【作者】 张静敏；

【导师】 安汝国；

【作者基本信息】 吉林大学 ， 生化与分子生物学， 2004， 博士

【摘要】 目的：近年来，肿瘤的发病率和死亡率呈明显上升趋势，严重威胁着人类健康。常规的化疗、放疗和手术疗法仍存在着各种问题，因此迫切要求寻找新的治疗手段。肿瘤免疫治疗亦是这些新的治疗手段之一。然而研制肿瘤治疗性疫苗，其前提是需要获得特异性肿瘤抗原。肿瘤细胞是正常细胞的“非己”化。肿瘤细胞在“非己”化过程中，必然会产生一些“非己”的成分。我们把肿瘤的这种“非己”成分限定为只存在于肿瘤细胞，而正常组织细胞不具有的细胞成分，通常称为肿瘤抗原。机体免疫系统可以识别任何“非己”的成份，并通过免疫应答将其排除。近些年来，肿瘤抗原研究的重要进展之一，就是人们对肿瘤抗原递呈和免疫识别有了新的了解，认识到机体免疫系统抗肿瘤主要方式是T淋巴细胞介导的免疫排斥反应，主要是CD8~+细胞毒性T淋巴细胞(CTL)通过其受体TCR识别MHCl类分子结合的肿瘤抗原肽而活化并发挥杀伤肿瘤细胞的效应，有人把这一认识概括为MHC一抗原肽一TCR模式，这里所说的抗原肽是CTL所识别的由8—10个氨基酸组成的线性肽段，又称肿瘤特异性抗原。到目前为止，一系列肿瘤抗原已相继被发现并得到确认，其中包括癌一睾丸抗原(Cancer Testis Antigen CTA)家族。该家族成员只在睾丸组织和一些肿瘤组织表达，在其他正常组织不表达。由于睾丸组织缺乏人类白细胞抗原(HLA)，所以，CTA是一类特异性强的肿瘤抗原。 CD109、RFP和BRDT基因与人肿瘤关系的研究国内外报道的较少，尤其CD109基因。为了探讨CD109、RFP和BRDT能否作为肿瘤治疗的潜在分子靶点，本研究全部采用临床病人的活体标本，运用Real-time PCR的方法，分析了CD109、RFP和BRDT在人、鼠正常组织及癌组织中的表达；试图为肿瘤免疫诊断与治疗提供基本的实验依据，因此具有非常重要的理论和实际意义。 方法：成年BALB／C鼠(18-20g)取自名古屋大学医学部实验动物中心，从AICHI癌症中心进行肿瘤手术切除的病人获得人正常组织及癌组织，组织样品立即冻于液氮中，然后保存于一80“C供提取RNA用。所有样品的取得均遵守现行的道德规范准则，并正式得到所有参与者和该研究各方的同意。该协议同时也得到AICHI癌症中心医院的同意。我们采用实时荧光定量RT一PCR研究了CD109、RFP和BRDT在肿瘤中的表达.结果: 一CDIOg基因的克隆及实时荧光定量RT一PCR检测其表达 ZA和ZB型多发性内分泌腺瘤(MEN)的发生主要是由RET原癌基因的点突变引起的。MENZA是以甲状腺髓样癌(MTC)和嗜铬细胞瘤为特点的，而MENZB呈现更加复杂的表型，它与甲状腺髓样癌，嗜铬细胞瘤以及发育异常，如粘膜神经瘤和马方综合症的骨骼改变等都相关。为了阐明MEN ZA和MENZB表型发生的机理，我们通过差异显示分析证实了一些在N工H 3T3细胞和用MENZA和MEN ZB突变的RET基因转染的NIH 3T3细胞(分别称为RET一MEN ZA或尺ET一MEN ZB)中的差异表达基因。我们发现了一个未知序列基因在NIH一RET(MENZB)细胞中表达上调，这一基因可能成为癌症治疗的一个新的分子靶点。 通过差异显示，我们检测到一个在N工H一RET(MEN ZB)细胞中表达上调的转录子。用它的eDNA片段，我们对NIH一RET(MEN ZB)细胞的CDNA文库进行筛选，并且获得了一个包含4329bp开读框的全长4717bp的CDNA克隆。它编码一个含有1442个氨基酸的蛋白，该蛋白含有一个22个氨基酸的信号肤和一个疏水的糖基化磷脂酞肌醇(GPI)锚定剪切位点，此外，在923一926位氨基酸上还存在一个硫脂键。经过测序证实这一cDNA编码一个与人CDIOg基因同源的小鼠CD109基因，人CD109基因是最近刁‘报道的。2巨球蛋白C3，C4和C5家族成员。 为了证实鼠CDIOg是一个GPI锚定细胞表面蛋白，我们将FLAG标记的鼠CDIOg基因eDNA克隆到表达载体pSR a 296PL上，然后转染COS7细胞。Westernblot分析表明，转染细胞表达一个180kDa的蛋白，该蛋白能够被抗FLAG单克隆抗体所识别，表明该蛋白为目的蛋白分子，即FLAG标记CD109融合蛋白。用抗FLAG单克隆抗体通过免疫染色观察到的FLAG标记CD109融合蛋白的表达。此外，当用能够剪切GP工锚定蛋白的特异性磷脂酞肌醇磷脂酶C(P工一PLC)处理转染细胞后，检测不到细胞表面CD109融合蛋白的表达，证实鼠CD109是一个Gpl锚定细胞表面蛋白。 应用Real一t ime PCR检测CD109基因在各种人和鼠成熟组织中的表达情况时发现它在正常组织中高表达于翠丸组织，在癌组织标本中检测到子宫颈鳞状细胞癌组织的CD 109基因表达水平高于正常子宫颈组织，差异极显著(P<O.ol)，子宫内膜腺癌组织的CD109基因表达水平高于正常子宫内膜组织，胃腺癌组织的CD109基因表达水平高于正常胃组织，食管鳞状细胞癌组织的CDIOg基因表达水平高于正常食管组织，而子宫颈鳞状细胞癌组织的CDIOg基因表达水平高于子宫内膜腺癌组织，差异极显著(P<0.01)，食管鳞状细胞癌组织的CD109基因表达水平高于胃腺癌组织，脑癌组织中的CD109基因平均表达水平高于正常脑组织. 二.实时荧光定量RT一PCR检测RFP基因表达 R即通过与RE’r原癌基因重排而最初被认定为癌基因。RFP属于B盒RING指蛋白家族，该家族蛋更多还原

【Abstract】 Objective: In recent years, morbidity and mortality of cancer are increasing obviously, therefore cancer becomes a serious threat to human health. There are still various kinds of problems in conventional chemotherapy , radiotherapy and operative treatment. Tumor immunotherapy is one of newly developed therapeutic methods. However, it is, important for us to get specific tumor antigen in order to develop tumor specific therapeutic vaccine. Tumor cell is "non- self" of normal cell. We define the component which express only in tumor cell but in normal cell as tumor antigen. Immune system can recognize any "non-self" component and remove them through immunological response. In recent years, significant progression in tumor antigen research is the new perspective that T lymphocyte-mediated response is the major manner by which the body to fight tumor cells, especially CD8+ cytotoxict lymphocytes. Tumor antigen is loaded with MHCI molecules and form the tumor peptide complexes which is recognized by T-cell receptor(TCR), the activated T cells will kill tumor cells expressingtumor peptides. Someone outline the pathway as MHC-antigen peptide-TCR mode, the antigen peptide is 8-10 amino acids recognized by CTL and also called tumor specific antigen. Up to date, a series of tumor antigens have been identified and confirmed, include Cancer Testis Antigen family, which express only in cancer testis tissues and tumor tissues but in normal tissues. There is not HLA in testis tissues, therefore CTA is a kind of specific tumor antigen.The relationship between CD109, RFP, BRDT and human tumor has been less reported, especially CD109. To explore whether CD109, RFP, BRDT may become potential molecular targets for new cancer therapy, in the present study tumor and normal tissue samples were obtained from patients undergoing tumor resection, the expression of CD109, RFP, BRDT in tumor and normal tissue was detected through real-time PCR. The present study tried to offer fundamental experiment base for immunological diagnosis and therapy of tumor and has very important theoretical and practical significance.Method: Tumor and normal tissue samples were obtained from patients undergoing tumor resection at Aichi cancer center hospital, the tissue samples were snap frozen in liquid nitrogen, and stored at -80?C until RNA extraction. All samples were obtained according to current ethics regulations. Informed consent was obtained from all subjects before their participation in the study. The protocol was approved by the Aichi cancer center hospital (Nagoya, Japan).We used real-time PCR to analyze CD109, RFP and BRDT expression in different tumor,Result:1. Investigation of Cloning and Expression of CD109 Gene Using Real-Time Fluorescence Quantitative RT-PCRGermline mutation of the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia(MEN)type 2A and 2B. MEN 2A is characterized by the development of medullary thyroid carcinoma(MTC) and pheochromocytoma whereas MEN 2B shows a more complex phenotype with association of MTC, pheochromocytoma, and developmental abnormalities such as mucosal neuroma, hyperganglionosis of the intestinal tract, and marfanoid skeletal changes. To elucidate the mechanisms of development of MEN 2A and MEN 2B phenotypes, we carried out differential display analysis of gene expression using NIH3T3 cells expressing the RET-MEN 2A and RET-MEN 2B mutant proteins. In this study, we cloned one of previously unidentified genes induced by RET-MEN 2B. It turned out to encode mouse orthologue of the human CD109 gene.Using its cDNA fragment, we screened the cDNA library constructed from RNA of NIH-RET(MEN2B)cells and obtained a cDNA clone of 4717 base pairs in length that contained a 4329 bp open reading frame. It encodes a protein of 1442 amino acids having an amino-terminal signal peptide of 22 amino acids and a carboxyl-terminal hydrophobic glycosyl-phosphatidylinositol (GPI)-anchored cleavage-addition site. In addition, a sequence for the thioester bond (CGEQ) was prese更多还原