白血病耐药相关膜蛋白SFPQ的发现及其耐药机制的研究

发布时间：2018-03-20 15:37

本文选题：白血病耐药　切入点：消减免疫　出处：《中国协和医科大学》2010年博士论文　论文类型：学位论文

【摘要】： 目前化学治疗仍是临床治疗白血病的主要手段,也是造血干细胞移植的基础。近年来尽管有多种新的药物和治疗方案推出,但大多数急性白血病患者最终因治疗失败而死亡。肿瘤细胞对抗癌药物产生抗药性是化疗失败的一个主要原因。导致治疗失败的原因是多方面的,其中多药耐药性(multi-drug resistance,MDR)是最重要的一种。MDR又称多药抗药性(pleiotropic durg resistnace),是指恶性肿瘤细胞对一种化疗药物产生耐药现象后,产生了对多种结构不同、作用靶点和作用机制各异的其他抗癌药物的抗药性。白血病耐药的产生是多因素的,随诱导药物、细胞种类、分化阶段、及细胞所处微环境的不同而表现出不同的耐药表型。虽然已有的研究揭示了一些肿瘤多药耐药的机制,但目前仍然不能完全解释肿瘤多药耐药现象并有效逆转肿瘤细胞的多药耐药。目前在白血病耐药的临床治疗方面主要以抑制P-糖蛋白功能的耐药逆转剂为主,但其临床效果也不理想,至今无一获得FDA批准上市。虽然急性白血病耐药的发生机制已经被广泛的研究,但耐药机制仍不明了,与白血病耐药相关的细胞膜新靶点的筛选方法仍未得以建立。事实上白血病患者耐药产生并非单一机制,此外,临床上患者间的个体差异也为治疗带来了困难。可见发现更多的白血病耐药相关的治疗靶点以实现多靶点联合检测与治疗、丰富白血病耐药机理从多角度全面阐明白血病耐药机制以综合制定白血病耐药治疗方案,建立治疗和预后指标是解决上述问题的对策。细胞膜蛋白暴露于细胞外表面,特别容易被抗体等试剂识别,作为靶点特异的检测、诊断和治疗的标志物；此外细胞膜蛋白还特别适于作为较为分散的血液肿瘤的治疗靶点。鉴于此,本文着力于寻找新的白血病耐药相关的细胞膜蛋白,考察其与白血病耐药的相关性并探讨其可能的机制。为寻找白血病耐药相关的细胞膜靶点,本研究首先应用消减免疫法,以人早幼粒细胞HL60活细胞作为耐受原诱导Balb/c小鼠免疫耐受,然后以阿霉素耐药株HL60/ADR细胞作为免疫原免疫小鼠,得到能特异识别差异表达的耐药株与敏感株HL60细胞膜表面蛋白的杂交瘤细胞库。通过对HL60和HL60/ADR两种靶细胞的选择性筛选和亚克隆培养、抗体的制备与纯化等过程,我们最终获得五株与两种靶细胞结合能力不同的单克隆抗体,这些抗体携带特定的耐药蛋白的标志信息。为了进一步获得相应抗原的信息,我们通过免疫沉淀和蛋白电泳技术分离敏感和耐药的HL60差异表达的细胞膜蛋白,并通过MALDI-TOF-MS鉴定抗原。最终我们获知单抗5D12的靶抗原为SFPQ,该蛋白高表达于敏感的HL60细胞膜表面,在阿霉素耐药株细胞膜其表达降低。通过siRNA干扰SFPQ,我们发现细胞膜高表达该蛋白的HL60细胞其膜SFPQ表达水平降低,由此确认SFPQ确为单抗5D12的靶抗原。此结果系首次发现SFPQ可表达在肿瘤细胞膜表面。通过实验我们发现5D12为有生物学活性的细胞膜蛋白SFPQ的特异性抗体。在以5D12作为研究SFPQ的功能的实验中我们发现,细胞膜SFPQ可增强细胞对阿霉素的敏感性。进一步的研究显示,细胞膜SFPQ可能通过抑制细胞增殖和克隆形成能力而减缓细胞生长,从而维持HL60细胞对阿霉素的敏感性。在对影响细胞增殖能力可能机制的研究中我们发现,细胞膜SFPQ通过诱导细胞凋亡和减少细胞S期比例减少DNA复制而抑制细胞增殖。该结果系首次报道HL60细胞膜表面蛋白SFPQ为一耐药相关蛋白。综上结果,我们认为细胞膜SFPQ可作为人早幼粒细胞白血病阿霉素耐药的新的细胞膜靶点,并且该靶点有可能成为检测和诊断该类白血病阿霉素耐药的新指标,为针对多因素耐药而采取的多靶点治疗和新的耐药机制的研究奠定基础。
[Abstract]:At present, chemotherapy is the primary treatment of leukemia, but also the basis for hematopoietic stem cell transplantation. In recent years, despite the introduction of new drugs and treatment programs, but most of the patients with acute leukemia died of treatment failure and death eventually. Tumor cells produce resistance to anticancer drugs is a major cause of chemotherapy failure causes. Treatment failure is in many aspects, including multi drug resistance (multi-drug, resistance, MDR) is one of the most important.MDR multiple drug resistance (pleiotropic durg resistnace), refers to the malignant tumor cell resistance to a chemotherapy drug, has a variety of different structures of other anticancer drug targets and the mechanism of different resistance.
Leukemia resistance is multifactorial, with the induction of drugs, cell types, cell differentiation stage, and the micro environment of different show different resistance phenotype. Although the existing research reveals the mechanism of multidrug resistance in some tumors, but is still can not fully explain the phenomenon of multidrug resistance and multi drug resistance to effectively reverse tumor cells. The clinical treatment for leukemia drug resistance mainly to the resistance reversal agent inhibits P- glycoprotein function, but its clinical effect is not ideal, one has been approved by the FDA. Although the pathogenesis of acute leukemia drug resistance has been extensively studied, but the mechanism of drug resistance is still unknown, yet the screening method of cell membrane associated with leukemia resistant new targets have been established.
In fact, drug resistant leukemia patients are not a single mechanism. In addition, the clinical individual differences between patients for treatment of difficult. It found more drug-resistant leukemia related therapeutic targets to achieve the target of joint detection and treatment, rich in drug-resistant leukemia from the multi angle comprehensive mechanism to clarify the mechanism of resistance to develop a comprehensive treatment of drug-resistant leukemia for the treatment and prognosis of leukemia, the establishment of indicators is the countermeasures to solve these problems. The cell membrane proteins are exposed on the outer surface of the cells, are particularly susceptible to antibody reagent identification, targeting specific marker detection, diagnosis and treatment; in addition treatment target cell membrane protein is particularly suited as scattered blood tumor in view of this, this paper focuses on the search for leukemia drug resistance associated membrane protein, to study its correlation with drug resistance of leukemia and to explore its possible The mechanism.
The cell membrane target for leukemia drug resistance related, in this study we used subtractive immunization, human promyelocytic HL60 living cells as tolerogen Balb/c induced immune tolerance of mice, then to adriamycin resistant HL60/ADR cell line used to immunize mice, obtain hybridoma cell library specific expression to identify differentially resistant strains with the sensitive strain of HL60 cell surface membrane protein. Through selective screening of HL60 and HL60/ADR two kinds of target cells and sub clone culture, preparation and purification process of antibody, we finally obtained five strains and two target cell binding ability of different monoclonal antibodies, these antibodies carrying resistance protein marker specific information.
In order to obtain the corresponding antigen, cell membrane protein expression of HL60 we isolated sensitive and resistant by immunoprecipitation and protein electrophoresis, and identified by MALDI-TOF-MS antigen. Finally we know target antigen monoclonal antibody 5D12 for SFPQ, the protein expression of HL60 cell membrane surface sensitive, reduce its expression in the cell membrane adriamycin resistant strains by siRNA interference. SFPQ, we found that the protein HL60 cell membrane SFPQ membrane expression level decreased, thereby confirming the target antigen of SFPQ mAb 5D12. This is the first discovery of SFPQ expressed on tumor cell surface membrane.
Through the experiment we found that the specific antibody 5D12 for biological activity of cell membrane protein SFPQ. 5D12 was used as the study on the function of SFPQ in the experiment we found that the cell membrane of SFPQ could enhance the sensitivity of cells to adriamycin. Further studies showed that the cell membrane SFPQ may through inhibition of cell proliferation and clone formation ability and slow down cell growth, thus maintaining the sensitivity of HL60 cells to adriamycin. The possible mechanism of effect on cell proliferation, we found that the inhibition of cell proliferation cell apoptosis induced by SFPQ and decreased the proportion of cells in S phase reduced DNA replication. This is the first report of HL60 cell membrane surface protein SFPQ as a resistance related protein.
In summary, we believe that SFPQ can be used as the cell membrane of human promyelocytic cell membrane new target myeloid leukemia adriamycin resistant, and the target may become a new indicator for the detection and diagnosis of the adriamycin resistant leukemia, lay the foundation for studying multi targets and take to several factors of drug treatment and new drug resistance the mechanism.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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