【作者】 孙静；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2006， 硕士

【摘要】 Bretscher和Cohn在T细胞活化双信号模型的基础上提出“协同刺激信号”，即T细胞的活化除了需要通过APC递呈MHC-抗原肽给抗原特异性T细胞提供第一信号外，还需要一系列协同刺激分子提供第二信号，才能使T细胞达到生理活化阈值产生正常的免疫应答。如果缺少共刺激分子提供的第二信号，将会导致T细胞的无反应性或特异性免疫耐受甚至凋亡。正性和负性协同刺激信号的调节及两者之间的平衡在机体免疫应答的整个过程中起着重要的调节作用。 共刺激分子分为TNF／TNFR和B7／CD28两大超家族。近几年，B7家族成员不断扩大，其中PD-L1是新近发现的共刺激分子之一。PD-L1具有IgV和IgC样区、跨膜区和胞浆区，具有广泛的组织表达谱，甚至在一些肿瘤细胞株上也有较高表达。PD-1是PD-L1的一个抑制性受体，主要表达在活化的T细胞上，PD-1／PD-L1途径在抑制T细胞增殖的同时，也影响很多细胞因子的分泌。同时，PD-L1在肿瘤细胞上的高表达，能减弱机体抗肿瘤免疫应答的作用，可能与肿瘤的免疫逃避机制密切相关，阻断此途径可增强机体的抗肿瘤免疫应答。大量研究显示PD-L1在机体免疫应答的调控方面发挥着重要的作用，PD-L1可能会成为肿瘤治疗中新的靶分子。更为重要的是，目前关于PD-L1对T细胞的生物学作用，不同的研究小组报道了截然不同的结果，一些研究表明，PD-L1还能作用T细胞促进其显著增殖和IL-2的分泌，具体机制尚不明了。 本研究课题首先利用天然高表达PD-L1的人乳腺癌细胞株MDA-MB-231细胞为免疫原免疫BALB／c小鼠，采用B淋巴细胞融合技术，将反复免疫后的小鼠脾脏细胞与小鼠骨髓瘤细胞SP2／0进行细胞融合，以转基因细胞L929／PD-L1作为阳性筛选细胞，以转空质粒的对照细胞L929／mock作为阴性对照细胞，经免疫荧光标记分析及抗体分泌阳性杂交瘤细胞的反复筛选，并经多次克隆化培养，最终获得1株持续、稳定分泌鼠抗人PD-L1单克隆抗体的杂交瘤细胞株，命名为10E10。经快速定性试纸法分析鉴定，10E10的重链为IgG1：而轻链为κ，可用于Western-blot和免疫组织化学分析。杂交瘤细胞株体外连续传代(40代)培养，液氮冻存半年后复苏，仍生长良好，稳定分泌抗体。更多还原

【Abstract】 Since it was proposed in 1970, the two-signal hypothesis for T lymphocyte activation has become widely accepted that T cells require two distinct signals for expansion. This model hypothesizes that peptides presented to antigen-specific T cells by the MHC molecule delivers the first signal, whereas costimulatory molecules (CMs) on T cell surface trigger the second signal. Costimulatory signals (positive or negative), play an important role in regulating the balance of immune responses.The most intensively characterized CMs are members of the B7 family. A number of new members (B7-H1, B7-H2, B7-H3, B7-DC) of the B7 family have recently been identified. It has been proposed that they regulate the function and differentiation of effector lymphocytes in the periphery. Recently, B7-H1 (PD-L1) and B7-DC (PD-L2) were identified as ligands for PD-1 with different binding properties. PD-L1 is expressed in a broad spectrum of tissues including some malignant tumors, suggesting the potential mechanism of immune evasion. Therefore, the enhancement of T cell responses via blockade of PD-1/PD-L1 pathway may be a potential therapeutic strategy for tumor treatment.BALB/c mice were immunized with human breast caicinoma cell line MDA-MB-231, which highly expresses PD-L1 molecule, and the splenocytes were fused with murine myeloma SP2/0 cells by the cell fusion hybridoma technique. By means of multiple cell subcloning and repeated screening with L929-PD-L1 as antibody screening positive cell and L929-mock as negative control, a hybridoma cell line (named as 10E10) stably secreting anti-PD-L1 monoclonal antibodies was selected out. Then the Ig isotype was identified with test paper. The heavry chain of 10E10 was mouse IgG1, and the light chain belonged to k. The mAb 10E10 could be used for Western-blot and immunohistochemistry assays. The hybridoma cells grew well after long-term culture in vitro (40 passages) and storage in liquid nitrogen.更多还原