【作者】 周桓；

【导师】 顾宗江； 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2005， 硕士

【摘要】 CD40分子属于肿瘤坏死因子受体(TNFR)超家族成员,为Ⅰ型跨膜糖蛋白。人CD40分子于八十年代中期被发现,分子量约为48KD,其基因定位于20q11-20q13.2,cDNA长约1.5KD编码277个氨基酸(AA),其中氨基末端193个AA构成胞外段,22个AA构成跨膜段,66个AA构成胞浆段。CD40分子介导的信号在特异性免疫应答中居于重要的上游位置。该分子广泛表达在免疫系统以及非免疫系统的许多类型的细胞表面,包括B细胞,树突状细胞,上皮细胞以及一些肿瘤细胞如B细胞淋巴瘤。B细胞淋巴瘤是起源于B淋巴细胞的恶性肿瘤,常全身广泛转移,表现为多器官侵犯。随着肿瘤的放、化疗技术的发展,大约50%-60%的B细胞淋巴瘤病人获得临床缓解。然而残余的恶性细胞导致的肿瘤复发常有发生。本室研制的激发型CD40单克隆抗体5cll对B细胞淋巴瘤的生物学行为有多重影响,包括:生长抑制;膜表面粘附分子表达变化;凋亡敏感性的提高等等。体外实验结果提示了5cll对B细胞淋巴瘤具有潜在治疗价值。 SCID小鼠即重症联合免疫缺陷(Severe Combined Immune deficiency)小鼠,1983年由美国学者Bosma首先发现于C.B-17近交系的小鼠,是由于位于16号染色体的单个隐性基因突变所致。SCID小鼠的所有T、B淋巴细胞功能测试均为阴性,对外源性抗原无细胞免疫及体液免疫应答,体内缺乏携带前B细胞、B细胞和T细胞表面标志的细胞。SCID小鼠由于T、B淋巴细胞的缺失,为人类淋巴系统在其体内的重建提供了可能,即SCID小鼠的人源化。这种人、鼠嵌合体为在小鼠体内有效地研究人类免疫系统的功能及其机制成为可能,同时也为肿瘤免疫学治疗的研究提供了一个独特的动物模型。 本课题的研究目的是建立人源化SCID小鼠人B细胞淋巴瘤模型,在此基础上探讨激发型抗人CD40单克隆抗体5cll联合CTL对B细胞淋巴瘤的免疫治疗作用。更多还原

【Abstract】 CD40, a member of TNFR superfamily, is a type I membrane glycoprotein with the Mr of 48KD. Human CD40 molecule was found in 1980s and encoded by a gene located at 20q11-20q13.2. Its cDNA with the length of 1.5KD encoded 277 amino acid (AA). Interaction by CD40/CD40L is a main signal during immune response. CD40 is expressed on many types of cells including B cells, dendritic cells, endothelial cells epithelial cells and also many carcinoma cells, such as B lymphoma cells. B lymphoma is malignant tumor mostly derived from B cells in lymphoid tissues. With the significant advance in chemotherapy and radiotherapy, about 50-60% patients suffering from this disease can achieve complete regression (CR). However, in many cases residual tumor cells always cause the recrudescence of the same disease. 5cll is an agonist monoclonal antibody prepared in our department. Treatment with 5c11 influenced biological behavior of B lymphoma cell line Daudi including proliferation inhibition, cell cycle arrest and enhancement of the sensitivity to apoptosis. Experimental results indicated the significant therapeutic potential of 5c11.The SCID mouse is of particular interest because of the absence of mature and functional T and B lymphocytes. Due to a defect in the recombinase system, linked to a nonsense mutation of DNA-dependent protein kinase catalytic subunit, SCID mice cannot successfully rearrange their immune receptor genes and consequently have no mature T or B cells. Thus, SCID mice can tolerate a graft with human cells and particularly those of purified peripheral blood mononuclear cells (PBMC) administered by intraperitonealinjection. The reconstituted SCID mice also called humanized SCID (hu-SCID) mouse, provide a unique model to particularly study tumor immunotherapy in an in vivo animal system.In this study we aimed to establish hu-SCID mouse human B lymphoma model and study the therapeutic effect of 5c 11, CTL or 5c 11 combined with CTL in this model.1. Establishment and identification of hu-SCID mouse medelSCID mice were treated by CTX to inhibit the hemocytopoiesis. With successive 4-day injection, human peripheral blood mononuclear cells (PBMC) were engrafted into SCID mice through intraperitoneal injection. After 4, 8 and 12 weeks of engraftment , peripheral blood, spleen and liver tissues of engrafted SCID mice were harvested. Human CD3+, CD19+ cells in peripheral blood were analyzed by fluorescence microscopy and FCM; human CD3+, CD19+ cells in spleen and liver tissues were detected by immune histochemistry; human IgG level in SCID mouse serum was measured by ELISA. Results showed that after engraftment of 4, 8 and 12 weeks, human CD3+, CD19+ cells in SCID peripheral blood were identified by fluorescence microscopy and the percents were 31%> 10% respectively determined by FCM. Through immune histochemistry human CD3+> ’ CD19+ cells were detected in mouse spleen but not in liver tissue. Furthermore the titer of human IgG in mouse serum was 390ug/mh 1 lOOug/ml and 1040|xg/ml at each time point respectively.2. 5cll promoted the cytotoxity of CTL to B lymphoma cell line Daudi cells in vitroHuman B lymphoma cell line, Daudi cells were treated with 5ug/ml CD40 mAb (5C11) for 24 hours. Annexin V/PI binding assay was used to analyze apoptosis, FACS to analyze expression of Fas (CD95). Human PBMC derived DCs was loaded with apoptotic Daudi cells and stimulated by 5c 11 for additional 48 hours to maturation. Tumor specific CTL was generated in vitro by autologous T cells co-cultured with mature DCs. Daudi cells were co-cultured with 5c 11, CTL or 5c 11 combined with CTL. DNA fragmentations were detected by JAM assay. The results showed that 5c 11 did not induce Daudi cells apoptosis, but could up-regulate the expression of CD95. CTL could lyse Daudi cellseffectively after co-cultured for 8 hours and the fragmentation of Daudi cells wassignificantly enhanced by the combination of 5c 11.3. Therapeutic effect of 5cll, CTL and Sell combined with CTL on B lymphomaB lymphoma model was established in humanized SCID mouse (hu-SCID) by subcutaneous injecting Daudi cells. 1 week and 3 weeks after transplantation, the mice were regarded as minimal tumor burden and extensive tumor burden mice respectively. Tumor bearing mice were treated with 5cll, CTL or 5cll combined with CTL respectively by intraperitoneal injection. Results showed that all the treatments could postpone the growth of tumor in vivo and significantly prolonged the survival of tumor bearing mice. Minimal tumor burden mice got the CR of 33% or 67% respectively when received CTL treatment or the combination treatment of 5cl 1 and CTL. Furthermore, 5cl 1 also promoted the secretion of IFN-y in vivo. CD25+ cells could be detected in the tumors of mice treated by CTL or 5c 11 combined with CTL, and the amount in combination treatment mice was more than that of CTL single treatment.更多还原