【作者】 郑永昌；

【导师】 赵玉沛； 张太平；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 研究背景:胰腺癌是一种起病隐匿、进展迅速、预后极差的消化道高度恶性肿瘤。早期诊断是胰腺癌以手术为主、联合运用多种方法的综合治疗体系的关键所在,血清肿瘤标记物的筛查是胰腺癌早期诊断研究的重要内容之一。本课题组提出了以肿瘤免疫理论为基础采用胰腺癌相关免疫原性膜抗原抗体作为胰腺癌诊断标记物的新构想,并设计出了胰腺癌相关免疫原性膜抗原的筛查策略。为了筛查胰腺癌相关免疫原性膜抗原进而确定胰腺癌血清抗体作为肿瘤标记物,前期实验通过运用“胰腺癌细胞株膜蛋白-免疫印记杂交-蛋白质组学”筛查方式发现,相比于正常人和慢性胰腺炎病人,胰腺癌病人体内特异性存在SLP-2和TOM40蛋白的血清抗体,从而提出SLP-2和TOM40可能为人胰腺癌相关免疫原性膜抗原,其血清抗体有可能被用作胰腺癌的肿瘤标记物,因此有必要在人胰腺癌组织中对SLP-2和TOM40的表达进行研究。研究目的:对人胰腺癌组织中SLP-2和TOM40的表达进行研究,从基因(转录)和蛋白(翻译)两个水平、细胞和组织两层面获取SLP-2和TOM40作为胰腺癌相关免疫原性膜抗原存在的客观证据;对SLP-2和TOM40进行生物信息学分析,在三株常用胰腺癌细胞系中对SLP-2和TOM40进行定位和相对表达强度研究,构建SLP-2基因RNA干扰的表达载体pGPU6/GFP/Neo-SLP-2-shRNA,为在胰腺癌中进一步开展SLP-2的研究打下一定基础。研究方法:采用Real time PCR和Western blot研究SLP-2和TOM40在胰腺癌中的表达情况,同时采用人胰腺癌组织芯片(TMA)进行SLP-2和TOM40的免疫组化染色,从组织水平获取SLP-2和TOM40作为人胰腺癌免疫原性膜抗原存在的客观证据;运用生物信息学网络平台NCBI、ASTD、Oncomine Research Platform、Gene Ontology、UCSCGenome Browser以及本地生物信息学分析软件ESvPred3D、ClustalX、TreeView对SLP-2和TOM40进行蛋白进化分析、基因可变剪接分析、电子表达谱分析、转录调控分析,同时进行三维结构预测和miRNA作用位点预测并分析SLP-2和TOM40可能的B细胞表位和CTL表位;对胰腺癌细胞株AsPC-1、BxPC-3、PANC-1进行SLP-2和TOM40的细胞免疫荧光染色,利用激光共聚焦显微镜研究其在胰腺癌细胞株中的相对表达强度和细胞定位;采用pGPU6/GFP/Neo构建用于SLP-2 RNA干扰研究的表达载体pGPU6/GFP/Neo-SLP-2-shRNA。实验结果:1.Real time PCR检测显示SLP-2和TOM40基因在10例正常胰腺组织和10例胰腺癌组织均存在表达,相对于正常胰腺组织,TOM40有高表达趋势。2.Western blot检测显示SLP-2和TOM40蛋白在10例正常胰腺组织和10例胰腺癌组织均存在表达;含75例胰腺导管腺癌的组织芯片免疫组化检测显示SLP-2和TOM40在胰腺癌细胞中存在表达,并且相对于正常胰腺组织,SLP-2和TOM40有高表达趋势。3.蛋白进化分析构建了SLP-2和TOM40系统进化树;基因调控分析提示SLP-2可能受到CEBP、ELK1等转录因子的调控,TOM40可能受到CDPCR3、PAX5等转录因子的调控;miRNA预测分析了可能调控SLP-2和TOM40的miRNA;三级结构预测获得了SLP-2可能的三级结构;抗原表位预测分析提示氨基酸序列“SSGSSRD”和“TSTSRSS”可能为SLP-2和TOM40较合理的B细胞表位,而氨基酸序列“ILEPGLNIL”和“TLNNWLATV”等可能为SLP-2和TOM40的CTL细胞表位。4.胰腺癌细胞株免疫荧光染色激光共聚焦显微镜观察显示,SLP-2主要位于胰腺癌细胞株AsPC-1、BxPC-3、PANC-1的细胞质,三株胰腺癌细胞株SLP-2的相对表达水平存在显著差异,其中PANC-1表达最高,而AsPC-1表达最低;TOM40主要位于胰腺癌细胞株AsPC-1、BxPC-3、PANC-1的细胞质,三株胰腺癌细胞株TOM40的相对表达水平存在显著差异,以PANC-1表达最高,而AsPC-1表达最低。5.预测了SLP-2 RNA干扰靶位点,挑选设计出了4个可能的干扰序列,利用其完成SLP-2RNA干扰载体pGPU6-GFP-Neo-SLP-2-shRNA的构建并成功转染胰腺癌细胞株SW1990。结论:SLP-2和TOM40在人胰腺癌组织中呈广泛性表达趋势,而病人血清内特异性存在其抗体,提示SLP-2和TOM40为人胰腺癌相关免疫原性膜抗原并在人胰腺癌中可能具有一定的代表性,为以SLP-2和TOM40血清抗体作为胰腺癌肿瘤标记物的新策略提供了一定的实验证据支持。更多还原

【Abstract】 BACKGROUNDThe human pancreatic cancer is one of the most deadly malignancies with silence onset,rapid progress and poor prognosis.Early diagnosis of pancreatic cancer plays a central role in the comprehensive therapy system which combined the surgery and other therapies such as chemotherapy and radiotherapy.The screening of serum tumor marker was considered to be one of the most hopeful methods to make a progress in pancreatic diagnosis research.Our lab presented a new concept to detect pancreatic cancer which planed to use the antibodies of immunogenic associated membrane antigens,and also presented the concept of pancreatic cancer associated immunogenic membrane antigens and designed a new screening strategy for the pancreatic cancer associated immunogenic membrane antigens.After the previous work of our research team which were based on the pancreatic cancer cell line extracted membrane proteins,Western blot and proteomics,the IgG of SLP-2 and TOM40 were detected in the serum of pancreatic cancer patient without in the normal persons and chronic pancreatitis patients,so the antibodies of SLP-2 and TOM40 were considered to have potential diagnosis value in pancreatic cancer,and the expression condition of SLP-2 and TOM40 in pancreatic cancer are needed to be further investigated.OBJECTIVETo study the expression of SLP-2 and TOM40 in human pancreatic cancer both in the transcript level and translation level,and to identify the SLP-2 and TOM 40 as pancreatic cancer associated immunogenic membranes,and also conduct some initial study of these two proteins which are associated with pancreatic cancer and do some basic work for the future investigation of the 2 proteins in pancreatic cancers.METHODSThe Real time PCR,Western blot and immunohistochemistry of human pancreatic cancer tissue microarray were used to detect the expression of SLP-2 and TOM40 in different levels of human pancreatic cancer.Bioinformatics analysis was conducted to both gene and protein of SLP-2 and TOM40 by the online database such as NCBI、 ASTD、Oncomine Research Platform、Gene Ontology、UCSC Genome Browser,and also the local bioinformatics software such as ESyPred3D,ClustalX,and TreeView.etc.The analysis included the evolution of protein,the gene alternative splicing and transcript, electronic expression spectrum,the prediction included miRNA and the antigen epitope of B cell and cytotoxic T lymphatic cell.The immunologic fluorescence cytochemistry method was conducted to the pancreatic cell line AsPC-1,BxPC-3,PANC-1 and the confocal laser scanning microscopy was used to identify the position of the SLP-2 and TOM40 and also the related expression levels in the 3 pancreatic cell lines.The vector pGPU6/GFP/Neo was used to construct the SLP-2 RNA interfering vector.RESULTS1.Real time PCR detected the expression of SLP-2 and TOM 40 in both 10 cases human pancreatic caner and 10 cases normal human pancreatic tissue,and the TOM40 had high expression level in pancreatic cancer tissue compared to the normal pancreatic tissue.2.Western blot detected the expression of SLP-2 and TOM40 in both 10 cases human pancreatic caner and 10 cases normal human pancreatic tissue;the immunohistochemistry of human pancreatic tissue microarray indicated the expression of SLP-2 and TOM40 in pancreatic cancer,the SLP-2 and TOM40 had high expression levels in pancreatic cancer tissues compared to the normal pancreatic tissues.3.The protein evolution trees of SLP-2 and TOM40 was constructed,and the transcript factor prediction analysis indicated that the SLP-2 might be regulated by CEBP and ELK1 ect,and the TOM40 might be regulated by the CDPCR3,PAX5 etc.The miRNA prediction presented the miRNA which might regulated the SLP-2 and TOM40,and the prediction 3-D structure of SLP-2 was made,and the antigen epitope of B cell and cytotoxic T lymphatic cell were predicted and presented.4.The confocal laser scanning microscopy observation of pancreatic immunologic fluorescence cytochemistry indicated that both SLP-2 and TOM40 was expressed in the cytoplasm of pancreatic cancer AsPC-1,BxPC-3 and PANC-1.The PANC-1 cell line had the highest expression level of both SLP-2 and TOM40,and the AsPC-1 cell line had the lowest expression level.5.The SLP-2 RNA interfering vector pGPU6-GFP-Neo-SLP-2-shRNA was constructed and the pancreatic cell line SW1990 was transfected successful.CONCLUSIONS1.SLP-2 and TOM40 were identified as the human pancreatic cancer associated immunogenic membrane antigens.2.The SLP-2 and TOM40 proteins mainly localized in the cytoplasm of pancreatic cell line AsPC-1,BxPC-3 andPANC-1,the expression of both SLP-2 and TOM40 had significant difference in these three pancreatic cell line,the PANC-1 had the highest expression level but the AsPC-1 had the lowest one.更多还原