【作者】 俞静；

【导师】 林茂芳；

【作者基本信息】 浙江大学 ， 内科学， 2005， 博士

【摘要】 树突状细胞(DC)是免疫系统强有力的抗原递呈细胞,在免疫应答中起关键的引导和调节作用,其成熟程度、亚群分类决定着免疫激活与免疫耐受之间的平衡,DC的成熟程度为引发T淋巴细胞依赖性免疫应答必需的条件之一。因此,探讨与DC相关的免疫耐受机理已成为移植研究工作者关注的热点之一。抑制性DC是移植免疫学提出的新概念,而未成熟DC则是其中之一。未成熟DC因其低表达协同刺激分子,诱导T淋巴细胞无能及凋亡,在免疫耐受中具有重要的意义。Hirano等报道,在异基因移植动物模型中,输注未成熟DC后不会产生排斥。因此,通过一定的手段诱导产生未成熟DC,使受者建立长期的免疫耐受,可能是一种有前途的治疗手段。 CD47分子是近些年发现的与机体免疫系统、神经系统有密切关系的膜糖蛋白,广泛地表达于所有组织的不同细胞上。已报道,在人DC有CD47抗原表达,本研究结果也显示,人未成熟DC和成熟DC表面均有CD47高表达(94%-98%)。CD47与其配体整合素、信号调节蛋白α(SIRP-α)、凝血酶敏感蛋白(TSP-1)相互作用对机体发挥着多重免疫调控作用,如CD47/SIRP-α可抑制中性粒细胞、单核细胞的迁移及黏附过程:CD47/TSP复合体可诱导T淋巴细胞、DC及单核细胞发生凋亡等,故运用生物手段在体外合成CD47的各种“模拟”配体以干预机体免疫已成为目前许多研究者研究的重点之一。国外有研究报道,采用人工手段合成的各种针对CD47抗原表位的抗体或抗体片段能诱导人T淋巴细胞无能并阻止DC成熟,因此本实验选用B6H12单抗与CD47分子相互作用,以观察是否能干扰DC的分化成更多还原

【Abstract】 Dendritic cells (DCs) are important functional antigen-presenting cells that play an essential role in initiating and modulating immune responses.The maturation and subtypes of DC determine the balance of immune activation and tolerance .Their mature states are crucial for the induction of T-cell-mediated immune reactions. "Tolerogenic DCs" stand for a new concept in the immunology of transplantation and immature DCs belong to this category.Characteristics of the low expressions of co-stimulatory molecules and the maintenance of T cell anergy have endowed immature DCs with important significance in immune tolerance. Hirano et al. have successfully induced permanent tolerance in an allograft model by intravenous injection of immature DCs. Therefore, the mechanism of T cell anergy related with DCs and research in applying immature DCs to establish donor-specific immune tolerance have become one of the new frontiers in the field of transplantation immunity.The CD47 Ag, an Ig superfamily transmembrane glycoprotein widely expressed in most cells and tissues(including DCs),has been found to be intimately related with the immune system and nervous system.Our data also showed the expressions of CD47 on immature and mature DCs were high ,which were about to 94%-98% level.The CD47 molecule has multiple modulations on the immune system with its ligands—integrins.SlRPs and TSPs.The CD47 ligands can serve to a"fine tune " the inflammatory response by regulating the rate of PMN and monocytes migration and inducing T cell.DCs and monocytes into apoptosis. Therefore synthesizing variable artifical ligands to mimic the effects of CD47 engagement to interfere with the immune response has become an new highlight to the immunologists.lt reported that the artifical mAb of CD47 molecule can make T cell anergy and inhibit the maturation of DCs.However ,none research relative to this subject has been proceeded in China. In this study, the soluble anti-CD47 mAb,B6H12, was selected as an ligand of CD47 molecule, whether any observed influence on DCs maturation and function,which retain DCs in "an relatively immature stage" ,could be ascribed to the effects of this CD47 ligation.Thus it may enrich the knowledge of the potential application of the engagement.In order to explore the influences of CD47 ligation on DCs maturation,function and cell signal transduction,we established a stable culture system of DCs in vitro firstly.The study aimed to enucleate the probable immune mechanisms of the integrin associated protein with its ligand and to find the latent signal transduction pathway related with the maturation of DCs.The results of the optical and transmission electron microscope revealed that the peripheral monocyte progenitors successfully developed into DCs with the combination of cytokine, GM-CSF, 1L-4 and lipopolysaccharide in our culture system of DCs. The immature DCs, appeared to have much relatively shorter processes on the surface and fewer organells in the cytoplasm compared with its counterpart — mature DCs,which turned to have even more abundant cytoplasmic extensions, more mitochondrias and less lysosomes,irregular large nuclei in the cytoplasm. DCs treated with anti-CD47 mAb showed a semblable outline with the immature DCs.The shorter dendrites and fewer organells distinguished them from the mature DCs and the control mAb treated DCs.Compared with the other two groups in which DCs were treated with or without the isotype control mAb, the expressions of CD80, CD86, CD83, CDla, HLA-DR in B6H12 mAb(10μg/ml)treated group decreased significantly via flow cytometry analysis.The differences were significant between groups, [CD80 (F=9.924, P<0.05) ,CD86 (F=l8.461, P<0.01 ) ,CD83(F=6.012, P<0.05),CDIa(F=58.021, P<0.01 ),HLA-DR(F=53.174, P<0.01 )], The phenotypes of DCs treated with or without B6H12 mAb were as follow: CD80~+(68.14±7.41)% vs(89.17± 8.59)%,CD86~+(67.33 + 4.71)% vs(87.27 + 3.56)%,CD83~+(40.08 + 14.80)% vs(72.77 ± 8.68)%,CDla~+(66.45±4.06)% vs (95.93 + 3.03) %, HLA-DR~+(40.67± 13.48)% vs(98.97± 1.01)%.Allo-MLR was更多还原