【作者】 李杰；

【导师】 王冠军；

【作者基本信息】 吉林大学 ， 内科学， 2004， 博士

【摘要】 慢性粒细胞白血病(CML)是骨髓多能造血干细胞恶性增殖性疾病。CML的95%以上患者存在ph染色体（即t(9:22)(q34:q11)），分子病理特征为9号染色体q34的c-abl原癌基因易位到22号染色体q11bcr基因的3’末端，形成bcr-abl融合基因，可表达具有酪氨酸激酶活性的蛋白（分子量为210kDa的融合蛋白）。大剂量化疗及骨髓移植可使CML患者的临床症状、血液学指标暂时缓解，但对残存恶性肿瘤细胞（微小残留病变MRD）清除则要依赖于有效的细胞免疫治疗。随着分子生物学和免疫学的深入研究，人们发现bcr-abl融合基因连接区编码的特殊氨基酸于正常人不表达，故为激活特异性T细胞免疫提供了特异性抗原。尽管如此，CML病人并未在自然情况下产生理想的免疫反应，主要因为还存在着抗原提呈、反应原性等问题，而抗原提呈是中心环节。 树突状细胞（dendritic cell ，DC）是目前发现的功能最强的一类专职抗原递呈细胞（APC），也是体内唯一能直接激活初始T淋巴细胞的APC，它能摄取各类抗原，利用其自身高表达的MHC分子、共刺激分子及粘附分子等，将共刺激信号传递给T细胞，从而诱导特异性CTL，放大免疫效应，因此有人称DC是架在CTL与肿瘤细胞之间的桥梁。已有人利用特异性抗原多肽致敏自体树突状细胞进行B细胞淋巴瘤、前列腺癌、多发骨髓瘤、黑色素瘤的抗肿瘤免疫治疗，收到良好疗效，并进入I期或II期临床实验研究。白细胞介素18（Interleukin-18，IL-18）是近年发现的重要的免疫调节因子之一，具有诱生IFN-γ,增强NK和CTL细胞活性、促进T细胞增殖活化、增强Fas介导的细胞毒作用等多种生物学功能，是一种应用前景较好的抗肿瘤细胞因子。DC可以促进T细胞增殖活化，分泌高水平的IL-12，从而主导Th1型免疫应答，并可以显著诱导机体产生抗原特异性CTL，特异性杀伤肿瘤细胞。IL-12与IL-18联合应用可促进Th1细胞产生IFN-γ，增强细胞免疫反应。因此,从理论上讲，将IL-18与DC联合应用可增强抗肿瘤作用。本研究拟将CML患者外周血CML细胞诱生成具有白血病源性的DC（具有 bcr-abl融合基因），并在形态、表型、功能上进行分析鉴定，检测其是否能对抗<WP=106>原进行有效提呈，是否能够激活T淋巴细胞产生特异性杀伤CML细胞作用。并将DC应用于CML主动免疫治疗，观察CML病人应用后的治疗效果，为将来进一步进行临床应用奠定基础。为研究IL-18在CML治疗中的作用，拟用携带IL-18基因的真核表达质粒转染CML-DC，于体外观察转染后的CML-DC是否能够增强特异性细胞毒T淋巴细胞（CTL）的杀伤活性和有效的NK细胞效应，探索增强抗肿瘤作用的有效方法。本实验中应用DC进行CML免疫治疗国内未见报道，国外只有个案报道，转染IL-18基因CML-DC抗白血病免疫研究结果国内外均未见报道。CML细胞体外诱生DC及其激活抗白血病免疫反应我们应用GM-CSF、TNF-α、IL-4，在体外与CML患者外周血白血病细胞共培养12-14天，利用流式细胞仪、透射电镜、扫描电镜和荧光显微镜对诱导细胞进行分子表型及细胞形态进行检测、观察CML细胞是否可诱生为DC；利用D-FISH和RT—PCR检测所诱导DC是否为慢性粒细胞白血病源性。利用MTT法检测CML DCs刺激淋巴细胞增殖反应和特异性细胞毒性，观察GM-CFU集落形成情况来判定DC激活的T淋巴细胞对慢性粒细胞白血病前体细胞的影响。利用显微镜对其形态进行观察，CML DCs呈大体积悬浮，随诱导时间延长，DC由未成熟状态过渡到成熟状态，细胞膜扩展成多方向的突起，呈面纱或叶状；电镜观察，表明CML DCs具有富含胞浆突起，胞浆内含大量线粒体，少量溶酶体，说明其运动功能活跃，吞噬功能变弱。透射电镜观察可见，CML DCs表面皱褶丰富，有不规则胞浆突起伸出，均具备DC典型的形态特征。应用流式细胞仪免疫荧光染色法检测CML PBMNC经诱导分化后的表型变化，结果显示，刚分离CML细胞HLA-DR、CD1a、CD86阳性细胞<5%，体外诱导12－14天后，CD1a阳性细胞占18.7±12.20％，HLA-DR阳性细胞占26.9±7.67％，CD86阳性细胞占18.0±8.07％，提示CML DCs高表达共刺激分子、MHC－II类分子，具备抗原提呈及激活T淋巴细胞的分子基础及功能。应用D-FISH对免疫磁珠纯化的DC检测表明，CML DCs细胞为bcr-abl融合基因阳性细胞（93.5%±1.4%），利用RT-PCR检测CML DCs有bcr-abl融合基因转录，证明了体外诱导的CML DCs为白血病源性。 <WP=107>在混合淋巴细胞反应（MLR）中，CML细胞和CML DCs对自体T淋巴细胞的刺激作用，后者明显强于前者，且随S／R的增加，刺激增殖作用增强（P<0.05）。在细胞毒实验中，DC＋－T和DC－－T对自体CML细胞具有杀伤作用，在3个不同效／靶比中，DC＋－T对CML细胞杀伤作用明显高于DC－－T（P<0.05）,且随效／靶比增高而增强。在DC＋－T对CML细胞、自体T淋巴母细胞、K562细胞、HL－60细胞杀伤作用比较中，对CML细胞杀伤作用明显强于其它靶细胞，且随效／靶比而增高，说明CML DCs激活的CTL对自体CML细胞有特异性杀伤作用。对K562细胞、HL－60细胞杀伤作用强于自体T淋巴母细胞，且随效／靶比增高而增强，此种现象可认为是非肿瘤特异性的移植物排斥反应。在集落培养实验中，DC＋－T组集落生成数明显少于DC－－T组及无T细胞共培养组更多还原

【Abstract】 ` Chronic myelogeous leukemia (CML) is a clonal disorder of a pluripotent hematopietic stem cell associated in over 95%of cases with the Ph chromosome. This cytogenetic abonormality results from a reciprocal translocation involving chromosome 9 band q34 and chromosome 22 band q11,forming a fusion gene on chromosome 22 from the protonco gene ABL and the BCR gene, both expressed in normal cells and encoding presumably nonimmmunogenic proteins. High –dose chemoradiotherapy and autologous bone marrow transplantation can induce transient remission in patients with CML. Both normal and malignant progenitors are present in this disease. Relapse after auotologous bone marrow transplantation is related to the presence of malignant cells in the marrow graft, which can probably be eliminated by cytotoxic immune therapy. Products of either the b3a2or b2a2 exon junction with abnormal tyrosine kinase activity, at the fusion point, of a codon or an amino acid not derived from either the bcr or abl proteins, which provide the potent specific antigen to T lymphocyte cell. However, CML cells don’t express co-stimulatory molecules, which makes T cell anergy.Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC) specialized in the initiation of primary immune responses.. They possess an exceptional capability to capture antigens, process and present antigenic peptide fragments, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to induce primary immune responses of both CD8+ and CD4+ T cells. Now data concerning cancer immune therapy have been shown to be capable of inducing a specific antitumor response in vitro and in vivo. Some clinical experiments are in their I stage and II stage ,such as mutiple myeloma, melaoma.Interleukin –18 (IL-18) is found to be an important immune regulatory cytokines in recent years. With many biological functions, including the ability to induce IFN-γ,augment the cytotoxic activity of NK and CTL cells, promoting <WP=111>proliferation of T cells and cytotoxic activity by Fas. IL-18 is an antitumor cytokine which has good applying prospect. Dendritic cells(DC) capable of improving proliferation and priming T cell., induing high levels of IL-12 and Th1 immune reaction . DC can also induce antigen-specific CTL and enhance specific kill tumors. Moreover Il-18 and IL-12 synergistially upregulate the production of IFN-γ by Th1 cells and improve immune reaction . In conclusion , combing IL-18 and DC can augment antitumor effect.In the study leukemia dendritic cells were induced from CML peripheral blood and the morphological, phenotypic properties and function of DC were determined .We detected whether Dc can effectively present antigen and na?ve T cell inducing specific effect of kill tumors. Then dendritic cells were vaccinate for patients with CML and the therapy effect were observed which will establish the basis for future clinical application. In order to study the effect of IL-18 on CML therapy, CML-DC were transfected with pVAX1-IL-18 plasmid .The effects of CML-DC transfected IL-18 on the specific CTL and NK were detected.. This will provide a method for improving antitumor effect .The research of CML vaccinated with DC has not been reported in our country. It is the first report of antileukemia effect by IL-18 transfected CML-DC .1. The study on dendritic cells derived from CML cells in vitro and antileukemia immune response activated by dendritic cells. In this study, we induced DCs from CML peripheral blood mononuclear cells by GM-CSF,TNF-(,IL-4 . In addition, the cells were detected for morphological and phenotypic properties by inversed microscope, electro- microscope and FACS. DC exhibited the typical morphology with thin cytoplasmatic process , Transmission electron microscopy revealed that the cell surface had many irregular dendrities, numerous mitochondria, but scanty lysosomes, ribosomes. and expressed high levels of HLA-DR,CD80,CD86,CD1a.CML cells incubated concurrently with <WP=更多还原