【作者】 刘媛媛；

【导师】 李力；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2007， 硕士

【摘要】 卵巢癌是最常见的女性恶性肿瘤之一,它的5年存活率低。其重要原因之一是肿瘤细胞的多药耐药现象。为了探讨卵巢上皮癌铂类耐药机理,本课题组前期采用FDD-PCR方法筛选卵巢上皮癌铂类耐药细胞和敏感细胞系中差异表达基因。结果显示①S4:Homosapiens mitochondrion②a17:5-methyltetrahydrofolate-homocysteinemethyltransferase reductase③actin-related protein 3 homolog mRNA④s16:rihosomal protein L35a mRNA⑤s20:replication protein A2 mRNA⑥s23:basictranscription factor 3(BTF3)mRNA⑦a5:thymosin,beta10(TMSB10)mRNA⑧a13:esterase D/formylglutathione hydrolase(ESD)⑨a26:mitochondrion基因片段为各种耐药细胞系共存且与亲本敏感细胞间有表达差异的基因。本实验进一步验证上述差异表达基因及P73、WWOX基因在卵巢癌耐药和敏感细胞(组织)的表达,并探讨其与卵巢癌多药耐药和卵巢癌临床病理之间的关系。最后采用RNA干扰阻断WWOX基因表达的体外实验研究。1、卵巢上皮癌铂类耐药细胞和敏感细胞系中差异基因的表达:同时运用RT-PCR和Northern blot方法检测上述9条差异表达基因在卵巢癌耐药和敏感细胞中的表达,结果Northern杂交仅仅得到的四条基因片段(a17、a32、s23、s16)的mRNA表达信息,没有检测到其余5条基因的表达情况。其中a17和a32仅仅在卵巢癌耐药细胞中表达,而S16、S23基因在卵巢癌某些敏感和耐药细胞中表达,两种方法验证的结果一致,暗示这些基因与肿瘤多药耐药有联系。根据他们的生物学特性,我们推测a17基因使药物代谢灭活增加导致了肿瘤的耐药;S16是通过导致细胞毒性损坏从而参与了多药耐药的发生。而因为没有关于a32和s23的生物学信息作为参考,我们尚不能推测其通过哪方面参与了多药耐药的发生。2、卵巢癌多药耐药差异表达基因在卵巢肿瘤组织中的表达及其临床意义:采用RT-PCR方法对卵巢肿瘤及对照组织中的上述基因表达进行了检测,旨在探讨这些差异表达基因的表达上调或下凋是否与临床多药耐药现象相吻合以及其临床意义。结果显示,S4、S16、S20、S23、a5、a26、a13、a32基因片段的高表达与卵巢癌化疗铂类耐药之间可能存在着某些联系。通过他们的生物学特征我们推测a13、a17通过参与药物代谢过程、a26通过抑制肿瘤细胞凋亡和改变线粒体膜渗透性、s16通过参与细胞毒性损坏、s20通过参与DNA损伤修复从而导致了多药耐药。而a5、a17、s4、a32、s23的功能尚未明确。且S4、S23、a5、a13、a32的高表达与卵巢癌的高转移和进展有关,其中我们推测a5高表达导致了肌动蛋白骨架的断裂导致了肿瘤细胞的转移。a17、a26、s16、s20与卵巢癌的临床病理特征无关,考虑这几种基因可以成为卵巢癌耐药的指标,却与进展无关。3、P73基因在卵巢肿瘤组织和卵巢上皮癌耐药细胞系中的表达及其临床意义:采用RT-PCR和northern blot方法检测P73基因在卵巢肿瘤组织中和铂类药物敏感和耐药的卵巢上皮癌细胞系中的表达变化,旨在探讨其与多药耐药的相关性和临床意义。结果显示P73的表达与卵巢癌的临床分期、组织学类型、病情进展程度及患者预后无关。P73基因在卵巢癌耐药细胞株中的表达为高,考虑是否同时存在着P53的突变,P53的突变异构体使P73的功能失活才导致的耐药。WWOX基因在卵巢肿瘤组织和卵巢上皮癌耐药细胞系中的表达及其临床意义:采用RT-PCR和northern blot方法检测WWOX基因在卵巢肿瘤组织中和铂类药物敏感和耐药的卵巢上皮癌细胞系中的表达变化,旨在探讨其与多药耐药的相关性和临床意义。RT-PCR结果显示WWOX基因在卵巢癌敏感细胞系A2780中表达丰度均高于耐药细胞系,认为WWOX不介导多药耐药。而WWOX在敏感细胞系SKOV3中不表达,但是相对应的耐药细胞表达却明显增高,这个结论却与之前的推论背道而驰。我们考虑与这两种细胞耐药性不同有关。Northern杂交的结果反应出WWOX基因在两种卵巢癌敏感细胞中表达丰度均大于耐药细胞株,暗示WWOX基因不诱导产生卵巢癌的多药耐药。WWOX在卵巢肿瘤及对照组织中的检测的结果显示WWOX在卵巢癌铂类耐药组织中的表达阳性率和表达量明显高于敏感组织,但是无统计学意义。本实验结果尚不支持WWOX作为一个抑癌基因的看法,但是提示WWOX基因的表达与卵巢癌的发生和进展有一定的关系,与国外部分观点符合。5、RNA干扰阻断WWOX基因表达的体外实验研究:本章采用针对WWOX基因的特异性小分子干扰RNA,转染进卵巢癌耐顺铂细胞株SKOV3/SB,从基因角度探索逆转卵巢癌细胞多药耐药的途径,以期恢复卵巢癌细胞对化疗药物的敏感性。进一步探讨WWOX是否作为抑癌基因,并且也将为WWOX基因参与肿瘤的基因诊断及治疗提供新的思路。转染了WWOX的干扰片段后,SKOV3-SB的耐药下降,说明了WWOX很可能参与了耐药。功能实验结果提示WWOX通过增强肿瘤细胞自身修复,抵御化疗药物的损伤的作用及发挥类似药物输出泵的作用参与了耐药的发生。RNAi结果与前一章结果相反,考虑与实验误差有关,因为瞬时转染虽然可以抑制基因的表达,但对于干扰的长期有效性尚无实验结论,需要在后续实验中通过长效表达载体实验中做进一步验证。从结果来看,虽然本实验结果验证了与卵巢癌多药耐药有关的一些基因,并且也推测了他们参与多药耐药的途径,但是这仅仅作为一种假设。我们还要进一步系统研究这些耐药相关因子在耐药发生过程中所起的作用,以期在未来建立对耐药性状具有准确预测能力的计算机耐药模型,对于临床上耐药卵巢癌患者的个体化治疗方案的制订和实施,从根本上解决耐药问题,是非常必要的。更多还原

【Abstract】 Ovarian cancer is one of the most common malignant tumor in women.The majority of ovarian cancers are diagnosed in their advanced stage and the relative 5-year survival rate is low.To explore the mechanism of multiple drug resistance in ovarian cancer,The differentiated gene expression have further determined by FDD-PCR(fluro differentiated display PCR)method by the project team in prior period.The results showed①S4:Homo sapiens mitochondrion②a17:5-methyltetrahydrofolate-homocysteine methyltransferase reductase③actin-related protein 3 homolog mRNA④s16:ribosomal protein L35a mRNA⑤s20:replication protein A2 mRNA⑥s23:basic transcription factor 3(BTF3)mRNA⑦a5:thymosin,heta10(TMSB10)mRNA⑧a13:esterase D/formylglutathione hydrolase(ESD)⑨a26:mitochondrion differentiat expressed in resistant and sensitive ovarian cancer cells,we further studied these differentiated gene and P73、WWOX expression in resistant and sensitive ovarian cancer cells,and investigated the relationship between these genes and the multi-drug resistance and clinical pathology in ovarian cancer.At last,we researched the WWOX gene expression in the cisplatin resistant cells.1、The different genes expressed in the cisplatin resistant and sensitive ovarian cancer cells:RT-PCR and Northern blot methods were applied to determine the different genes expressed in the cisplatin resistant and sensitive ovarian cancer cells.The result showed only four genes(a17、a32、s23、s16)were investigated by Northern blot.the a17 and a32 only expressed in the drug-resistant ovarian cancer cell,but s16 and s23 gene expressed in some drug-resistant and sensitive ovarian cancer cells,the result implied these genes has the connection with the drug resistance,we presumed the a17 induce the drug resistance by increasing the drug metabolization,the s16 lead to the drug resistance by increasing the cell toxicity.Because we didn’t have the biological message of the a32 and s23 gene,so we can’t speculate the function.2、The expression and clinical value of these different genes in the ovarian cancer organization:RT-PCR method was applied to verify the gene expression in the tumor organization and the nomal organization,in order to probe which the expression of these different genes have the relationship in the clinical drug-resistance or not,and to analyze the clinical significance.The resulti showed that the high expression of S4、S16、S20、S23、a5、a26、a13、a32 genes hayed the connection with the cisplatin reststance in the ovarian cancer.We presumed that a13\a17 induce the drug resistance by increasing the drug metabolization,a26 lead to the drug resistance by inhibiting cancer cell atopotisize and change the penetrability of mitochondrial membrane,s16 conduce the resistance by increasing the cell toxicity,s20 cause the resistance by restorating the DNA damage.But we can’t speculate the function of a5、a17、s4、a32、s23.Besides,the high expression of S4、S23、a5、a13、a32 haved the connection with the metastasis and progress in the ovarian cancer,a17、a26、s16、s20 haven’t the association with clinical pathology.3、P73 gene express in the ovarian cancer organization and drug-resistance cell:RT-PCR and Northern blot methods were applied to determine the different genes expressed in the cisplatin resistant and sensitive ovarian cancer cells and organization,in order to investigate the relationship between P73 and the multidurg resistance and the clinical value.The result showed that there is a distant connection between the gene and clinical pathology.P73 expressed highly in the drug-resistance ovarian cancer,we considered that whether had the p53 mutation or not,the mutation induced the drug-resistance to occur.4、wwox gene express in the ovarian cancer organization and drug-resistance cell:RT-PCR and Northern blot methods were applied to determine the different genes expressed in the cisplatin resistant and sensitive ovarian cancer cells and organization,in order to investigate the relationship between wwox and the multidurg resistance and the clinical value.The RT-PCR result showed that WWOX expressed highly in the ovarian cancer drug-sensitive cell A2780 than the drug-resistanceA2780/SB/KB/TS,the result hints that wwox cann’ t induce the multi-drug resistance,hut wwox expressed highly in the drug-resistance cell SKOV3/SB/KB/TS than drug-sensitive,the result was opposite to the prior conclusion,we considered that the phenomenon be related to the two cells’ respective resistance indexes.Norther blot showed wwox expressed in the drug-sensitive cell highly than the durg-resistance cell,the conclusion implied that wwox hadn’t have the relation to the chemotherapy resistance in ovarian cancer.Besides,wwox expressed highly in the cisplatin ovarian cancer organization,but it haven’ t the statistics value.The research result haven’ t consider wwox is a tumor suppressor,but suggest wwox has the relation with the occur and progress in the ovarian cancer.5、wwox gene’s research in vitro by RNAi:RNA interference(RNAi)was used to explore the ways to reverse the multidurg-resistance in ovarian cancer,in order to restore the chemotherapy sensation.And further stugy whether wwox is a tumor suppressor or not,the research will provide the new train of thought for the gene diagnose and treatment.The result showed that wwox siRNA reduced the resistance indexes of SKOV3 SB,supposed that wwox had induced the drug-resistance by restorating the DNA damage、resisted chemotherapy drug’ s damage and played a important role in drug pumb.But the result will be further vertificate by expression vector.From our result,we have come to the conclusion that some gene which are in relationship with multidrug resistance in ovarian cancer,and presumed the way these gene induced the drug-resistance,But it is only a hypothesis,we felt it reasonable to suggest that efforts should be made to construct an ideal computer model of drug resistance,with all possible concerned factors identified, verified,and included,which would be the essential preparation for future individualized management and satisfying reverse technique for those patients with resistant malignancy of ovary.更多还原