血清中可溶性B7-H1检测方法的建立及临床意义研究
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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。
Objective: B7-H1 is one of the B7 costimulatory molecule family members. It hardly express on the surface of normal tissues. But it expresses in the tumor tissues on a high level, and the expression of the constitutive B7-H1on the tumor tissues surface is related to the oncogenesis, tumor development and prognosis. B7-H1 plays an important role in the primary reaction, activated stage of naive T cells and the secondary reaction of memory T cells respectively by the combination with its inhibitory receptors. B7-H1 induces the apoptosis of tumor antigens specific CTL and the anti-apoptosis of tumor cells. It plays an important role in tumor immunologic escape, multidrug resistence, virus infection, inflammation, anto-immune disease and the tolerance of implant. B7-H1 is a potential marker of tumor. Due to the recently research of B7-H1 were all limited to the constitutive B7-H1 on the surface of tumor cells and tissues, our preliminary research had demonstrated there was soluble B7-H1 in the serum of cancer patients, the subject planned to ascertein the existence of soluble B7-H1 in serum, then research its function and the relationship between the contents and the development of cancer; We will prepare the anti-B7H1 monoclonal antibody as there is only one such antibody on sale, establish a convinent clinical method to detect the soluble B7-H1 in serum by sandwich ELISA. Methods: First, we prepared the anti-human B7-H1 monoclonal antibody by B cell hybridoma technique. That laid the foundation to detect the soluble B7-H1 in serum. Then, we hypodermic injected SP2/0 cells into Babl/c mice and induced the formation of solid tumor. Detected the expression of soluble B7-H1 in the serum of tumor-burdened mice by these ways: (1) Ascertained the existence of soluble B7-H1 in serum of tumor-burdened mice by Dot blot. (2) Measured the molecular weight of soluble B7-H1 fragments by Western blot, and discussed the relationship between relative content of B7-H1 and the span of tumor-burdened time, tumors weight for the research of soluble B7-H1 in cancer patients serum. We finally detected the soluble and constitusive B7-H1 of lung cancer patients in these ways: (1) Ascertained the existence of soluble B7-H1 in serum of lung cancer patients. (2) Measured the molecular weight of soluble B7-H1 fragments by Western blot. (3) Established the clinical detection of soluble B7-H1 method and detected 10 lung cancer patients soluble B7-H1 contents. (4) Immunohistochemical stained the lung cancer tissues to ascertain the expression of constitutive B7-H1. Results: Injected human B7-H1IgV region mixed with adjuvant into Babl/c mice to induce the immune response, and acquired one anti-human B7-H1 monoclonal antibody with a high affinity. Serum of tumor-burdened mice detection results: (1) Dot blot results showed there do have soluble B7-H1 in tumor-burdened mice serum and with different contents respectively. (2) Western blot showed the molecular weight of the fragment was 37kD, but there didn’t have obvious relativity from the contents to the span of tumor-burdened time and the tumors weight. Serum and tissues of lung cancer patients detection results: (1) Dot blot results showed there do have soluble B7-H1 in lung cancer patients serum and with different contents respectively. (2) Western blot showed the molecular weight of the fragment was 42kD. (3) Established the method to detect the content of soluble B7-H1, and detected 10 lung cancer patients serum. But we couldn’t appraise the relativity from the contents to oncogenesis and development of cancer provisionally because of the lack of case imformations. (4) The immunohistochemical stain of tumor tissues results showed constitutive B7-H1 widely expressed in endochylema in tumor cells. Conclusion: Successfully prepared a anti-human B7-H1 monoclonal antibody. Established a soluble B7-H1 detection method, and successfully confirmed the presence of soluble B7-H1, laid the foundation for clinical detection. The relativity between the soluble B7-H1 and tumor development still needs further investigation.
Objective: B7-H1 is one of the B7 costimulatory molecule family members. It hardly express on the surface of normal tissues. But it expresses in the tumor tissues on a high level, and the expression of the constitutive B7-H1on the tumor tissues surface is related to the oncogenesis, tumor development and prognosis. B7-H1 plays an important role in the primary reaction, activated stage of naive T cells and the secondary reaction of memory T cells respectively by the combination with its inhibitory receptors. B7-H1 induces the apoptosis of tumor antigens specific CTL and the anti-apoptosis of tumor cells. It plays an important role in tumor immunologic escape, multidrug resistence, virus infection, inflammation, anto-immune disease and the tolerance of implant. B7-H1 is a potential marker of tumor. Due to the recently research of B7-H1 were all limited to the constitutive B7-H1 on the surface of tumor cells and tissues, our preliminary research had demonstrated there was soluble B7-H1 in the serum of cancer patients, the subject planned to ascertein the existence of soluble B7-H1 in serum, then research its function and the relationship between the contents and the development of cancer; We will prepare the anti-B7H1 monoclonal antibody as there is only one such antibody on sale, establish a convinent clinical method to detect the soluble B7-H1 in serum by sandwich ELISA. Methods: First, we prepared the anti-human B7-H1 monoclonal antibody by B cell hybridoma technique. That laid the foundation to detect the soluble B7-H1 in serum. Then, we hypodermic injected SP2/0 cells into Babl/c mice and induced the formation of solid tumor. Detected the expression of soluble B7-H1 in the serum of tumor-burdened mice by these ways: (1) Ascertained the existence of soluble B7-H1 in serum of tumor-burdened mice by Dot blot. (2) Measured the molecular weight of soluble B7-H1 fragments by Western blot, and discussed the relationship between relative content of B7-H1 and the span of tumor-burdened time, tumors weight for the research of soluble B7-H1 in cancer patients serum. We finally detected the soluble and constitusive B7-H1 of lung cancer patients in these ways: (1) Ascertained the existence of soluble B7-H1 in serum of lung cancer patients. (2) Measured the molecular weight of soluble B7-H1 fragments by Western blot. (3) Established the clinical detection of soluble B7-H1 method and detected 10 lung cancer patients soluble B7-H1 contents. (4) Immunohistochemical stained the lung cancer tissues to ascertain the expression of constitutive B7-H1. Results: Injected human B7-H1IgV region mixed with adjuvant into Babl/c mice to induce the immune response, and acquired one anti-human B7-H1 monoclonal antibody with a high affinity. Serum of tumor-burdened mice detection results: (1) Dot blot results showed there do have soluble B7-H1 in tumor-burdened mice serum and with different contents respectively. (2) Western blot showed the molecular weight of the fragment was 37kD, but there didn’t have obvious relativity from the contents to the span of tumor-burdened time and the tumors weight. Serum and tissues of lung cancer patients detection results: (1) Dot blot results showed there do have soluble B7-H1 in lung cancer patients serum and with different contents respectively. (2) Western blot showed the molecular weight of the fragment was 42kD. (3) Established the method to detect the content of soluble B7-H1, and detected 10 lung cancer patients serum. But we couldn’t appraise the relativity from the contents to oncogenesis and development of cancer provisionally because of the lack of case imformations. (4) The immunohistochemical stain of tumor tissues results showed constitutive B7-H1 widely expressed in endochylema in tumor cells. Conclusion: Successfully prepared a anti-human B7-H1 monoclonal antibody. Established a soluble B7-H1 detection method, and successfully confirmed the presence of soluble B7-H1, laid the foundation for clinical detection. The relativity between the soluble B7-H1 and tumor development still needs further investigation.
引文
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