鼠抗人4-1BBL单克隆抗体制备及其生物学功能的研究

发布时间：2018-04-12 07:23

本文选题：4-1BBL + 双向信号　；参考：《苏州大学》2010年硕士论文

【摘要】： 4-1BBL(CD137L)是Ⅱ型跨膜糖蛋白分子,与其受体4-1BB同为共刺激分子TNF/TNFR超家族中的重要成员。4-1BBL分子表达在各种活化的免疫细胞表面,如:B细胞、巨噬细胞、树突状细胞、活化的T细胞等免疫细胞,但在未活化的T细胞上不表达。另有研究显示,在某些肿瘤细胞表面也有4-1BBL分子的表达。4-1BBL的表达与恶性血液病有关:如恶性血液病患者的血清中可溶性4-1BBL(s4-1BBL)含量异常升高;另外,在某些T和B肿瘤细胞系,虽然4-1BBL/4-1BB呈较低的构成性共表达,但4-1BBL/4-1BB信号均可促进白血病细胞增殖并参与白血病细胞耐受抗肿瘤药物的机制。4-1BBL分子与其受体4-1BB结合后可介导双向信号。研究表明,两者相互作用产生的正向信号可促进活化的T细胞增殖,这一过程并不依赖CD28信号;而通过膜4-1BBL传递的逆向信号可以抑制活化T细胞增殖并诱导其凋亡;4-1BBL逆向信号还能诱导单核细胞活化,促进其IL-6、IL-8和TNF-α的分泌以及M-CSF的产生,并延长其生存;逆向信号还能刺激CD34+造血干细胞来源的DC成熟等,如4-1BBL逆向信号可以上调DC细胞上CD86、CD83的表达以及IL-12的分泌。因此,4-1BBL/4-1BB介导双向信号在T淋巴细胞、单核细胞及树突状细胞(DC)介导的免疫调节中发挥重要的作用。但这种4-1BB/4-1BBL双向信号在免疫应答和免疫调节中的作用和确切机理尚待深入研究和全面理解。为此,制备特异性抗人4-1BBL分子单克隆抗体,具备重要的研发价值。本实验制备了一株鼠抗人4-1BBL单克隆抗体39A8,能够识别多种肿瘤细胞株,同时以表达4-1BBL分子的人单核白血病细胞株U937、THP-1及胶质瘤细胞株675321为研究对象,初步研究了单克隆抗体39A8对肿瘤细胞体外生长的影响。本论文共分为两个部分。 1.鼠抗人4-1BBL单抗的杂交瘤细胞株的制备以基因转染细胞L929/4-1BBL作为免疫原,免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术,将免疫小鼠的脾脏和小鼠骨髓瘤细胞SP2/0进行细胞融合,用HAT选择培养基培养。以L929/4-1BBL为阳性筛选细胞株,以L929细胞为阴性对照,通过间接免疫荧光标记法对杂交瘤细胞株进行筛选。用有限稀释法进行阳性杂交瘤的亚克隆化培养。经过多次亚克隆及反复筛选,最终得到一株持续分泌抗人4-1BBL单克隆抗体的杂交瘤细胞株(39A8),经快速定性试纸法鉴定,39A8的重链IgG2a,轻链为κ链。杂交瘤细胞株经体外连续传代培养,液氮冻存半年后复苏,仍生长良好,稳定分泌抗体。 2.鼠抗人4-1BBL单克隆抗体的体外生物学特性研究间接免疫荧光法标记U937、THP-1、Hela、PC-3、Daudi、M231、SHG44等细胞,用流式细胞术分析细胞膜荧光标记百分率和荧光强度。结果显示,4-1BBL能不同程度地在某些肿瘤细胞株上表达。将不同浓度的4-1BBL单抗分别加入到天然表达4-1BBL分子的U937、THP-1、675321细胞的培养体系中,分别在培养24h、48h、72h和96h,观察细胞的生长状态、计数细胞数量并绘制生长曲线。研究结果显示:4-1BBL单抗39A8可促进上述三种细胞的生长与增殖。综上所述,本研究成功制备了一株特异性的鼠抗人4-1BBL单克隆抗体,进而鉴定了其生物学特性;利用抗人4-1BBL单抗初步探讨了4-1BBL逆向信号对肿瘤细胞的生物学效应。因此,这株功能性鼠抗人4-1BBL单克隆抗体的成功制备为其以后的生物学功能和信号途径的研究以及临床诊断试剂盒的研制提供了有效的物质基础。
[Abstract]:4-1BBL (CD137L) is a type II transmembrane glycoprotein, and its receptor 4-1BB is an important member of the.4-1BBL molecular TNF/TNFR superfamily expression on the surface of various activated immune cells such as B cells, macrophages, dendritic cells, activated T cells and other immune cells, but not expressed in non activated T cells. Other research shows that in some tumor cells also have the expression of.4-1BBL 4-1BBL molecules associated with malignant hematological diseases: such as serum of patients with malignant hematological diseases in 4-1BBL (s4-1BBL) levels were higher; in addition, T and B in some tumor cell lines, while 4-1BBL/4-1BB was a low CO expression, but 4-1BBL/4-1BB signal can promote two-way signal mediated molecular mechanism of.4-1BBL proliferation of leukemia cells and leukemia cells in tolerance of antitumor drugs after binding to its receptor 4-1BB. The results show that two The positive signals generated by the interaction can promote the proliferation of activated T cells, this process is not dependent on the CD28 signal; and reverse signal transfer through the membrane of 4-1BBL can inhibit the activation of proliferation and induce apoptosis of T cells; 4-1BBL reverse signaling can induce monocyte activation and promote its IL-6, IL-8 and TNF- secretion and M-CSF the produce, and prolong its survival; reverse signal can stimulate CD34+ hematopoietic stem cell derived DC mature, such as 4-1BBL reverse signal can up regulate DC cell CD86, CD83 expression and secretion of IL-12. Therefore, 4-1BBL/4-1BB mediated bidirectional signaling in T lymphocytes, monocytes and dendritic cells (DC) mediated immunity play an important role in the regulation of 4-1BB/4-1BBL. But the effect of this two-way signal in immune response and immune regulation and the exact mechanism still needs further research and comprehensive understanding. Therefore, the preparation of specific anti Human 4-1BBL monoclonal antibody, has important research value. A mouse anti human 4-1BBL monoclonal antibody 39A8 was prepared in this experiment, it can identify a variety of tumor cell lines, and 4-1BBL expression in human monocytic leukemia cell lines U937, THP-1 and glioma cell line 675321 as the research object, we studied the influence of monoclonal 39A8 antibody on the growth of tumor cells in vitro. This paper is divided into two parts.
Preparation of hybridoma cell line of 1. mouse anti human 4-1BBL monoclonal antibody
In transfected cells L929/4-1BBL as immunogen, BALB/c mice were immunized with B lymphocyte hybridoma technique, the immunized mouse spleen and mouse myeloma cell SP2/0 cell fusion by HAT selective medium. L929/4-1BBL was used as positive screening cell line, L929 cells as negative control. The hybridoma cell lines were screened by indirect immunofluorescence positive hybridoma by limited dilution of a Longhua culture. After many times of subcloning and repeated screening, finally get a continuous secretion of anti human 4-1BBL monoclonal antibody hybridoma cell line (39A8), the rapid qualitative identification method, the heavy chain IgG2a 39A8, light chain kappa chain. Hybridoma cell lines in vitro cultured and cryopreserved in liquid nitrogen after half a year, still grew well and stably secreting antibodies.
In vitro biological characteristics of 2. mouse anti human 4-1BBL monoclonal antibodies
Indirect immunofluorescence labeling of U937, THP-1, Hela, PC-3, Daudi, M231, SHG44 cell, analysis of cell membrane fluorescence labeling percentage and fluorescence intensity by flow cytometry. The results showed that 4-1BBL at different level of expression in some tumor cells. Different concentrations of 4-1BBL were added to the natural expression of 4-1BBL monoclonal antibody molecular U937 culture system of THP-1675321 cells, respectively in cultured 24h, 48h, 72h and 96h, observe cell growth state and the number of cells and the growth curve. The results showed that 4-1BBL mAb 39A8 could promote the growth and proliferation of the three cell types.
In summary, a strain specific mouse anti human 4-1BBL monoclonal antibody was prepared successfully in this study, and then identified its biological characteristics; using anti human 4-1BBL monoclonal antibody to investigate the biological effects of 4-1BBL on tumor cell reverse signal. Therefore, this strain was successfully functional mouse anti human 4-1BBL monoclonal antibody provides a material basis the prepared for the research the biological function and signaling pathways in the future and development of clinical diagnostic kit.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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