和p27表达的影响,探讨其调控胆管癌细胞增殖的作用和机制。方法1、MTT比色法观察不同浓度蟾蜍灵(0.1μM、1μM、10μM)作用于人胆管癌细胞QBC939,分别观察24小时和48小时,观察其对细胞增殖的影响。2、乳酸脱氢酶(LDH)试验检测实验最大浓度蟾蜍灵毒性。观察蟾蜍灵是否是通过其毒性抑制了细胞增殖。3、流式细胞术检测不同浓度蟾蜍灵(0.1μM、1μM、10μM)作用人胆管癌细胞QBC939 24小时后细胞周期的变化。4、RT-PCR法测定不同浓度蟾蜍灵(0.1μM、1μM、10μM)作用于人胆管癌细胞QBC939 24小时后RT-PCR法测定cyclin E、p27 mRNA水平变化。5、Western blot法测定不同浓度蟾蜍灵(0.1μM、1μM、10μM)作用于人胆管癌细胞QBC939 24小时后cyclin E、p27蛋白水平变化。结果1、MTT比色法结果表明蟾蜍灵对人胆管癌细胞QBC939生长具有较强的抑制作用,并在一定范围内具有时间和浓度依赖性。2、蟾蜍灵不是通过其毒性作用抑制QBC939细胞增殖。3、流式细胞仪检测经不同浓度蟾蜍灵作用24小时后,人胆管癌细胞QBC939细胞周期阻滞在G0/G1期,具有良好的量效关系。4、RT-PCR法测定显示,经不同浓度蟾蜍灵作用24小时后,人胆管癌细胞QBC939中cyclin E mRNA表达量下降,p27 mRNA表达量升高。5、Western blot测定显示,经不同浓度蟾蜍灵作用24小时后,人胆管癌细胞QBC939中cyclin E蛋白表达量下降,p27蛋白表达量升高。结论蟾蜍灵可能是通过减少人胆管癌细胞QBC939 cyclinE的表达,增加p27的的表达,阻滞细胞增殖周期,抑制了细胞增殖,达到抗肿瘤作用。更多还原

【Abstract】 Cholangiocarcinoma is derived from the intrahepatic or extrahepatic bile duct epithelial tumors. In the liver primary tumors, the incidence of cholangiocarcinoma is lower than the liver cancer. There is a high degree of malignancy with occult its incidence, clinical symptoms and signs appeared later. which accounts for less 2% of all human malignancies, accounting for 3% of gastrointestinal cancer. In 1840, Durand Fardel firstly proposed cholangiocarcinoma, In recent years, the incidence and mortality of cholangiocarcinoma upward trend, the survival of 5-year is less than 5%. More tend to comprehensive treatment based on surgery, even only part of the early surgical treatment for patients. Since the special position of cholangiocarcinoma, the bile duct tumors have not been completely blocked in patients with subclinical before. So it is difficult to make an early diagnosis. Most of the clinical patients are treated in end of stage. Because of the advanced or metastatic of the tumors, about 80% of cholangiocarcinoma patients are only treated with palliative treatment. The conventional methods of cholangiocarcinoma are not sensitive to radiotherapy and chemotherapy. It is great significance to explore a new and effective therapeutic approach to improve its prognosis.Monomer bufalin, the traditional Chinese medicine, originates from the Chinese big toad and the black frame toad ear the gland and the skin gland secretion’s size, the molecular formula is C24 H34 O4, relative molecular mass is 386.5. A great many of researches have indicated that this ingredient has lots of medical functions, such as anticancer treatment, cardiotonic effect, toxicity and anaesthesia. The machines of induction tumour cell differentiation and perishe are possible to suppresses the leukemia cell’s multiplication through the disturbance of mitotic cycle, the changing of perishes in related gene, such as bcl-2 family, inhibit the abnormal activation of MAPK and so on. There have been a lot of studies reported in liver cancer, stomach cancer, the colon cancer, however, only few of reports have concerned cell’s function and the machine-made aspect of bile duct cancer. We use the bufalin monomer function in human bile duct cancer cell, named QBC939, to examine the suppressing of cyclin E, P27 mRNA and the protein expression. The purpose of this stuy is to explore the influence of bufalin on the bile duct cancer cell, its possible molecular mechanism and then provide the theory basis for the clinical therapy.ObjectiveThe aim of this study is to observe the effect of bufalin on the expression of P27 and Cyclin E in cholangiocarcinoma cell line QBC939, investigate the pathways of bufalin in inhibiting tumor cell proliferation, and explore its antitumor mechanism.Methods1. Cholangiocarcinoma cell line QBC939 was cultured by routine method, and then treated with different concentrations of bufalin (0.1μM, 1μM, 10μM).The proliferation apoptosis and cell cycle of QBC939 cells were investigated by MTT and Flow cytometry.2. Bufalin toxicity was assayed by Lactate dehydrogenase (LDH).3. RT-PCR and Western blot were performed to measure the expression of P27 and Cyclin E at gene and protein level.Results1. MTT assay results showed that of bufalin on the growth of human cholangiocarcinoma cell QBC939 inhibited, and in a certain range of time-and concentration-dependent. 2. Bufalin does not through its toxic effects to inhibition of cell proliferation QBC939.3. With different concentrations of bufalin for 24 hours, cholangiocarcinoma cell line QBC939 cell cycle arrest in G0/G1, with a good dose-effect relationship.4. By RT-PCR and Western blot assay, we found that bufalin at different concentrations for 24 hours, in cholangiocarcinoma cell line QBC939 decreased expression of cyclin E, p27 expression increased.ConclusionBufalin may inhibit the proliferation of QBC939 cells by down-regulating the expression of Cyclin E and up-regelating the expression of P27, blocking cell cycle.更多还原