【作者】 臧彩红；

【导师】 王庆瑞；

【作者基本信息】 郑州大学 ， 药理学， 2011， 硕士

【摘要】 背景和目的：肝细胞癌(hepatocellular carcinoma,HCC)是一种高发性肿瘤,其发病率在全世界位列第五,而在肿瘤导致的死亡中位列第三。在北美和西欧一些国家中,每100 000人中大约有10人会因为患有肝癌而导致死亡,而在亚洲和非洲一些国家中,每100 000人中会有50-150因此而死亡。尽管在过去的50年中,对肝癌的治疗大有改进,但肝癌细胞容易对一些化疗药物产生多药耐药(multidrug resistance, MDR)而导致治疗效果一直不佳。MDR是指肿瘤细胞对一种抗肿瘤药物产生抗药性的同时对其他结构和作用机理不同的抗肿瘤药物也产生交叉抗药性。其中产生多药耐药的一个很重要原因是P-糖蛋白(P-glycoprotein, P-gp)的过度表达。P-gp是膜转运系统超家族成员之一,定位于细胞膜。Loo TW等等发现P-gp由两个相似的部分组成,每一部分都有六个跨膜区和一个核苷酸结合域。另外,Loo TW等通过对P-gp二硫键的分析,发现近其质膜侧的第六跨膜区和第八跨膜区具有膜转运功能,据此认为P-gp具有通道蛋白性质。Kimura Y等发现在真核生物中ABC转运子(ATP-binding cassette transporter)具有运输调节通道功能,而P-gp是ABC转运子超家族中最主要的成员,P-gp主要通过ATP的水解而实现其运输功能。许多文献都报道了P-gp是介导肿瘤耐药的重要因素,在许多肿瘤耐药细胞中,如肝癌耐药细胞、肺癌耐药细胞、乳腺癌耐药细胞、白血病耐药细胞等,均可检测到P-gp的高表达,其介导肿瘤耐药的机制主要为P-gp能够识别抗肿瘤药物,并且利用ATP产能,把抗肿瘤药物从细胞内不断地泵出到细胞外,从而使抗肿瘤药物在细胞内不能达到所期待的浓度,降低药物的疗效。为克服MDR需寻找有效的逆转剂,有相关文献表明一些逆转剂如维拉帕米和环孢素A等可逆转肿瘤耐药,但这些药物应用于体内会产生很大的毒性从而限制其临床应用。盐酸千金藤碱(cepharanthine hydrochloride, CH)是从防已科千金藤属植物的块茎中提取分离出来的一种双苄基异喹啉类生物碱单体化合物千金藤[14]与盐酸成盐而得,具有多种生物学活性。目前,有文献表明其具有体外逆转多柔比星等一些抗肿瘤药物产生的耐药性,但对于逆转肝癌多药耐药的体内研究,比较少见,尤其从P-gp ATP酶活性方面来研究其机制的文献更是少见。在临床上,有观察发现,肝癌患者更易对多柔比星(adriamycin, ADR)、顺铂(cis-dichlorodiamineplatinum, CDDP)5一氟尿嘧啶(5-flurouracil,5一FU)产生耐药,本课题在模拟临床FAP(ADR+CDDP+5-FU)方案[15-16]建立BALB/c小鼠肝癌多药耐药模型基础上[17],研究CH联合FAP对P-gp ATP酶活性的影响,从而进一步探讨CH联合FAP对肝癌多药耐药的逆转作用及其机制。材料与方-法：在建立BALB/c小鼠肝癌多药耐药的模型基础上,应用MTT法测定CH和维拉帕米对H22/FAP耐药细胞的无毒剂量；MTT法测定肝癌H22/FAP细胞对多柔比星(adriamycin, ADR),顺铂(cis-dichlorodiamineplatinum, CDDP),和5-氟尿嘧啶(5-fluorouracil,5-FU)的耐药性,并计算其耐药倍数(resistance index,RI)；观察用药后BALB/c小鼠的生存时间,并计算小鼠生命延长率,评价CH联合FAP对肝癌多药耐药的逆转作用；试验用雌性BALB/c小鼠腹腔接种H22/FAP肝癌细胞(每只0.2ml,1×105个细胞/m1),随机分组,尾静脉给药,连续用药7天后,按参考文献法制备质膜；用考马斯亮蓝法取20ug上述步骤中的质膜,并用定磷法检测P-gp ATP酶的活性；结果：1、MTT法检测CH与VER对H22/FAP的无毒剂量(抑制率<10%)：结果显示,CH和VER剂量分别为1.0mg/ml,0.15mg/ml时,对H22/FAP细胞的抑制率<10%；2、用MTT法检测ADR, CDDP,和5-FU对H22肝癌细胞的IC50分别为：0.12±0.01,0.10±0.02,6.36±0.23；ADR, CDDP,和5-FU对H22/FAP肝癌多药耐药细胞的IC50分别为：3.33±0.07,1.02±0.21,71.23±0.24:H22/FAP肝癌多药耐药细胞对ADR,CDDP,和5-FU的耐药倍数分别为27.75,10.20,11.20,证实实验所用的H22/FAP细胞为一多药耐药细胞。3、对小鼠生命延长率的评价发现：CH10mg/kg,CH5mg/kg,VER1.25mg/kg组均可明显延长H22/FAP MDR小鼠的生存时间,生存天数分别为29.46±2.33,31.08±2.42,29.21±1.47,与单纯用化疗药的FAP组(20.89±1.21)对比,对小鼠的生命延长率分别为40.1%,48.8%,39.8%,有统计学意义(P<0.05)；4、通过对体内P-gp ATP酶活性的检测发现,CH联合FAP能有效提高基础ATP酶活性,Km大约为为1.11mg/kg,Vmax大约为184nmol／(min·mg)结论：1、通过对小鼠生命延长率的实验发现,CH联合FAP能明显延长BALB/c小鼠生存时间,逆转H22/FAP肝癌细胞的耐药；2、CH逆转耐药的机制之一可能为通过提高P-gp ATP酶活性,抑制P-gp的外排,增加细胞内化疗药物的浓度。更多还原

【Abstract】 Backgrounds and ObjectiveHepatocellular carcinoma (HCC) is the fifth most common disease in the world and the third major cause of cancer-related deaths. The incidence of this tumor type ranges from approximately 10 cases per 100 000 in North America and Western Europe to 50-150 per 100 000 in Asia and Africa. Despite improvement in liver surgery,patient prognoses after surgical resection for hepatocellular carcinoma(HCC) remain unsatisfied.One of the obstacles in managing post-operative recurrence is multidrug resistance (MDR) to chemotherapy. Multidrug resistance (MDR) is a phenomenon encountered in cancer treatments in which the tumors become resistant to a variety of cytotoxic chemotherapeutic agents. The molecular basis for one major type of MDR is the overexpression of the P-glycoprotein (P-gp), a plasma membrane glycoprotein with molecular masses ranging from 130 to 170 kDa, confering resistance to a broad range of commonly used chemotherapeutic drugs and other hydrophobic agents. Anticancer agents, such as anthracycline derivatives including adriamycin(ADR),cis-dichlorodiamineplatinum (CDDP), and 5-fluorouracil (5-FU), have been used for these treatments. HCC, however, is often resistant to these drugs. The key for succeeding in chemotherapy against HCC is highly dependent on how we can control drug resistance. Cepharanthin hydrochloride (CH), is a natural bisbenzy- lisoquinoline alkaloid extracted from the roots of Stephania cepharantha and salificatied with Hydrochloric Acid. Recently, cepharanthine has been reported to potentiate the activity of some anticancer agents including ADR and to restore the effect of anticancer drugs in multidrug resistant cancer cells.It is, therefore, fascinating to consider the potential benefit of CH when treating HCC mice and the study demenstrates the positive effect of Ch in overcoming MDR by inhibiting the drug efflux through p-gp.Materials and MethodsDrug sensitivity testing with [3-(4,5-dimethylthi-azol-2-yl)-2,5 diphenyl-terazolium bromide] MTT assay showed that CH significantly enhanced cytotoxicity of adriamycin(ADR), cis-dichlorodiamineplatinum (CDDP),and 5-flurouracil(5-FU), but only in resistant cells, while,using the ATP-hydrolisis,we showed that compared with VER,CH increased ATPase activity approximately with a Km of 1.11mg/kg and Vmax of 184nmol/(min-mg).Life-prolongation test showed that CH combined with ADR,CDDP,5-FU significantly prolong the survival time of the mice.Results1、Cell toxicity test showed the small toxicity of CH and VER on BALB/c mice is 1.0mg/ml and 0.15mg/ml respectively;2、Statistical analysis with the MTT method showed resistance index (RI) of H22/FAP tumor cells (RI(H22/ADR)=27.75,RI(H22/CDDP)=10.20,RI (H22/5-FU)=11.20), and it supported powerfully that a HCC multidrug resistance model was established successfully. IC50 of H22/ADR cells (≈3.33) increased by 26.75 fold vs the group of H22 cells (≈0.12), respectively with H22/CDDP cells increased by 9.20 fold and H22/5-FU increased by 10.20 fold.3、CH10mg/kg,CH5mg/kg,VER1.25mg/kg in combination with FAP prolonged the life of BALB/c mice with the life prolongation rate about 40.1%,48.8%,39.8% (P<0.05,vs FAP group);4、In vivo, CH increased ATPase activity approximately with a Km of 1.11mg/kg and Vmax of 184nmol/(min·mg).Conclusions1、The data showed CH combined with FAP agents prolonged the life of mice significantly.2、One of the mechanism is that CH combined with FAP can increase the activity of P-gp ATPase.更多还原