【作者】 梅玫；

【导师】 鲁重美； 陈原稼； 邓大君； 张业；

【作者基本信息】 中国协和医科大学 ， 消化内科学， 2009， 博士

【摘要】 第一部分散发性胰岛素瘤中错配修复基因MLH1、MSH2失活导致微卫星不稳定及其临床意义研究目的散发性胰岛素瘤的分子发病机制未明。目前仍缺乏可靠的用于鉴别胰岛素瘤良恶性的分子标志物。本课题第一部分的研究目的是明确胰岛素瘤是否发生错配修复基因失活,从而导致微卫星不稳定;错配修复基因的异常以及微卫星不稳定是否可用于鉴别该类肿瘤的良恶性和判断预后。实验方法用PCR方法检测61例胰岛素瘤的微卫星不稳定,MLH1基因的杂合缺失和启动子甲基化。用变性高效液相色谱分析方法测定外显子突变并DNA测序验证。用免疫组织化学染色方法检测MLH1和MSH2蛋白在肿瘤及瘤旁组织中的表达。分别用单因素和多因素分析方法对实验数据与患者的临床病理学资料进行相关性分析。实验结果在58个散发性胰岛素瘤中,MLH1和MSH2蛋白表达下降分别为38%和16%。散发性胰岛素瘤发生MLH1基因启动子高甲基化和基因杂合缺失分别为32%和50%。MLH1基因启动子高甲基化或同时发生启动子高甲基化和基因杂合缺失与蛋白的表达下降显著相关（P值分别为0.041和0.027）。散发性胰岛素瘤中未发现MSH2基因杂合缺失。1/3的散发性胰岛素瘤发生高频的微卫星不稳定,MLH1或MSH2蛋白表达下降与高频的微卫星不稳定显著相关（P=0.006和0.049）。MLH1和MSH2蛋白表达同时下降也与高频的微卫星不稳定显著相关（P=0.004）。与临床病理相关分析显示,高频的微卫星不稳定、MLH1蛋白表达下降或MLH1和MSH2蛋白表达同时下降与肿瘤良恶性显著相关(P值分别为2.6×10^-5、0.008和0.018)。高频的微卫星不稳定、MLH1蛋白表达下降以及同时发生MLH1和MSH2蛋白表达下降均为患者未即时治愈的独立影响因素（P值分别为0.002、0.011和0.030）。实验结论在散发性胰岛素瘤中MLH1基因的失活与杂合缺失和基因启动子高甲基化有关。MLH1和MSH2基因的表达下降以及高频的微卫星不稳定可作为鉴别胰岛素瘤的良恶性以及辅助判断患者的预后的分子标志物。错配修复基因的异常可能在胰岛素瘤的发生中起一定的作用。第二部分α-internexin在胰腺内分泌肿瘤中的异常表达及其临床意义研究目的胰腺内分泌肿瘤的分子发病机制未明。目前仍缺乏可靠的用于鉴别胰腺内分泌肿瘤良恶性的分子标志物。课题第二部分的研究目的是明确胰腺内分泌肿瘤是否存在α-internexin的表达及其表达调控机制;探讨α-internexin的表达是否可作为分子标志物用于鉴别该类肿瘤的良恶性和判断预后。实验方法用免疫组织化学染色方法检测242例胰腺内分泌肿瘤α-internexin的表达,其中部分标本用免疫印迹法及反转录PCR法检测α-internexin蛋白及mRNA的表达进行确认。免疫印迹法检测17株细胞系该蛋白的表达。分别用反转录PCR、DNA测序和染色质免疫沉淀检测5-aza-dC作用前后人肿瘤细胞系α-internexin的mRNA表达、启动子甲基化及组蛋白修饰状态。分别用单因素和多因素分析方法对实验数据与临床病理学资料进行相关性统计分析,Kaplan-Meier法用于生存分析。实验结果238个胰腺内分泌肿瘤中有132个肿瘤（55.4%）表达α-internexin蛋白,而对照组53个胰腺组织标本中仅1个标本（2%）表达该蛋白(P=9.97×10^-14)。α-internexin的表达率在功能性与无功能性胰腺内分泌肿瘤之间有很显著地差异(71.4%vs.35.4%,P=2.36×10^-8)。在恶性肿瘤、发生转移的肿瘤中α-internexin蛋白表达明显下降（P值分别为0.016、0.003）。α-internexin蛋白表达下降与患者因肿瘤死亡或肿瘤未治愈的不良预后均显著相关（P值分别为0.025或0.017）。在多种人肿瘤细胞系中,α-internexin蛋白表达下降与其启动子甲基化显著相关（P=0.002）。但在正常胰腺和胰腺内分泌肿瘤的组织中发现α-internexin基因的启动子处于非甲基化状态,提示该基因在胰腺内分泌肿瘤中的表达调控与启动子甲基化与否无明显相关。实验结论α-internexin表达下降可作为鉴别胰腺内分泌肿瘤的良恶性以及辅助判断患者的预后的分子标志物。α-internexin启动子甲基化是多种肿瘤细胞系中调控α-internexin蛋白表达的重要机制,但与胰腺内分泌肿瘤中α-internexin蛋白的表达无明显相关。更多还原

【Abstract】 Purpose The molecular pathogenesis of sporadic insulinomas is unknown.There is a lack of biomarker to distinguish benign and malignant insulinoma.The aim of the first part of this study is to identify if mismatch repair genes are inactivated in insulinomas,which induce the microsatellite instability, and if the genetic/epigenetic alterations of the gene（s） as well as microsatellite instability could be used to distinguish benign and malignant insulinoma and to predict the outcome of the patients.Experimental Design We detected microsatellite instability,allelic typing and promoter methylation by PCR.Denaturing high performance liquid chromatography（DHPLC） was used to analyze exon mutations which were further confirmed by sequencing.Expression of MLH1 and MSH2 gene in 61 insulinomas and paired normal tissues were tested by immunohistochemical staining.The data were correlated with clinical-pathological characteristics,and were analyzed by both univariate and multivariate statistical methods.Results We found expression of MLH1 and MSH2 protein was reduced in 38%and 16%of 58 sporadic insulinomas,respectively.Promoter methylation and loss of heterozygosity（LOH） of MLH1 gene was found in 32% and 50%of sporadic insulinomas,respectively.Promoter methylation or both methylation and LOH of MLH1 were significantly associated with reduced expression of MLH1（P=0.041 and P=0.027,respectively）.LOH of MSH2 gene was not found in sporadic insulinomas.A high rate of microsatellite instability （MSI-H） was found in 33%of sporadic insulinomas,which was correlated with the reduction of MLH1 or MSH2（P=0.006 and P=0.049,respectively）.Reduced expression of both MLH1 and MSH2 were significantly correlated with MSI-H （P=0.004）.Reduced expression of MLH1 or both MLH1 and MSH2 were significantly associated with tumor malignancy（P=0.008 or P=0.018）.Although the rate of reduced expression of MSH2 in malignant tumors was higher than in benign tumors,the statistic difference was not significant（P=0.072）.MSI-H was significantly associated with tumor malignancy and incurable insulinoma(P=2.6×10^-5 and P=0.002,respectively).Conclusion Allelic loss and promoter methylation contribute to the inactivation of MLH1 gene in sporadic insulinomas.Assessing alterations of MLH1 and MSH2 gene,as well as MSI-H,could be used to distinguish benign from malignant insulinomas and to predict the outcome of patients.The genetic/epigenetic silencing of MLH1 gene may play an important role in tumorigenesis in a subset of the tumors. Purpose The molecular pathogenesis of pancreatic endocrine tumors is unknown.There is a lack of biomarker to distinguish benign and malignant pancreatic endocrine tumors.The aim of the second part of this study is to identify ifα-internexin expressed in pancreatic endocrine tumors,as well as analyzing the molecular mechanisms regulating the expression ofα-internexin; and to find if the alteration of the expression ofα-internexin could be used to distinguish benign and malignant pancreatic endocrine tumors and to predict the outcome of the patients.Experimental Design Expression ofα-internexin in 242 pancreatic endocrine tumors were tested by immunohistochemical staining,and part of the tumors were also tested the expression of mRNA and protein ofα-internexin by RT-PCR and western blot,respectively.We also detected the protein expression ofα-internexin in 17 cell lines by western blot.Before and after treated with 5-aza-dC,the mRNA,promoter methylation and histon modification ofα-internexin in cell lines were detected by RT-PCR,sequencing and chromatin immunoprecipitation analysis,respectively.The data were correlated with clinical-pathological characteristics,and were analyzed by both univariate and multivariate statistical methods.Kaplan-Meier was used to analyse the cumulative survival.Results We found 132 of 238（55.4%） pancreatic endocrine tumors expressedα-internexin,whereas only one of 53 pancreatic tissues expressed the protein(P = 9.97×10^-14).The percentage of the expression ofα-internexin was significantly higher in functional tumors than in non-functional ones(71.4%vs. 35.4%,P = 2.36×10^-8).Reduced expression ofα-internexin was significantly correlated with malignant tumors or tumors with metastasis（P=0.016 and P=0.003,respectively）.Besides,reduced expression ofα-internexin was also significantly correlated with patients’ outcome of dead with tumors or survival with tumors（P=0.025 and P=0.017,respectively）.Promoter methylation was significantly associated with reduced expression ofα-internexin in 14 tumor cell lines（P=0.002）.However,promoter unmethylation status was found in both pancreatic endocrine tumors and normal pancreatic tissues,which indicated that promoter methylation was not play a role in the regulation mechanism of the expression ofα-internexin in pancreatic endocrine tumors.Conclusion Reduced expression ofα-internexin could be used to distinguish benign from malignant pancreatic endocrine tumors and to predict the outcome of patients.Promoter methylation contributes to the inactivation ofα-internexin gene in many kinds of tumor cell lines,but it may not play an important role in pancreatic endocrine tumors.更多还原