【作者】 洪波；

【导师】 陈功祥；

【作者基本信息】 浙江大学 ， 临床检验诊断学， 2015， 硕士

【摘要】 背景与目的：胰腺癌是一种以起病隐匿、生长迅速并早期转移为特征的消化系统恶性肿瘤,是死亡率最高的恶性肿瘤之一,目前尚无有效的治疗措施,其发病率在世界范围内呈逐年上升趋势。由于胰腺组织的解剖位置关系,早期症状不明显且缺乏有效的检测手段,大多数患者就诊时已为中晚期,预后差,5年生存率不足5%。早期发现与早期诊断是改善胰腺癌患者预后的有效措施之一。因此,寻找早期诊断、特异性强、灵敏度高的肿瘤分子标记物对于改善胰腺癌患者的预后具有重要的临床意义,同时也能为胰腺癌新型靶向治疗技术提供依据。B7-H4(也称为B7S1或B7x)是新近发现的B7家族成员,能抑制T细胞增殖、细胞因子产生和细胞周期的进行,从而抑制T细胞介导的免疫应答。B7-H4在肿瘤免疫应答中起重要作用。B7-H4mRNA的在脾、肺、胸腺、胎盘、肝、肾等正常组织中均有表达,然而,免疫组化显示,B7-H4蛋白在正常外周组织中几乎没有阳性表达,而在一些肿瘤组织中有丰富表达,例如乳腺癌、卵巢癌、肾癌、前列腺癌和非小细胞肺癌,并与肿瘤进展密切相关。鉴于目前尚无可用于多种方法检测B7-H4表达的抗体出售,本研究以稳定表达B7-H4的3T3-B7-H4细胞为免疫原,制备了能稳定分泌特异性单克隆抗体的杂交瘤细胞株,并对其进行了生物学特性分析。同时,应用免疫组织化学染色检测B7-H4在胰腺癌组织中的表达,探讨B7-H4的表达与胰腺癌临床病理特征之间的关系,分析B7-H4在胰腺癌诊疗及预后判断等方面的潜在价值。方法：1.以稳定表达B7-H4胞外段的3T3-B7-H4细胞为免疫原,免疫Babl/C小鼠；应用常规杂交瘤技术将免疫的小鼠脾细胞与Sp2/0小鼠骨髓瘤细胞融合,使用间接ELISA方法筛选分泌抗B7-H4单克隆抗体的杂交瘤细胞株。并对所获得的单克隆抗体进行亚型鉴定,使用Western blotting、免疫沉淀等方法对单克隆抗体特异性进行鉴定。2.利用制备的小鼠抗B7-H4单克隆抗体,通过免疫组化(IHC)等方法分析B7-H4在胰腺癌组织中的表达特征,并评价其与临床病理特征的关系。结果：1.获得1株亚型为IgM、轻链为κ型的针对B7-H4的鼠源单克隆抗体：特异性抗B7-H4的单抗3E8能用于免疫沉淀(IP)及IHC等方法检测组织及细胞中的B7-H4表达。2.免疫组化显示：胰腺癌组织中B7-H4在胞浆和(或)胞膜弥漫表达,表达量显著高于正常胰腺组织(P<0.05)。B7-H4在临床肿瘤分级较高患者的胰腺癌组织及有淋巴结转移患者的胰腺癌组织中表达显著增强。结论：成功获得了特异性抗B7-H4的单克隆抗体,B7-H4在胰腺癌组织中表达上调,并与患者肿瘤分级和淋巴结转移密切相关。更多还原

【Abstract】 Background and purpose:Pancreatic cancer is an insidious onset, characterized by fast growth and early Metastatic malignant tumors of the digestive system, is the one of the highest cancer mortality, there is currently no effective treatment measures, its incidence is an upward trend in the world. Due to the anatomical position of the pancreatic tissues, no obvious early symptoms and the lack of effective means of detection, most patients have advanced, poor prognosis, the5-year survival rate of less than5%. Early detection and early diagnosis is one of the effective measures to improve the prognosis of patients with pancreatic cancer. Therefore, looking for early diagnosis, with high sensitivity and specificity, cancer molecular markers to improve the prognosis of patients with pancreatic cancer has important clinical implications, will also provide the basis for new targeted therapy for pancreatic cancer technology.B7-H4(also called B7S1or B7x) is a newly-discovered members of the B7family, inhibits T-cell-mediated immune response through inhibiting T cell proliferation, cytokine production, and cell cycle[3-6]. B7-H4played an important role in tumor immune response. B7-H4mRNA was expressed in spleen, lung, thymus, placenta, liver, kidney, and other normal organization[3]. However, B7-H4protein was not expressed in normal tissues detected with immunohistochemistry (IHC)[3]. B7-H4was expressed in a high level in some tumor tissues, such as breast cancer, ovarian cancer, kidney cancer, prostate cancer and non-small cell lung cancer, and is closely related to tumor progress[5,7-11]. Given that there is no anti-B7-H4antibody which can be used for a variety of methods to detecting the expression of B7-H4, this research prepare the mouse monoclonal antibodies (mAbs) against B7-H4through preparing3T3-B7-H4cells as a immunogen which can stably express B7-H4. And the antibody’s biological properties were analyzed. And the expression and clinical significance of B7-H4in pancreatic cancer tissue was investigated.Methods:1. Balb/c mice were immunized with3T3-B7-H4cells which expressed extrinsic B7-H4. Then the spleen cells of the immunized mice were fused with Sp2/0myeloma cells by conventional hybridoma techniques. An indirect ELISA using lysate of3T3-B7-H4as antigen was established to screen antibody-producing hybridoma cell lines. Western blotting and Immunoprecipitation (IP) were applied to characterize the mAb.2. Immunohistochemical (IHC) staining was used to detect the expression of B7-H4in pancreatic cancer tissue. And the relationship between the expressions and pathology was evaluated.Results:1. We obtained a hybridoma cell line secreting mAb against B7-H4. The subclasse of this mAb was IgM, and light chain was Kappa. Western blotting and IP showed that the mAb specifically recognized B7-H4.2. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The B7-H4was diffuse expressed in cytoplasma and/or membrane of the pancreatic cancer tissue, which was much higher than that expressed in normal pancreatic tissue (4.00±1.44vs.1.12±0.78, P<0.01). The expression of B7-H4in the pancreatic cancer tissue was higher in patients with higher tumor grade (6.10±0.72vs.3.55±1.12, P<0.01) compared with lower tumor grade or lymph node metastatic carcinoma (6.14±0.66vs.3.70±1.25, P<0.01). The expression level of B7-H4was not related to the patients’ age and gender.Conclusions:mAb against B7-H4with high activity and specificity was prepared successfully. The expression of B7-H4in pancreatic cancer tissue was increased. The abnormal expression of B7-H4in pancreatic cancer is closely related with the tumor grade and lymph node metastasis.更多还原