【作者】 毕林涛；

【导师】 高长斌；

【作者基本信息】 吉林大学 ， 内科学， 2009， 博士

【摘要】 肿瘤坏死因子相关的凋亡诱导配体( tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)是肿瘤坏死因子超家族的新成员。TRAIL可诱导肿瘤细胞发生快速、高效的凋亡反应,而对正常细胞无明显毒性。除诱导实体瘤细胞凋亡外,目前研究还表明,TRAIL可诱导淋巴瘤、多发性骨髓瘤、白血病等细胞凋亡。然而体外试验也观察到血液系统恶性肿瘤对重组TRAIL产生耐药的现象,该问题的出现阻碍了TRAIL在临床上的进一步应用。TRAIL治疗淋巴瘤也面临着耐药的困扰。本实验以淋巴瘤细胞系Raji、Jurkat作为研究对象,应用化疗药物顺铂、pCMV-TRAIL表达载体、顺铂联合pCMV-TRAIL表达载体处理上述细胞,观察不同处理方法下细胞的生长状况,以MTT法测定细胞存活率,流式细胞仪及TUNEL染色分析细胞凋亡率,RT-PCR检测细胞周期相关蛋白mRNA表达的变化,免疫印迹法检测凋亡信号,探讨TRAIL联合化疗药物顺铂是否具有协同作用,并试图阐明TRAIL耐药及协同作用的可能机制,为淋巴瘤的治疗提供新的策略及理论依据。研究结果显示pCMV-TRAIL表达载体及顺铂联合应用处理淋巴瘤细胞系Raji及Jurkat,其细胞存活率均明显低于单药处理组及对照组,而细胞凋亡率则明显高于单药处理组及对照组。RT-PCR检测凋亡相关蛋白cyclin E、CDK2、p27 mRNA表达的变化:DDP化疗组、pCMV-TRAIL重组质粒转染组和联合用药组cyclin E、CDK2表达均低于空质粒转染组和对照组;而p27表达则高于空质粒转染组和对照组。通过免疫印迹法检测survivin、caspase-3、NF-κB等凋亡信号,pCMV-TRAIL重组质粒转染组和联合用药组中caspase-3表达明显增高,而survivin及NF-κB表达则显著降低,且联合用药组NF-κB表达低于pCMV-TRAIL重组质粒转染组。因此,我们认为TRAIL与化疗药物顺铂治疗B、T细胞淋巴瘤具有协同作用,其机制可能与cyclin E、CDK2、p27等调控细胞周期因子以及Survivin、caspase-3、NF-κB等凋亡因子的改变有关。更多还原

【Abstract】 The Non-Hodgkin lymphomas (NHLs) are a diverse group of hematologic cancers which encompass any lymphoma other than Hodgkin lymphoma.Lymphoma is a type of cancer derived from lymphocytes, a type of white blood cell. Many subtypes of non-Hodgkin lymphoma have been described. Non-Hodgkin lymphomas are treated traditionally by combinations of chemotherapy, immunotherapy and radiation, et al.However, the function of these treatment is limited as well as side effect.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a new member in the family of tumor necrosis factors. The death receptors belong to a subset of tumor necrosis factor receptor superfamily members and include Fas (CD95), tumor necrosis factor receptor (TNFR)-1, death receptor 3 (DR3/wsl-1), death receptor 4 (DR4, TRAIL-R1), and death receptor 5 (DR5, TRAIL-R2). The respective ligands bind to these receptors causing trimerization of the receptors and apoptosis. Two of these receptors, DR4 and DR5, have aroused considerable interest and research because of their potential for the treatment of the hematological malignancies. This is due to the observation that combination of TRAIL and DR4 or DR5 selectively can induce apoptosis in tumor cells. There are two major pathways for apoptosis, the mitochondrial or intrinsic apoptotic pathway and the death receptor or extrinsic apoptotic pathway. The intrinsic apoptotic pathway is typically initiated by DNA damage, which causes the upregulation of p53, an increase in the bax:bcl-2 ratio, and the release of cytochrome c from between the inner and outer mitochondrial membranes into the cytosol. Cytochrome c binds to and activates apoptosis-activating factor-1 (Apaf-1) in the cytosol in a process that requires deoxyadenosine triphosphate (dATP), and both caspase 9 and 3 are subsequently activated with the induction of apoptosis. The extrinsic apoptotic pathway is triggered by binding of the death receptor ligands to their cognate receptors. The death receptors for TRAIL, DR4 and DR5, contain cysteine rich repeats in the extracellular domain that bind to TRAIL causing receptor trimerization and subsequent apoptosis. Therefore, TRAIL is promising in treating the hematological malignancies including NHL. However, resistance to TRAIL in tumor cells have blocked its usage in clinical work. Studies have been done to value the combination of TRAIL with chemotheray and to explore the mechanism of TRAIL-resistance.Cis-platinum complexes is a platinum-based chemotherapy drug used to treat various types of cancers, including sarcomas, some carcinomas (e.g. small cell lung cancer, and ovarian cancer), lymphomas and germ cell tumors. It was the first member of its class, which now also includes carboplatin and oxaliplatin. Platinum complexes are formed in cells, which bind and cause crosslinking of DNA; ultimately triggering apoptosis. Combination of cisplatinum with TRAIL have been proved to be useful to treat some tumors, such as cervical carcinoma, laryngocarcinoma, and so on. Given all the above, in order to investigate the synergistic molecular mechanism of Cisplatin enhancing the TRAIL-induced apoptosis of lymphoma Raji and Jurkat cell lines and provide a novel treatment model of the TRAIL in lymphoma, we design this study. The human lymphoma cell line Raji and Jurkat was treated with Cisplatin, TRAIL, respectively, or in combination. The cytotoxic effect and the rate of apoptosis were determined by MTT and flow cytometry,respectively; the expression of cyclin E(317bp)CDK2(546bp)and p27(237bp)mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR); and the expression of signal transduetion proteins,such as Caspase-3,NF-κB and Survivin Was detected by Western blot.We get the results that combination with subcytotoxic dose of Cisplatin could reverse the sensitivity of raji and jurkat cells to TRAIL-induced apoptosis,and could down·regulate the expression of cyclin E,CDK2 and survivin, NF-κB; up·regulate the expression of Caspase3 and p27.In conclusion, Cisplatin could sensitize raji and jurkat cells to TRAIL-induced apoptosis.更多还原