趋化因子研究进展2

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,趋化因子受体在不同亚类,T,细胞中的表达谱,Nature Immunology, 2008 , 9(9), 970-980,（2）,CKR,在,DC,迁移和功能成熟中具有不同的表达模式:,非成熟,DC,：,CCR1,，,2,，,5,，,CXCR4,，,易于向炎症部位聚集，摄取抗原；,成熟,DC,：,CCR4,，,7,，,CXCR4,，,负载抗原,向淋巴组织迁移。,The EMBO Journal (2008), 110,Nonsignaling Scavenger Chemokine Receptor-like Molecules Adjust the Chemokine Environment,Richard M. Ransohoff1, Immunity, 2009 (31), 711-721.,趋化因子及其受体在免疫系统的表达和功能,Ye H. Oo et al, Journal of Autoimmunity 34 (2010) 4554.,(四),CKR,的信号传导途径,Domanska UM et al, Rev Oncol Hematol. 2010 Aug 13. Epub ahead of print .,（一）趋化作用；,（二）上调整合素的表达, 活化白细胞；,（三）促进细胞脱颗粒和生物活性物质释放:,CCL2,：,Ba,释放组织胺；,CXCL8,：,Neu,产生活性物质，呼吸爆发；,（四）调节血管生成：,ELRCXC,、,CCL2,促进血管生成；,CXCL10,、,CCL21/SLC,抑制血管生成。,三、趋化因子的基本功能,趋化因子的基本功能,趋化因子引起的细胞形态改变,CXCL8/IL-8,的作用,（五）调控免疫细胞分化、发育、成熟、归巢、相互作用等。,趋化因子参与,T,细胞发育,趋化因子及其受体在免疫应答中的作用,趋化因子参与免疫细胞在周围免疫器官的定位,四、,趋化因子与疾病,CKR,与疾病,疾病相关趋化因子受体在免疫细胞中的表达,Richard Horuk, Nature reviews, drug discovery, 2009, Jan, 8, 23-33,AIDS:,获得性免疫缺陷综合症,（,Acquired Immunodeficiency Syndrome, AIDS,）,CD4,分子和,CCR5,、,CXCR4,等,CKR,是,HIV-1,感染人体细胞,所必须的受体；,针对,CCR5,的小分子抑制物,Maraviroc,已获,FDA,批准并于,2007,年,8,月上市。,（一）,CKs,与,AIDS,CCR5,、,CXCR4,是,HIV,感染人体细胞的共受体,HIV,感染人体细胞的过程,Chem Biol Drug Des 2008; 72: 97110,It is currently believed that 90% HIV strains use CCR5 (macrophage tropic) for initial infections, and the infected viruses undergo mutations that allow them to use CXCR4 (T cell tropic) as the infection progresses to AIDS.,Entry of HIV into host cells requires the formation of an entry complex including the viral envelop glycoprotein gp120, CD4 and either CCR5 or CXCR4 receptors,。,CCR5,、,CXCR4,和,HIV,感染,Tian Jin et al, Cytokine 44 (2008) 18.,CCR5,缺失突变对嗜,M,HIV,病毒感染有抵抗性,Targets a host protein, CCR5, rather than a viral target;,Binding of maraviroc to CCR5 results in blocking HIV-1 attachment to the coreceptor and prevents the virus from entering CD4+ cells.,Maraviroc, the first US Food and Drug Administrationapproved drug from a new class of antiretroviral agents,Therapeutics and Clinical Risk Management 2008:4(2) 473485.,辉瑞公司生产；,副作用：肝脏或心脏损伤，出现其他感染或罹患,癌症的风险增加；,适应症：,FDA,强调该药仅适用于对其他抗艾药已,产生耐药性的患者，而不适用于刚确诊的患者。,它是自,2003,年以来研发成功的首个抗艾新药；也是首个趋化因子相关药物。,Selzentry,（,Maraviroc,）,1,、哮喘：,CCL3,，,5,，,7,，,11,，,22,等在靶细胞的迁,移、聚集、活化中发挥关键作用;,2、肿瘤：,CXCR4,与,CXCL12,、,CCR7,与,CCL21,在肿瘤细胞向,骨、淋巴结等器官的转移中起重要作用；,3,、自身免疫病：,MS,和,RA,等；,4,、动脉粥样硬化：,CX3CR1,基因缺陷人群对该病不易感,；,5,、移植排斥。,（二） 趋化因子与其它疾病,(1) inducing leukocyte infiltration to tumors and regulating immune functions, with emphasis on tumor-associated macrophages (CCL2, CCL5), T cells (CXCL9, CXCL10), dendritic cells (CCL19, CCL20, CCL21) and NK cells (CX3CL1);,(2) directing the homing of tumor cells to specific metastatic sites (the CXCL12CXCR4 axis);,(3) regulating angiogenic processes (mainly the ELR+CXC and non-ELRCXC chemokines);,(4) acting directly on the tumor cells to control their malignancy-related functions.,Multifaceted roles of chemokines in malignancy,肿瘤组织表达的趋化因子受体,Ann Richmond, et al. Pigment Cell Melanoma Res. 2009, 22; 175186.,Due to genetic alterations, epigenetic regulation and hypoxia in tumors, over 23 different cancer types overexpress CXCR4, therapies that target CXCR4 may be applicable to many cancer types.,In prostate cancer, CXCR4 expression relates to increasing tumor grade.,CXCR4 is highly expressed in breast cancer tissue, with low expression in normal breast tissue. CXCL12 is highly expressed at sites of breast cancer metastasis. CXCR4 is much more highly expressed in bone metastases than in visceral metastases. CXCR4 has an important role in the homing of tumor stem cells to the bone marrow, which is an area enriched with CXCL12.,Pivotal role of CXCL12/CXCR4 axis in bone metastasis,Antitumor activity induced by blocking CXCL12/CXCR4 pathway,Antibody to CXCR4 significantly reduces the number of bone metastasis of prostate cancers in an in vivo murine animal.,CTCE-9908(Chemokine therapeutics, vancouver, BC, Canada) is a peptide analog of CXCL12 that acts as a competitive antagonist of CXCR4. in a mouse model, treatment with it did not reduce the frequency of metastasis, but did decrease both the tumor burden of breast cancer in the bone and other organs, and also of the primary breast .,Plerixafor (MD3100, Genzyme Corporation, Cambridge, Ma, usa) is a CXCR4 antagonist that promotes hematopoietic stem cells to mobilize from the bone marrow into the bloodstream, and is approved for use in autologous transplantation.,Clinical trials of this agent are also underway, mainly for hematologic malignancies. Another potential strategy is to combine CXCR4 antagonists with other drugs, such as bisphosphonates, as these drugs are likely to have a synergistic action because zoledronic acid also inhibits CXCR4 expression on breast cancer cells.,Onishi, T.,et al. Nat Rev Clin Oncol. 2010 Aug 31. Epub ahead of print,KW-0761, a defucosylated humanized anti-CCR4 antibody, exerts a strong antibody-dependent cellular cytotoxic effect.,ATL: adult T-cell leukemia-lymphoma; PTPL: peripheral T-cell lymphoma .,KW-0761, finished phase I study for ATL and PTPL,Yamamoto K et al. J Clin Oncol. 2010 Mar 20;28(9):1591-8.,Ishii T et al. Clin Cancer Res. 2010 Mar 1;16(5):1520-31.,（三）趋化因子拮抗剂的种类,1、小分子化合物：与受体的穿膜区结合，最有希望进入临床应用；,2、修饰的趋化因子：具有结合受体的能力，但失去启动信号转导的功能；失去与,GAG,结合的能力；,3、抗体；,4、趋化因子结合蛋白：尚有待证实人类是否存在。,作为趋化因子受体拮抗剂的小分子化合物的结构,趋化因子受体拮抗剂的研发,Richard Horuk, Nature reviews, drug discovery, 2009, Jan, 8, 23-33,趋化因子受体拮抗剂的专利分布,趋化因子、受体及其与疾病的关系,五、趋化因子的常用研究方法,（一）趋化实验,(Chemotaxis Assay),1、体内实验,：,皮下、腹腔、静脉注射，观察细胞,浸润情况；繁琐。,2、体外实验：,最常用的方法是,Boyden,小室法，也称微孔滤膜,法。48孔趋化小室，上层放靶细胞，下层放待检测因子，两,层之间有聚炭膜隔开；简单易行。,细胞类型 膜孔径 趋化时间,Neu: 3um,30,min,；,T,、,B:,5,um,120,min,；,Mo: 8um 90min,反应结束后，洗掉非特异结合细胞，固定、染色，记数。,记数方法：,高倍镜下每孔随机选5个视野，取平均值。,趋化活性表示方法：,通常以趋化指数（,chemotatic index, CI,）,表示,。,CI,指实验组细胞数与对照组细胞数（三个复孔,的平均值）的比值，,CI,大于2有意义。,Boyden,小室示意图,A,、,微孔滤膜法示意图,B,、,内皮细胞趋化结果,（二）钙流实验,(Calcium Mobilization Assay),多数,CK,与受体结合后, 胞内,Ca2+,；,Indo-1 AM,或,Fura-2 AM,等是,Ca2+,螯合剂,EGTA,的衍生物，可特异高亲和,Ca2+,；,将靶细胞与荧光探针孵育；,检测荧光强度变化，反映,CK,活性。,CK,与,CKR,结合后,出现受体一过性内化,；,构建,CKR,与,EGFP,的融合表达载体，转染细胞，与待检测因子作用；,观察荧光定位改变。,（三）受体内化试验,(Receptor Internalization Assay),靶细胞与,PTX,共孵育；,趋化试验；,检测,PTX,是否可阻断靶细胞的趋化运动。,（四）,PTX,阻断试验,(PTX Blocking Assay),（五）功能实验,(Functional Assay),CXC,CKs:,弹性蛋白酶、葡萄糖醛酸酶释放检测、呼吸,爆发实验；,MIP-1,，,：,用骨髓造血干细胞集落形成实验；,CC CKs:,组织胺释放。,（六）交叉去敏感实验,Cross D,esensitization Assay,Ying Wang, Dalong Ma, et al, Life sciences, 2006, 78: 614-621.,B,：,B,淋巴细胞；,T,：,T,淋巴细胞；,Neu,：嗜中性粒细胞；,E:,嗜酸性细胞；,Ba:,嗜碱性细胞；,MC:,肥大细胞；,BMC,：骨髓细胞；,Th2,：,2,型辅助性,T,淋巴细胞；,Mo:,单核细胞；,M,:,巨噬细胞；,HPC:,造血前体细胞。,Abbreviations,思 考 题,1,、结合自己的专业，阐述趋化因子及其受体的功能及可能的临床应用。,Thank you for your attention!,