本文選題：B7-H1 + 單克隆抗體��； 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 目的:B7-H1是B7共刺激分子家族的成員之一,在正常的組織表面幾乎不表達(dá),但在腫瘤組織確是高表達(dá)的,而且腫瘤組織表面的組成型B7-H1的表達(dá)還與腫瘤的發(fā)生發(fā)展及預(yù)后相關(guān)。B7-H1通過(guò)與其抑制性受體結(jié)合,分別在初始T細(xì)胞的初級(jí)反應(yīng)階段和活化階段,記憶性T細(xì)胞的再次反應(yīng)階段起到重要的負(fù)性調(diào)節(jié)作用。B7-H1可誘導(dǎo)腫瘤抗原特異性CTL的凋亡,誘導(dǎo)腫瘤細(xì)胞的抗凋亡作用,在腫瘤的免疫逃逸、多藥耐藥、病毒感染、炎癥、自身免疫疾病和移植物耐受等多種病理情況下發(fā)揮著重要作用,是腫瘤的潛在靶標(biāo)和治療靶點(diǎn)。由于目前對(duì)B7-H1的研究均限于細(xì)胞或組織表面組成型表達(dá)的B7-H1分子,而我們的預(yù)試驗(yàn)發(fā)現(xiàn)腫瘤患者血清中有可溶性B7-H1的表達(dá),本課題擬通過(guò)證實(shí)血清中B7-H1可溶性片段的存在,探討其功能和與腫瘤發(fā)生發(fā)展之間的相關(guān)性;由于市售抗B7-H1單抗僅有一種,我們通過(guò)制備B7-H1的單克隆抗體,建立較為簡(jiǎn)便的B7-H1的夾心ELISA血清學(xué)臨床檢測(cè)方法。方法:首先,我們通過(guò)B細(xì)胞雜交瘤技術(shù)制備鼠抗人B7-H1單克隆抗體,為檢測(cè)血清中可溶性B7-H1片段奠定基礎(chǔ)。然后,我們將SP2/0細(xì)胞皮下注射Babl/c小鼠,構(gòu)建實(shí)體瘤模型,通過(guò)以下方法檢測(cè)荷瘤小鼠的血清中可溶性B7-H1的表達(dá):(1)采用Dot blot方法確定荷瘤小鼠血清中是否含有可溶性B7-H1;(2)采用Western blot方法鑒定可溶性B7-H1的分子量,以及其相對(duì)含量和小鼠荷瘤時(shí)間、瘤重的相關(guān)性,為研究腫瘤患者的可溶性B7-H1與其病程的相關(guān)性提供依據(jù)。最后,對(duì)肺癌患者血清及組織中可溶性和組成型的B7-H1進(jìn)行了檢測(cè),包括:(1)采用Dot blot方法確定肺癌患者血清中是否含有可溶性B7-H1片段;(2)采用Western blot方法確定可溶性B7-H1片段的分子量;(3)建立可溶性B7-H1的夾心ELISA臨床檢測(cè)方法,并對(duì)10例腫瘤患者血清的可溶性B7-H1含量進(jìn)行測(cè)定。(4)對(duì)肺癌病人的腫瘤組織進(jìn)行免疫組化染色以確定其有組成型B7-H1的表達(dá)。結(jié)果:用人源性B7-H1IgV樣區(qū)混合佐劑免疫Babl/c小鼠,最終獲得了一株高親和力的單克隆抗體。對(duì)荷瘤小鼠的血清檢測(cè)結(jié)果:(1)Dot blot結(jié)果顯示荷瘤小鼠血清中確含有可溶性B7-H1片段,且不同個(gè)體的含量不同;(2)Western blot結(jié)果顯示,可溶性片段的分子量約為37kD,但其相對(duì)含量與荷瘤時(shí)間和瘤重?zé)o明顯相關(guān)性。對(duì)肺癌患者血清和腫瘤組織的檢測(cè):(1)Dot blot結(jié)果顯示肺癌患者血清中確含有可溶性B7-H1片段,且不同個(gè)體的含量不同;(2)Western blot結(jié)果顯示,可溶性片段的分子量約為42kD;(3)建立了夾心ELISA檢測(cè)血清中可溶性B7-H1含量的檢測(cè)方法,并分析10例患者血清中可溶性B7-H1的含量,但由于缺少臨床病例資料,暫時(shí)還無(wú)法對(duì)此含量和癌癥發(fā)生發(fā)展的關(guān)系作出評(píng)價(jià);(4)對(duì)采血對(duì)象的腫瘤組織免疫組化結(jié)果顯示,腫瘤組織胞漿中有組成型的B7-H1表達(dá)。結(jié)論:成功地制備了一株單克隆抗體。建立了血清可溶性B7-H1的檢測(cè)方法,并成功證實(shí)了可溶性B7-H1片段的存在,為臨床檢測(cè)奠定了基礎(chǔ)�？扇苄訠7-H1與腫瘤發(fā)生發(fā)展以及預(yù)后的相關(guān)性仍需進(jìn)一步研究。
[Abstract]:Objective: B7-H1 is one of the members of the B7 costimulatory family, almost non expression on the normal tissue surface, but it is highly expressed in the tumor tissue, and the expression of the constituent B7-H1 on the surface of the tumor is associated with the development and prognosis of the tumor, and.B7-H1 is combined with its inhibitory receptor, in the primary T cells, respectively. At the stage and activation stage, the rereaction stage of the memory T cells plays an important negative regulatory role,.B7-H1 can induce the apoptosis of tumor antigen specific CTL, induce the anti apoptosis effect of tumor cells, the immune escape of the tumor, multidrug resistance, virus infection, inflammation, autoimmune disease and graft tolerance, and so on. It plays an important role as the potential target and therapeutic target of tumor. Since the current research on B7-H1 is limited to the B7-H1 molecules expressed in cell or tissue surface, our pre test found the expression of soluble B7-H1 in the serum of the tumor patients. This topic is to explore the existence of B7-H1 soluble fragments in the serum of the patients. Function and the correlation with the development of tumor; because of the only one kind of anti B7-H1 monoclonal antibody sold in the market, we establish a simple B7-H1 sandwich ELISA serological detection method by preparing the monoclonal antibody of B7-H1. First, we prepared the anti human B7-H1 monoclonal antibody by B cell hybridoma technique, to detect the serum The basis of soluble B7-H1 fragment was established. Then, we subcutaneously injected SP2/0 cells into Babl/c mice to construct a solid tumor model and detected the expression of soluble B7-H1 in the serum of the tumor bearing mice by the following methods: (1) the Dot blot method was used to determine whether the serum contained soluble B7-H1 in the tumor bearing mice; (2) the soluble Western blot method was used to identify the soluble B7-H1. The molecular weight of sex B7-H1, its relative content and the correlation of tumor time and tumor weight in mice provide the basis for the study of the correlation between soluble B7-H1 and its course of disease in cancer patients. Finally, the soluble and constituent B7-H1 in serum and tissue of lung cancer patients were detected, including: (1) Dot blot method was used to determine the sera of patients with lung cancer Whether or not contains soluble B7-H1 fragments; (2) Western blot method was used to determine the molecular weight of soluble B7-H1 fragments; (3) a sandwich ELISA clinical detection method for soluble B7-H1 was established and the content of soluble B7-H1 in serum of 10 patients with tumor was measured. (4) immunohistochemical staining of the tumor tissues of the patients with lung cancer to determine its group The expression of formed B7-H1. Results: a human B7-H1IgV like region mixed adjuvant was used to immunization of Babl/c mice and a high affinity monoclonal antibody was finally obtained. The results of serum detection for tumor bearing mice: (1) Dot blot results showed that the serum contained soluble B7-H1 segments in the tumor bearing mice, and the content of different individuals was different; (2) Western blot knot. The results showed that the molecular weight of the soluble fragment was about 37kD, but the relative content had no significant correlation with the tumor bearing time and tumor weight. The detection of serum and tumor tissue in the patients with lung cancer: (1) Dot blot results showed that the serum of the lung cancer patients had soluble B7-H1 fragments, and the contents of different bodies were different; (2) the Western blot results showed the solubility. The molecular weight of the fragment was about 42kD; (3) the determination of soluble B7-H1 content in serum by sandwich ELISA was established, and the content of soluble B7-H1 in serum of the patients was analyzed, but the relationship between the content and the development of cancer was not evaluated for the lack of clinical data. (4) the tumor tissue immunization of the blood samples. The histochemical results showed that there was a composition of B7-H1 expression in the cytoplasm of the tumor. Conclusion: a monoclonal antibody was successfully prepared. The detection method of soluble B7-H1 in serum was established and the existence of soluble B7-H1 fragment was successfully confirmed. The correlation between soluble B7-H1 and the development of tumor and prognosis of tumor was established. Further research is still needed.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

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