血清中可溶性B7-H1检测方法的建立及临床意义研究

发布时间：2018-06-13 00:39

本文选题：B7-H1 + 单克隆抗体　；参考：《第四军医大学》2010年硕士论文

【摘要】： 目的:B7-H1是B7共刺激分子家族的成员之一,在正常的组织表面几乎不表达,但在肿瘤组织确是高表达的,而且肿瘤组织表面的组成型B7-H1的表达还与肿瘤的发生发展及预后相关。B7-H1通过与其抑制性受体结合,分别在初始T细胞的初级反应阶段和活化阶段,记忆性T细胞的再次反应阶段起到重要的负性调节作用。B7-H1可诱导肿瘤抗原特异性CTL的凋亡,诱导肿瘤细胞的抗凋亡作用,在肿瘤的免疫逃逸、多药耐药、病毒感染、炎症、自身免疫疾病和移植物耐受等多种病理情况下发挥着重要作用,是肿瘤的潜在靶标和治疗靶点。由于目前对B7-H1的研究均限于细胞或组织表面组成型表达的B7-H1分子,而我们的预试验发现肿瘤患者血清中有可溶性B7-H1的表达,本课题拟通过证实血清中B7-H1可溶性片段的存在,探讨其功能和与肿瘤发生发展之间的相关性;由于市售抗B7-H1单抗仅有一种,我们通过制备B7-H1的单克隆抗体,建立较为简便的B7-H1的夹心ELISA血清学临床检测方法。方法:首先,我们通过B细胞杂交瘤技术制备鼠抗人B7-H1单克隆抗体,为检测血清中可溶性B7-H1片段奠定基础。然后,我们将SP2/0细胞皮下注射Babl/c小鼠,构建实体瘤模型,通过以下方法检测荷瘤小鼠的血清中可溶性B7-H1的表达:(1)采用Dot blot方法确定荷瘤小鼠血清中是否含有可溶性B7-H1;(2)采用Western blot方法鉴定可溶性B7-H1的分子量,以及其相对含量和小鼠荷瘤时间、瘤重的相关性,为研究肿瘤患者的可溶性B7-H1与其病程的相关性提供依据。最后,对肺癌患者血清及组织中可溶性和组成型的B7-H1进行了检测,包括:(1)采用Dot blot方法确定肺癌患者血清中是否含有可溶性B7-H1片段;(2)采用Western blot方法确定可溶性B7-H1片段的分子量;(3)建立可溶性B7-H1的夹心ELISA临床检测方法,并对10例肿瘤患者血清的可溶性B7-H1含量进行测定。(4)对肺癌病人的肿瘤组织进行免疫组化染色以确定其有组成型B7-H1的表达。结果:用人源性B7-H1IgV样区混合佐剂免疫Babl/c小鼠,最终获得了一株高亲和力的单克隆抗体。对荷瘤小鼠的血清检测结果:(1)Dot blot结果显示荷瘤小鼠血清中确含有可溶性B7-H1片段,且不同个体的含量不同;(2)Western blot结果显示,可溶性片段的分子量约为37kD,但其相对含量与荷瘤时间和瘤重无明显相关性。对肺癌患者血清和肿瘤组织的检测:(1)Dot blot结果显示肺癌患者血清中确含有可溶性B7-H1片段,且不同个体的含量不同;(2)Western blot结果显示,可溶性片段的分子量约为42kD;(3)建立了夹心ELISA检测血清中可溶性B7-H1含量的检测方法,并分析10例患者血清中可溶性B7-H1的含量,但由于缺少临床病例资料,暂时还无法对此含量和癌症发生发展的关系作出评价;(4)对采血对象的肿瘤组织免疫组化结果显示,肿瘤组织胞浆中有组成型的B7-H1表达。结论:成功地制备了一株单克隆抗体。建立了血清可溶性B7-H1的检测方法,并成功证实了可溶性B7-H1片段的存在,为临床检测奠定了基础。可溶性B7-H1与肿瘤发生发展以及预后的相关性仍需进一步研究。
[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.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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