【作者】 朱见；

【导师】 田兴松；

【作者基本信息】 山东大学 ， 外科学， 2008， 硕士

【摘要】 第一部分甲状腺癌中EG-1 mRNA的表达与预后的相关研究目的EG-1(endothelial derived gene-1,基因库序列号为AF358829)是Zhang L等于2002年在研究肿瘤条件液培养下的脐静脉内皮细胞对照正常情况下的脐静脉内皮细胞,发现的一种具有显著性差异的基因。目前在甲状腺癌方面,国内外尚未有EG-1的相关研究。本试验通过RT-PCR的方法,检测EG-1在甲状腺癌组织中表达情况,并进一步分析EG-1的表达和甲状腺癌的预后的关系。判断EG-1在评价甲状腺癌预后方面可能存在的价值。方法病理标本随机选取山东省立医院2000-2002年52例甲状腺癌石蜡包埋组织块,病理结果均经病理科专家进一步证实。52例甲状腺癌标本,其中乳头状癌36例,滤泡状癌8例,未分化癌5例,髓样癌3例。均有完整随访资料,随访截止日期为2007年5月,中位随访时间为70月(8-87月)。随访期间死于其他原因、失访及到随访截止时间仍生存者,均计为删失数据。同时取15例良性甲状腺组织(结节性甲状腺肿)作为对照。用RT-PCR方法检测甲状腺癌及良性甲状腺组织中EG-1mRNA的表达,并进一步分析比较甲状腺癌患者EG-1基因表达组和不表达组的生存资料。采用SPSS 10.0软件进行统计学分析,甲状腺癌与良性甲状腺组织中EG-1mRNA表达差异比较应用卡方检验。生存资料的单因素生存分析应用Kaplan-Meier法,采用Log-rank检验,多因素分析用Cox比例风险回归模型,Wald检验,检验标准均以p＜0.05为有统计学意义。结果1、EG-1基因在甲状腺组织中的表达:52例甲状腺癌组织中,38例甲状腺癌组织中检测到EG-1 mRNA,表达率73.08%。15例甲状腺良性结节中,4例检测到EG-1 mRNA,表达率为26.67%。两者比较,有统计学差异(x~2=10.720,p＜0.05)。2、单因素生存曲线分析的Log-rank检验,甲状腺癌患者EG-1表达组和未表达组总生存曲线分析存在差异(p=0.025),同时年龄、淋巴结转移,肿瘤分期,病理类型不同组别间总生存曲线比较均有统计学差别。性别间总生存曲线比较无统计学差别。3、多因素COX回归分析,研究发现EG-1表达、年龄、淋巴结转移、肿瘤分期、病理类型均为预测甲状腺癌生存率的独立预后因素,其中EG-1表达组死亡风险与EG-1未表达组相比明显增高(RR值4.947,p=0.008)。4、本实验同时评估目前甲状腺癌预后的几个评分系统,通过单因素生存曲线分析的Log-rank检验,发现AMES、AGES和MACIS各分组间生存曲线存在统计学差异。AMES、AGES和MACIS评分系统在评估甲状腺癌预后方面是可行的。结论及其意义EG-1基因在甲状腺癌中存在高表达,其表达阳性的患者生存率差于阴性者,单因素及多因素生存分析均表明其是甲状腺癌的一个重要预后因素,可能成为一个较好的分子生物学标记。通过对该基因表达的检测,有助于我们对甲状腺癌患者的预后提供更确切地评价,从而制定合理的复诊时间,指导我们的临床工作。本实验为首次检测甲状腺癌中EG-1基因的表达情况,并进行甲状腺癌中该基因的表达与患者临床预后的相关研究,为甲状腺癌的预后评估提供一个可能的分子生物学标记。为下一步研究EG-1基因在肿瘤发生发展中的作用机制提供重要的研究基础。第二部分乳腺癌中EG-1 mRNA的表达与预后的相关研究目的目前乳腺癌相关癌基因已经发现很多,有的已经证实在乳腺癌临床诊治中有一定价值,但始终没有发现十分关键的基因。Liu等于2002年在研究肿瘤条件液培养下的脐静脉内皮细胞对照正常情况下的脐静脉内皮细胞,发现了一种具有显著性差异的基因,他们将之命名为EG-1。其与内皮细胞、上皮细胞的增生激活状态相关,可能在肿瘤血管发生中起重要作用。EG-1在乳腺癌临床中的研究尚未有报道,本研究旨在探讨其在乳腺癌中的表达情况与乳腺癌预后的相关性。方法取山东省立医院1995年10月至1999年8月间的有完整随访资料的乳腺癌手术切除石蜡包埋标本,均行根治性手术加腋窝淋巴结清扫,经病理学确诊为浸润性乳腺癌,所有标本均用免疫组化法检测ER、PR。从中随机抽取72例病历资料及石蜡标本,采用RT-PCR技术,检测EG-1基因在72例乳腺癌及18例良性乳腺组织中的表达,同时采用免疫组化法测定72例乳腺癌石蜡包埋标本p53、Her-2及VEGF蛋白表达情况。随访截止日期为2006年10月1日,中位随访89(32～123)个月。在随访期间,38例(52.7%)出现复发转移(远处转移25例,局部复发13例),其中有26例(36.1%)患者死于乳腺癌,全部病例中有4例(5%)死于心血管疾病或意外等其他原因,至随访截至仍生存者为42例(58.3%)。随访期间死于其他原因、失访或研究截止仍生存者,均计为删失数据。采用SPSS10.0软件进行统计学分析,采用Chi-square检验乳腺癌与良性乳腺组织EG-1基因表达的差异,单因素生存分析应用Kaplan-Meier法,采用Log-rank检验,多因素分析用Cox比例风险回归模型,Wald检验,检验标准均以p＜0.05为有统计学意义。结果1.72例乳腺癌组织中,51例(71%)的乳腺癌组织中可检测到EG-1 mRNA,18例良性乳腺组织中,4例(24%)可检测到EG-1 mRNA,乳腺癌组织EG-1 mRNA显著高于良性乳腺组织(x~2=17.604,p＜0.01)。2.EG-1mRNA与生存期单因素生存分析表明,EG-1表达不同的患者间总生存时间(OS)和无病生存时间(DFS)差异均有统计学意义(p＜0.05)。3.Cox模型多因素回归分析显示,淋巴结转移数、EG-1mRNA、Her-2蛋白是预测总生存率和无复发生存率的独立预后因素,而VEGF蛋白只是预测无复发生存的独立预后因素。结论及其意义EG-1基因在乳腺癌中有高表达,EG-1表达组乳腺癌患者预后差,EG-1是乳腺癌潜在的预后标记物及治疗靶点。本实验通过检测乳腺癌中EG-1基因的表达情况,并进一步研究乳腺癌中该基因的表达与患者临床预后的相关研究,为乳腺癌的预后评估提供一个可能的分子生物学标记。并为进一步研究EG-1基因在乳腺癌发生发展中的作用机制提供重要的研究基础。更多还原

【Abstract】 Objective Zhang et al performed SSH(suppression subtractive hybridization)on control HUVECs(human umbilical vein endothelial cells)versus HUVECs exposed to tumor-conditioned media.They found that a novel cDNA(GenBank Accession No. AF358829)is differentially expressed in endothelial cells on Northern analysis,and named it endothelial-derived gene-1(EG-1).There is still no research about correlation between EG-1 and thyroid cancer all over the world.In this research,we use RT-PCR to investigate the expression of EG-1 in thyroid cancer,further more,we analyze the relationship between gene expression and clinical prognosis of the patients.In order to find a good predictor for evaluating the prognosis of thyroid cancer.Methods Archived specimens of thyroid cancer patients who were treated in Shandong provincial hospital between Jan 2000 and Aug 2002 and were followed up routinely were obtained.All the patients were confirmed diagnosis of thyroid cancer by post operation pathology.52 cases which were randomly selected to be brought into the study were examined EG-1 expression by RT-PCR,15 cases of benign tumors were the control.The deadline of follow-up was May,2007,the median follow-up time was 70(8～87)months.Data of patients who were died of other causes, or out of follow-up,or survived till the end of follow-up were calculated censored. SPSS 10.0 software was used to statistical analysis.Kaplan—Meier curve was used to analyze monofactors survival,and cox proportion ratio regression model was used to analyze poly factors survival.Statistical significance was defined by p＜0.05.Results(1)EG-1 expression in 38 cases of malignant and 4 cases of benign thyroid tissues at the mRNA levels were evaluated by RT-PCR.The EG-1 expression was much higher in malignant tissue than in the corresponding benign tumor tissue at the mRNA levels.(2)Monovariate survival analysis showed that overall survival of EG-1 positive patients were significantly shorter than EG-1 negative patients.Meanwhile, the overall survival between different groups of age,lymphonode metastatic,tumor grade,and pathologic type were significantly different.(3)In a multivariate Cox model in which overall survival was evaluated,EG-1 provided independent significant predictive power,as well as age,lymphonode metastatic,tumor grade,and pathologic type.(4)To evaluate the value of three scoring systems in predicting the progonosis of thyroid cancer,in this test,Monovariate survival analysis showed they were all useful.Conclusions EG-1 is preferentially expressed in thyroid cancer and may be used as an important prognostic marker of it. Objective At present,a large number of breast cancer related oncogenes have been found and some of them have been demonstrated to be useful to the clinical investigation.However,the oncogene playing critical role in the progress of breast cancer has not been found so far.Liu et al performed SSH(suppression subtractive hybridization)on control HUVECs(human umbilical vein endothelial cells)versus HUVECs exposed to tumor-conditioned media.They found that a novel cDNA is differentially expressed in endothelial cells on Northern analysis,and named it endothelial-derived gene-1(EG-1).Their data suggested that EG-1 was associated with a stimulated state in endothelial and epithelial cells,and may have a role in tumor angiogenesis.There is still no research about correlation between EG-1 and breast cancer clinical characteristics,although the laboratory study have proven many results.Herein,we will do some preliminary work for future research.This study is designed to demonstrate expression of EG-1 in breast cancer and to correlate the EG-1 expression level with prognosis of breast cancer.Methods Archived specimens of breast cancer patients who were treated in Shandong provincial hospital between Oct,1995 and Aug,1999 and were followed up routinely were obtained.All the patients received radical breast cancer surgery and axillary lymph node dissection,and were all confirmed diagnosis of invasive breast cancer by post operation pathology.72 cases which were randomly selected to be brought into the study were examined EG-1 expression by RT-PCR,18 cases of benign tumors were the control.Immunohistochemistry were used to examine the expression of p53、Her-2 and VEGF protein.The deadline of follow-up was 1 st Oct,2006,the median follow-up time was 89(32～123)months.During the period of follow-up,38(52.7%)patients occurred relapse(25 cases of distant relapse,13 case of local relapse),among which,26(36.1%)patients died from breast cancer.4(5%) patients died of cardiovascular diseases and accidents.42(58.3%)patients survived till the end of follow-up.Data of patients who were died of other causes,or out of follow-up,or survived till the end of follow-up were calculated censored.SPSS 10.0 software was used to statistical analysis.The analysis of Chi-square test was performed for comparison of EG-1 expression between breast cancer specimens and benign tumor specimens.Kaplan—Meier curve was used to analyze monofactors survival,and cox proportion ratio regression model was used to analyze poly factors survival.Statistical significance was defined by p＜0.05.Results 1.EG-1 expression in 72 cases of malignant and 18 cases benign breast tissues at the mRNA levels were evaluated by RT-PCR.The EG-1 expression was much higher in malignant tissue than in the corresponding benign breast tissue at the mRNA levels(χ~2=17.604,p＜0.01).2.Monovariate survival analysis showed that overall survival and disease free survival of EG-1 positive patients were significantly shorter than EG-1 negative patients(p＜0.05).3.In a multivariate Cox model in which overall survival and disease free survival were evaluated,EG-1 provided independent significant predictive power,as well as positive lymph node numbers and Her-2 protein.However,protein VEGF only predicted disease free survival.Conclusions The EG-1 expression is up-regulated in breast cancer.It may be an important tumor biomarker and therapeutic target.更多还原