【作者】 王蕾；

【导师】 白海；

【作者基本信息】 兰州大学 ， 内科学（专业学位）， 2015， 硕士

【摘要】 多发性骨髓瘤(multiple myeloma, MM)是以异常浆细胞增生,最终导致单克隆免疫球蛋异常聚集的B系恶性肿瘤,近年来,MM的发病率在包括我国在内的许多国家都成上升趋势,除随着社会老龄化及社会环境因素改变致使本病实际发病率确有增加外,也和医务人员对本病的认识提高、诊断技术的进步是检出本病例数增加的缘故,而目前常用的基础治疗无法实现治愈,且受制于患者脏器功能、年龄、经济等多方面因素。因此急需寻找新的更加安全、有效、靶向性强、副作用轻的治疗方法。虽然第一代免疫调节剂沙利度胺,第二代免疫调节剂来那度胺,蛋白酶体抑制剂硼替佐米的出现显著改善了MM患者的临床预后,但随着病程的延长,大部分患者最终仍是走向复发、耐药。泊马度胺(Pomalidomide, CC-4047)的药物结构和沙利度胺类似,是由美国Celgene公司研发的第三代免疫调节剂。泊马度胺能减少例如白介素-6、肿瘤坏死因子-α等促进瘤细胞存活的细胞因子、下调MM细胞关键功能信号等发挥抗骨髓瘤作用。泊马度胺可直接刺激患者T淋巴细胞,增加IFN-γ等Thl型细胞因子的分泌,产生具有肿瘤特异性的Thl型反应。国外临床研究结果提示,泊马度胺联合地塞米松治疗复发、难治性患者能使部分患者获得缓解,延长患者生存期,且相较于第一代、第二代免疫调节剂药理作用更强,耐受性更好,毒性更低更安全。但仍有部分患者对治疗缺乏有效的临床反应,面对这部分患者的治疗,现有的基础治疗非常有限。T淋巴细胞在肿瘤免疫应答中起主要作用,但肿瘤可通过多种机制逃避T淋巴细胞识别和杀伤,抑制T细胞功能。嵌合抗原受体是一种合成的跨膜蛋白,经基因修饰后,靶向作用于肿瘤细胞,和既往的T细胞过继免疫治疗相比,CAR-T细胞的最大优势在于其杀伤性不受MHC限制,靶向性强。本研究选取以慢病毒为载体转染T细胞,表达嵌合抗原受体的T淋巴细胞可靶向性的与MM细胞表面高水平表达的CD138抗原相结合,产生明显的抗骨髓瘤细胞效应,增加骨髓瘤细胞凋亡。本研究通过观察不同浓度下的泊马度胺体外作用于MM细胞株RPMI8226、U266,观察泊马度胺在体外对RPMI8226、U266凋亡、细胞周期等影响。并进一步应用CD138-CAR-T、泊马度胺联合CD138-CAR-T细胞体外杀伤RPMI8226、U266。第一部分泊马度胺对多发性骨髓瘤细胞株U266及RPMI8226的影响目的：通过体外实验探讨不同浓度的泊马度胺对人多发性骨髓瘤细胞株U266、RPMI8226增殖、凋亡的影响,NF-κB的表达变化,凋亡信号Caspase-8、核转录因子IRF-4以及CRBN基因的表达变化。方法：将泊马度胺分别作用于骨髓瘤细胞U266、RPM18226,采用流式细胞术检测不同浓度下细胞的凋亡率、细胞周期分布、胞核NF-κB的表达变化；RT-PCR检测IRF-4、Caspase-8、及CRBN mRNA表达量的变化。结果：和对照组(药物浓度为0 ug/ml)相比,泊马度胺对RPMI8226细胞株无任何明显作用(P>0.05),结果不随药物浓度改变。与对照组(药物浓度为0 ug/ml)相比,流式结果显示泊马度胺可将U266细胞阻滞于GO/G1期,诱导细胞凋亡,其作用呈浓度依赖性(P<0.05)。泊马度胺作用48h后,U266细胞IRF-4、 CRBN mRNA表达均下降(P<0.05),Caspase-8表达上调(P<0.05)。结论：泊马度胺可降低U266细胞IRF-4、、CRBN mRNA表达,上调Caspase-8表达,诱导细胞发生凋亡,可将细胞阻滞于G0/G1期；但泊马度胺对多发耐药的细胞株RPM18226无明显的作用。第二部分泊马度胺联合CD138-CAR-T对多发性骨髓瘤细胞株RPMI8226、U266的体外杀伤作用目的：体外观察CD138-CAR-T对人MM细胞株RPMI8226和U266细胞的靶向杀伤作用,探讨泊马度胺对CD138-CAR-T细胞分泌IFN-γ及其杀伤作用的影响。方法：采用CFSE/7AAD双标法检测CD138-CAR-T及CD138-CAR-T联合泊马度胺对RPMI8226、U266细胞的杀伤活性。ELISA法检测CD138-CAR-T细胞分泌IFN-γ的变化。结果：CD138-CAR-T细胞作用18 h后,对RPMI8226、U266细胞的杀伤活性分别为(55.2±3.9)%、(85.1±2.4)%,对照组分别为(7.0±1.5)%、(12.5±2.1)%,差异均有统计学意义(P值均<0.01)；与CD138-CAR-T细胞组比较,CD138-CAR-T细胞联合泊马度胺(2.5 ug/ml)作用18h后,对RPMI8226、U266细胞的杀伤活性无明显变化(P>0.05)。与CD138-CAR-T细胞组比较,CD138-CAR-T细胞和埘细胞共培养组IFN-γ分泌水平显著增高；与共培养组比较,加入泊马度胺后能显著促进IFN-γ的释放,差异有显著统计学意义(P<0.01)。结论：CD138-CAR-T细胞对U266细胞、多发耐药细胞株RPMI8226有明显的杀伤作用；CD138-CAR-T细胞与瑚细胞共培养能促进CD138-CAR-T分泌IFN-γ;加入泊马度胺共培养后能促进CD138-CAR-T细胞分泌IFN-γ。更多还原

【Abstract】 Multiple myeloma is the proliferation of abnormal plasma cells, eventually leading to monoclonal immunoglobulin abnormal accumulation of B-lineage malignancies. Because of the aging of population and progress of diagnose, the incidence is climbing, and the most commonly used chemotherapy and stem cell transplantation were unable to achieve a cure, and subject to the patient’s age, organ function and other factors. So we need to find another therapeutic method which is more safe、effective、targetable and low side effect. Immunotherapy is an ideal therapeutic method. Although the appearing of the first generation immunomodulatory agent thalidomide, the second generation immunomodulatory agent lenalidomide and proteasome inhibitor bortezomib improved clinical outcomes in patients with MM, but with the extension of the course, the majority of patients disease eventually relapse. It is the third generation immunomodulator which developed by the company of Celgene. February 8,2013, the US Food and Drug Administration (FAD) approved Pomalidomide used for the treatment of relapsed, refractory multiple myeloma patients. Pomalidomide can reduce cytokines such as interleukin-6, tumor necrosis factor-a which can promote tumor cell survival, down the key function signals of MM cells. Immune dysfunction is characteristic of MM, MM patients had varying degrees of immune surveillance and response defects. It can directly stimulate T lymphocytes, increase the secretion of IFN-γ and other Thl-type cytokines. Foreign clinical studies suggest that, Pomalidomide joint dexamethasone treatment of recurrent, refractory patients can make some patients in remission, prolong survival. Compared to the first generation and second-generation immunomodulatory agents, its pharmacological effect is stronger, better tolerated, less toxic and safer. But there is still a lack of effective treatment in some patients for clinical response, existing treatment is very limited basis, the rapid development of modern cancer immunotherapy brings hope for cancer patients. T lymphocytes plays a major role in tumor immune response, but the tumor can escape identify and kill through a variety of mechanisms, and inhibit T cell function. T lymphocytes based adoptive immunotherapy has made some clinical effect, but not very satisfactory. Chimeric antigen receptor is a transmembrane protein synthesis, after genetic modification, it can target to the tumor cells, compared with previous adoptive T cell immunotherapy, the biggest advantage of CAR-T cells is unlimited by MHC. Our study is to be intended to transducer T cells with a lentiviral vector encoding a chimeric antigen receptor, the genetically modified T cells stably expressing the chimeric antigen receptor specific for CD 138, which is highly expressed on myeloma cells surface, can induce intensified anti-myeloma immune response, and reinforce the apoptosis of myeloma cells. T cells can be easily expanded to large numbers and readily available on demand in standardized quality for adoptive therapy. In this study, different concentrations of pomalidomide acting on MM cell lines RPMI8226, U266,to investigate the effect in vitro on RPMI8226, U266, and further observe the apoptosis of CD138-CAR-T, pomalidomide combines CD138-CAR-T cells on RPMI8226, U266 in vitro. Experimental study includes the following two parts:Part ⅠEffect of Pomalidomide on human Multiple Myeloma cells RPMI8226 and U266Objective:This study investigated the effects of pomalidomide (CC-4047) on the proliferation, apoptosis of human multiple myeloma (MM) cells U266 cell and RPMI8226 cell, the change of apoptosis signal protein Caspase-8、IRF-4 and CRBN mRNA expression.Methods:The two type of MM cells were reacted with pomalidomide separately and cellular morphology change was observed using light microscope; apoptosis rate, cell cycle and expression change of NF-kB were analyzed with flow cytometer; the mRNA expression level change of IRF-4, Caspase-8 and CRBN were examined by RT-PCR.Result:Pomalidomide did not shown significant effects on the cellular proliferation, cell cycle, morphology and NF-kB expression level in RPMI8226 cell (P>0.05). While for U266 cells, Pomalidomide arrested the cell cycle in G0/G1 phase and induced cellular apoptosis in a dosage-dependent manner (P<0.05); the mRNA expression level of IRF-4 and CRBN were decreased. Caspase-8 was up regulation in U266 cells (P<0.05).Conclusion:It is concluded that Pomalidomide is capable of induce U266 cell apoptosis, and arrest cell cycle in GO/G1 phase, whereas show no effect to RPMI8226 cell.Part ⅡThe cytotoxity of Pomalidomide combined CAR-T for multiple myeloma cell RPMI8226 and U266Objective:This study aimed to investigate the cytotoxity of CD138-CAR-T cells on human multiple myeloma cell RPMI8226 and U266 cells,to explore the effect of pomalidomide on CD138-CAR-T cells and the cytotoxity of CD138-CAR-T on RPMI8226 and U266 cells.Methods:The cytotoxity of CD138-CAR-T and CD138-CAR-T combined pomalidomide on RPMI8226 and U266 was detected by CFSE/7AAD. The cells were co-cultured with target cells at 5:1, after incubation for 18h, the supernatant were collected and used for ELISA assays.Results:After 18h, the cytotoxity of CD138-CAR-T on RPMI8226 and U266 was significantly higher than control(P<0.01). There was no significantly change on the cytotoxity of pomalidoide combined with CD138-CAR-T on RPMI8226 and U266.The result showed that co-cultured system contributed to a markedly increased production of IFN-γ, after adding pomalidomide to the co-cultured system, it can significantly enhanced the production of IFN-γ, compared with CD138-CAR-T.Conclusion:CD138-CAR-T had significantly cytotoxity on U266 and RPMI8226, co-culture can promote the secretion of IFN-γ. Pomalidomide can promote CD138-CAR-T cells secrete IFN-γ.更多还原