【作者】 马俊明；

【导师】 贾连顺；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 研究背景:脊索瘤(chordoma)是一种起源于胚胎残余脊索组织的原发性、低度恶性骨肿瘤,主要分布于人体中轴骨,约50%发生于骶尾部,约35%发生在枕骨斜坡部位。脊索瘤在生物学特性方面呈现缓慢、局部侵袭性生长,发生远处转移较晚且多见于肺部。脊索瘤破坏脊柱骨质,造成椎节不稳、塌陷及脊髓、神经受压,导致顽固性疼痛、脊髓神经功能障碍和瘫痪,严重影响患者生活质量。外科手术干预,在切除病灶、解除神经压迫、恢复脊柱稳定方面具有不可替代的作用。因脊柱部位局部解剖结构复杂,肿瘤往往比邻重要血管神经结构,手术难以彻底切除病灶。加之脊索瘤对放化疗均不敏感,尽管临床上多采取手术切除并配合术后放、化疗等综合治疗,脊柱脊索瘤总体预后仍较差,术后局部复发率高,患者多死于局部肿瘤病灶的反复复发。据统计,脊索瘤患者的死亡率在63%左右,5年生存率为50%,10年生存率约为28%。预后判断在正确制定脊柱脊索瘤治疗策略的过程当中起着重要的作用。因脊索瘤总体发病率相对不高,临床上缺乏较大宗的有关预后因素的分析的研究报道。随着分子生物学的发展,国内外肿瘤相关基因的研究已有了长足的进展。大量研究表明,肿瘤相关基因的突变、扩增及异常表达等改变常与肿瘤的预后密切相关。在乳腺癌、肺癌、肝癌等原发性肿瘤的研究相继发现了与之预后紧密相关的分子标志物,并成功应用于临床预后判断,有的甚至在后续研究中成为新治疗方法的靶点。而目前国内外在脊索瘤方面的研究不多,鲜见肿瘤相关基因与脊柱脊索瘤关系的相关报道。肿瘤的发生、发展是一种多基因、多因素参与的复杂过程。预后相关指标的筛选常需要大样本量、多因素的研究。传统方法需对多个样本重复操作、工作量大、耗时长、费用高,且由于实验条件不均一而使结果的准确性受到影响。组织芯片技术的出现为医学病理学以及分子流行病学提供了一种高通量、大样本以及快速的分子水平的分析工具,又克服了传统病理学技术、蛋白、基因芯片技术存在的某些缺陷,使人类第一次能够有效利用成百上千份自然或处于疾病状态下的组织标本分别在基因、基因转录和相关表达产物生物学功能3个水平上进行研究,并具有节约组织原材料,保证实验条件一致性,减小实验误差的优点。研究表明组织芯片技术稳定、可靠,其特点对于大样本标本的基因表达与预后分析具有极其重要的价值,并已应用于多种肿瘤的相关研究。钙粘蛋白及连环蛋白家族成员在细胞黏附过程中起着极其重要的作用。大量的研究表明,以上蛋白的异常表达情况往往和肿瘤的局部侵袭、转移能力有着直接的联系。P120ctn作为连环蛋白家族的新成员,近来成为各种肿瘤分子学机制研究的热点。但有关其在脊索瘤发生、发展过程中表达情况及意义,在国内外的研究中尚未见报道。研究目的:针对脊柱脊索瘤,以胎儿正常椎间盘脊索组织为对照,构建脊柱脊索瘤组织芯片;检测钙粘蛋白E和连环蛋白家族成员α-catenin,β-catenin,γ-catenin,P120-catenin的脊柱脊索瘤组织芯片的表达情况;通过统计学分析探讨相关蛋白及临床病理指标与脊柱脊索瘤预后之间的关系。研究方法:选取上海长征医院及温州医学院附属第一医院自1997年1月至2007年1月手术切除且临床病理及随访资料完整的脊柱脊索瘤40例标本,以及9周至25周胎儿椎间盘组织标本5例,以组织阵列仪构建样本直径1.0mm 9行×10列90个位点的组织微阵列蜡块,连续切片制作脊柱脊索瘤组织芯片。采用免疫组织化学SP法,检测钙粘蛋白E和连环蛋白家族成员α-catenin,β-catenin,γ-catenin,P120-catenin在脊柱脊索瘤组织芯片的表达,观察、评定各基因蛋白异常表达率及异常表达的情况。应用SPSS统计软件分析上述基因在脊柱脊索瘤组织与正常脊索组织中的表达差异,各基因表达相互间关系及与脊柱脊索瘤生物学行为的关系,Kaplan Meier生存分析及Log-Rank时序检验分析各检测指标及临床病理指标与脊柱脊索瘤预后的关系。采用Cox模型回归分析肿瘤复发相关基因和临床病理指标的回归系数,探询与脊索瘤局部复发相关的影响因素。结果:1.制作完成的组织微阵列蜡块完整、无裂缝,组织排列整齐,组织芯与受体蜡块融合致密,连续切片制成脊柱脊索瘤组织芯片50张,组织芯片平均利用率97.3%。2.5例胎儿椎间盘正常脊索组织中,E-cad及连环蛋白主要表达在细胞与细胞的连接处,即胞膜部位的阳性表达,而胞核内无上述基因表达。40例脊索瘤标本呈现出上述基因胞膜表达的减弱或缺失;P120ctn和E-cad在脊索瘤与正常脊索组织中胞膜部位表达存在显著差异(P＜0.05)。部分脊索瘤标本出现上述基因的“胞核内转位”:α-catenin 14例,β-catenin 2例,γ-catenin 13例,P120ctn 9例,E-cad4例。3.经统计学分析,α-catenin与γ-catenin异常表达率之间,以及E-cadherin与P120-catenin异常表达率之间呈现显著相关,P＜0.05。4.单因素分析显示,α-catenin表达与年龄之间,γ-catenin和P120-catenin表达与病理分级之间,E-cadherin的表达与椎节数目之间有显著相关。logisitic回归分析显示,女性患者β-catenin异常表达率高于男性患者,年龄是α-catenin异常表达的危险因素,KPS评分是β-catenin异常表达的保护因素,肿瘤病理分级是r-catenin和P120-catenin异常表达的危险因素,肿瘤侵犯椎节的数目是E-cadherin异常表达的危险因素。5.生存分析:Kaplan-Meier单因素分析和Log Rank时序检验显示,肿瘤平均复发时间在γ-catenin、P120-catenin、E-cadherin的不同表达水平及病理分级的不同状态下与存在显著差异;患者的生存期在P120-catenin和E-cadherin的不同表达水平状态下存在显著差异。COX模型回归分析显示,只有P120-catenin和病理分级是脊柱脊索瘤复发的独立预测因素。结论:1.组织芯片技术应用于脊柱脊索瘤研究稳定、可靠。2.在脊柱脊索瘤的发生、发展过程中,E-cadherin、α-catenin,β-catenin,γ-catenin,P120-catenin均出现了不同程度的异常表达。γ-catenin及P120-catenin异常表达与不同病理分级,E-cadherin异常表达与不同的累及椎节数目之间存在显著差异,提示他们与脊索瘤的去分化、侵袭力增强有关。3.γ-catenin,P120-catenin,E—cadherin及病理分级与脊柱脊索瘤局部复发有关;E—cadherin和P120-catenin与脊柱脊索瘤患者生存期有关,可作为临床观察脊柱脊索瘤预后的有意义的检测指标;P120-catenin,病理分级是脊柱脊索瘤局部复发的独立预测因素。降低P120-catenin的异常表达,可能有助于降低脊柱脊索瘤的局部复发率。更多还原

【Abstract】 Background Chordoma is a kind of low malignant,primarily bone tumor,and it arises from residue of the notochord.Chordoma mostly locates in the axis with nearly 50%in the sacrum,35%in the clivus.Chordoma has the character of biological behavior with slowly,infiltrating progression,and metastasis to lung in late cases. Conditions that occurs with spinal chordoma such as osteolysis of bone,collapse of vertebrae,instability of spine column and compression of spinal cord often lead to severe pain,neurofunctional impairment even plegia,then result in decrease of life quality of patients.Surgical treatment takes an irreplaceable role in the treatment of spinal chordoma with removing of lesions,decompression of cord,and reconstruction of spine.Because the complicated anatomic structure of the spine,tumors likely to be around by some vital nerv vasorum structure,which lead to the difficulty in the total excising the tumors.Also due to not sensitive to most radiotherapy and chemotherapy, chordoma has a high level of local recurrence rate and a poor prognosis after surgical treatments,even surgical treatment combined with radiotherapy and chemotherapy is adopted in clinical treatments.Patients may dead of the tumor with the recurrence again and again.According to some statistics report,patients with chordoma have a total mortality of 63%with survival rate of 50%in 5 years and 28%in 10 years. Reliable prediction is the key to determine suitable strategy for spinal chordomas. Because the total disease incidence is not high relatively,there are few study data reported of factors which affecting the prognosis of spinal chordoma.With the development of molecular biology,rapid progress was achieved in research of tumor-related genes.Many studies indicated close relationship between prognosis of tumor and mutation or abnormal expression of gene.Molecular biomarkers with prognostic value were identified in research of primary tumors such as breast cancer,lung cancer and liver cancer,and some of them even became new therapy target in subsequent research,while studies on chordoma were seldom and report about the correlation between the genes and tumors for the spinal chordoma is rare.The genesis and progression of tumor is regarded as a complicated course that multiple gene and multiple factor participates the process.Large sample and multiple genes research are needed to screen prognostic genes.Repetitive operation on multiple samples,large workload,long time and high costs were spent during a traditional method and the result could be affected by different experiment condition. The innovation of tissue chip provides a powerful tool with which high-throughput, large sample and rapid analyses on thousands of natural or pathologic tissue samples in three levels including gene,genetic transcription and biological function of expression product can be put into reality.It has been proved in many studies that tissue microarray is a reliable and stable method with the advantage of saving tissue and equal experiment condition.The value of tissue microarray technology in large sample and multiple gene analyses of prognostic research is enormous and it has been used in many studies on tumors.Cadherins and catenins play an important role in the course of adherence of cell to cell.Much research have indicate that the expression of the proteins as mentioned has a direct relationship to the invasiveness and metastasis of the tumors.As a new family member of the catenins,P120ctn became the key-point of research about molecule mechanism of tumors.But the expression and the correlating significance in chordomas had not been reported in the native or foreign literatures.Objective To construct tissue chip of spinal chordoma with normal fetal notochord as control,and detect the expression of E-cadherin and the catenins family members thatα-catenin,β-catenin,γ-catenin,P120-catenin in the tissue array of chordoma,then to detect the correlation of the exppresion of proteins and the recurrence of this tumor with the clinical and pathological data combination by statistic analyses.Methods A total of 40 cases of spinal chordoma underwent surgical treatment in Changzheng Hospital and the first affiliated hospital of WenZhou medical university from Jan.1997 to Jan.2007 with complete clinical and follow up data,as well as five specimens of normal fetal notochord ranged from 9 weeks to 25 weeks was included in this study A 9-row and 10-column tissue microarray consisting of 90 samples with 1.0mm diameter was built by a tissue microarrayer.Tissue chips of spinal chordoma were obtained from serial sections of tissue microarray.The expression of E-cad and catenins were detected on the tissue chips by immunohistochemistry to detect the abnormal expression of the proteins.The SPSS statistical software was used in analyses on different exppresion between chordoma tissue and normal notochord,relationship between each gene expression and biological behavior of spinal chordomas.The significance of different gene expression and clinical data,which correlates to prognosis of patients was determined by Kaplan Meier survival analyses and Log-Rank test.Cox model multivariate analyses were used to detect the correlation factors about tumor recurrence.Results 1.The tissue microarray built in this study is integrated well with a high availability of 97.3%,and 50 tissue chips were produced from it.2.For the five specimens of normal fetal notochord,the expression of proteins mainly appeared on the point of cell-cell conjunction,means cytomembrane,and no stain could be detected on the nucleus.For the 40 specimens of spinal chordoma,the spare or total loss of expression is the majority.Significance correlation were found in P120ctn and E-cad when comparing the result of chordomas with that of notochord(P＜0.05). Some of the specimens of chordoma presented with the manifest of"transposition into nucleus",including 14 specimens ofα-catenin,2 specimens ofβ-catenin,13 specimens ofγ-catenin,9 specimens of P120ctn,and 4 specimens of E-cad.3.Based on the result of statistics,there is significance correlation between abnormal expression of proteins,such asα-catenin andγ-catenin,E-cadherin and P120-catenin. 4.Mono-factor analysis revealed that significance difference occurred between factors,such asα-catenin and age,γ-catenin and pathological grading, P120-catenin and pathological grading,E-cadherin and vertebral quantity.The result of Logisitic Statistics revealed that the abnormal expression rate ofβ-catenin in females is higher than it in males,KPS score is the protect factor for the abnormal expression ofβ-catenin,age is the risk factor of the abnormal expression ofα-catenin,pathological grading is the risk factor of the abnormal expression of both r-catenin and P120-catenin.5.Kaplan-Meier analysis and Log Rank check revealed that significance difference occurred among factors about mean free survival time of patients,such as different expression ofγ-catenin、P120- catenin,E-cadherin and different levels of pathological grading.While,significance difference occurred among the different level of factors about total survival time of patients,such as P120-catenin and E-cadherin.COX model analysis revealed that P120-catenin and pathological grading were the independent pretest factors of the recurrence of the spinal chordoma.Conclusion 1.The applying of the technique of tissue microarray in the study of spinal chordoma was well preserved and stable.2.In the process of carcinogenesis and progression of spinal chordomas,abnormal expression of E-cadherin,α-catenin,β-catenin,γ-catenin,P120- catenin occurred in different degrees.Significance occurred between the abnormal expression ofγ-catenin and pathological grading,the abnormal expression of P120-catenin and pathological grading,and different level of pathological grading and vertebral quantity of involvement,which revealed that these factors may be correlating to the enhancement of dedifferentiation and invasiveness of spinal chordoma.3.Significant correlation occurred between the different levels ofγ-catenin,P120-catenin,E—cadherin and pathological grading and recurrence of the spinal chordoma,as well as between the different levels of E—cadherin,P120-catenin and survival time of patients.So these factors may be regarded as useful index in detecting the prognosis of spinal chordoma. P120-catenin and pathological grading were the independent pretest factors of the prognosis in spinal chordoma.更多还原