本文選題：突變體 + CD40��； 參考：《蘇州大學(xué)》2007年碩士論文

【摘要】： CD40是分子量為45～50KD的Ⅰ型跨膜糖蛋白,屬于腫瘤壞死因子受體(TNFR)超家族成員,表達(dá)于多種抗原遞呈細(xì)胞、內(nèi)皮細(xì)胞、上皮細(xì)胞、造血前體細(xì)胞、成纖維細(xì)胞以及某些腫瘤細(xì)胞。通過(guò)單克隆抗體激發(fā)CD40分子不僅可以直接作用于表達(dá)CD40分子的腫瘤細(xì)胞,其介導(dǎo)的信號(hào)還可在多個(gè)環(huán)節(jié)影響腫瘤發(fā)生、發(fā)展,如調(diào)控細(xì)胞增殖/凋亡,增強(qiáng)其它治療手段的敏感性,此外還能通過(guò)調(diào)節(jié)適應(yīng)性和固有免疫應(yīng)答而起到抗腫瘤作用。然而由于CD40在體內(nèi)正常組織的廣泛表達(dá),三株抗人CD40單抗雖然已進(jìn)入美國(guó)臨床前期試驗(yàn),但其存在的副作用和安全性問(wèn)題仍引起高度關(guān)注。本科室以往的研究發(fā)現(xiàn)一些腫瘤細(xì)胞株和新鮮腫瘤細(xì)胞表達(dá)CD40突變體(CD40mu)(CAC→CAA,78His→78Gln) (NCBI Assay Id: ss23134804,Reference SNP Id:rs17177493),該突變位點(diǎn)位于CD40胞外段第二個(gè)富含半胱氨酸的區(qū)域,這一區(qū)域是CD40與CD40L(CD154)相互作用的重要區(qū)域�；蜣D(zhuǎn)染細(xì)胞的構(gòu)建進(jìn)一步證實(shí)了該突變體與抗體的結(jié)合能力與野生型CD40截然不同,CD40mu胞外段一個(gè)氨基酸的突變可導(dǎo)致局部結(jié)構(gòu)改變而形成新的抗原表位,該抗原表位為CD40mu所特有,CD40mu在腫瘤中的表達(dá)特點(diǎn)和生物學(xué)意義以及單抗激發(fā)CD40mu對(duì)腫瘤細(xì)胞生物學(xué)行為的影響有待進(jìn)一步研究。 1.分泌鼠抗人CD40mu單抗的雜交瘤細(xì)胞株的制備 利用已成功構(gòu)建的基因轉(zhuǎn)染細(xì)胞L929/CD40mu為免疫原,免疫BALB/c小鼠,采用B淋巴細(xì)胞融合技術(shù),將反復(fù)免疫后的小鼠脾臟細(xì)胞與小鼠骨髓瘤細(xì)胞SP2/0進(jìn)行細(xì)胞融合,以突變體基因轉(zhuǎn)染細(xì)胞L929/CD40mu為陽(yáng)性篩選細(xì)胞,以野生型CD40基因轉(zhuǎn)染細(xì)胞L929/CD40wt為陰性篩選細(xì)胞,通過(guò)間接免疫熒光標(biāo)記法對(duì)雜交瘤細(xì)胞株進(jìn)行篩選,經(jīng)多次克隆化培養(yǎng),最終獲得四株持續(xù)、穩(wěn)定分泌鼠抗人CD40mu單克隆抗體的雜交瘤細(xì)胞株,分別命名為3G5、5H6、3B9和2B6,前三株抗體的重鏈為IgG1,2B6的重鏈為IgG2a,輕鏈均為κ鏈。四株抗體均可用于免疫組織化學(xué)分析,其中3G5和2B6可用于Western-blot檢測(cè)。雜交瘤細(xì)胞株經(jīng)體外連續(xù)傳代(40代)培養(yǎng),液氮凍存半年后復(fù)蘇,仍生長(zhǎng)良好,穩(wěn)定分泌抗體。 2.鼠抗人CD40mu單克隆抗體的體外生物學(xué)特性研究 運(yùn)用成功制備的單抗檢測(cè)發(fā)現(xiàn),部分惡性B細(xì)胞株及上皮性腫瘤細(xì)胞株表達(dá)CD40mu,其在新鮮腫瘤組織中也有高水平的表達(dá)。四株抗體識(shí)別的抗原位點(diǎn)不同,對(duì)細(xì)胞株的識(shí)別譜也不相同。繼而采用抗CD40mu單抗對(duì)表達(dá)CD40mu的人卵巢癌細(xì)胞株HO8910和人白血病細(xì)胞株K562細(xì)胞的體外實(shí)驗(yàn)初步表明,單抗2B6能夠促進(jìn)HO8910細(xì)胞和K562細(xì)胞的生長(zhǎng),促使HO8910細(xì)胞進(jìn)入S期的比例增加和細(xì)胞因子IL-6的分泌,而另一株單抗3G5則能夠誘導(dǎo)表達(dá)CD40mu分子的細(xì)胞株HO8910細(xì)胞凋亡。 綜上所述,本研究成功制備了四株特異性鼠抗人CD40mu單克隆抗體,并初步探討了CD40mu在腫瘤中的表達(dá)及其生物學(xué)意義,證實(shí)了腫瘤細(xì)胞表達(dá)突變的CD40分子;不同的單抗激發(fā)表達(dá)該突變體的腫瘤細(xì)胞后引起的生物學(xué)效應(yīng)也不同,或促進(jìn)細(xì)胞增殖或誘導(dǎo)其凋亡。這些特異性抗體的獲得為深入探討腫瘤細(xì)胞表達(dá)CD40mu分子的生物學(xué)意義提供了物質(zhì)基礎(chǔ),也為靶向CD40mu的腫瘤生物治療提供了新思路。
[Abstract]:CD40 is a type I transmembrane glycoprotein with a molecular weight of 45 to 50KD. It belongs to the member of the tumor necrosis factor receptor (TNFR) superfamily. It is expressed in a variety of antigen presenting cells, endothelial cells, epithelial cells, hematopoietic progenitor cells, fibroblasts and some tumor cells. Using monoclonal antibodies to stimulate CD40 molecules can not only directly affect the expression of CD40. Molecular tumor cells, its mediated signal can also affect the occurrence and development of tumors in many links, such as regulating cell proliferation / apoptosis, enhancing the sensitivity of other therapies, in addition to regulating adaptive and inherent immune responses. However, three anti human CD strains are widely expressed in normal tissues in the body. 40 McAbs have entered preclinical trials in the United States, but their side effects and safety problems still attract high attention. Previous studies in the undergraduate laboratory found that some tumor cell lines and fresh tumor cells expressed CD40 mutants (CD40mu) (CAC to CAA, 78His to 78Gln) (NCBI Assay Id: ss23134804, Reference SNP). The mutation loci is located in second cysteine rich regions of the CD40 extracellular segment. This region is an important region of the interaction between CD40 and CD40L (CD154). The construction of gene transfected cells further confirms that the binding ability of the mutant to the antibody is very different from that of the wild type CD40. The mutation of one amino acid in the extracellular segment of CD40mu can lead to the local structure. A new epitope is formed, the epitope of the antigen is unique to CD40mu. The expression and biological significance of CD40mu in the tumor and the effect of CD40mu on the biological behavior of the tumor cells need to be further studied.
Preparation of 1. hybridoma cell lines secreting mouse anti human CD40mu monoclonal antibody
Using the successfully constructed gene transfected cell L929/CD40mu as immunogen, immunized BALB/c mice and using B lymphocyte fusion technique, the spleen cells after repeated immunization were fused with the murine myeloma cells SP2/0, and the mutant gene transfected L929/CD40mu was the positive screening cell, and the transfection of the wild type CD40 gene was fine. The cell L929/CD40wt was negative screening cells, and the hybridoma cell lines were screened by indirect immunofluorescence. After multiple cloning and culture, four hybridoma cell lines that secreted the anti human CD40mu monoclonal antibody were obtained, which were named 3G5,5H6,3B9 and 2B6 respectively. The heavy chain of the first three antibodies was IgG1,2B6 to Ig. G2a, light chain is kappa chain. Four antibodies can be used in immunohistochemical analysis, in which 3G5 and 2B6 can be used for Western-blot detection. Hybridoma cell lines are cultured in vitro (40 generations), and the liquid nitrogen is resuscitated for half a year after the cryopreservation. It still grows well and secretes antibodies steadily.
In vitro biological characteristics of 2. mouse anti human CD40mu monoclonal antibody
Using the monoclonal antibody test prepared successfully, some malignant B cell lines and epithelial tumor cell lines expressed CD40mu, and they also expressed high levels in the fresh tumor tissues. The antigen loci identified by the four antibodies were different and the identification spectrum of the cell lines were different. Then, the anti CD40mu monoclonal antibody was used for the human ovarian cancer cell line HO expressing CD40mu. In vitro experiments of 8910 and human leukemia cell line K562 cells showed that McAb 2B6 could promote the growth of HO8910 cells and K562 cells, increase the proportion of HO8910 cells into S phase and the secretion of cytokine IL-6, while another monoclonal antibody 3G5 can induce apoptosis of HO8910 cells that express CD40mu molecules.
To sum up, four monoclonal anti human CD40mu monoclonal antibodies were successfully prepared, and the expression and biological significance of CD40mu in the tumor were preliminarily discussed, and the CD40 molecules expressed in the tumor cells were confirmed, and the biological effects caused by different monoclonal antibodies to express the mutant tumor cells were different, or promoted. The acquisition of these specific antibodies provides a material basis for exploring the biological significance of the expression of CD40mu molecules in tumor cells, and also provides a new way of thinking for the biological treatment of tumor targeting CD40mu.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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相關(guān)期刊論文 前2條

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