人骨肉瘤相关蛋白质组分析及癌基因、抑癌基因、凋亡相关基因表达差异的基因芯片研究
摘要
骨肉瘤(Osteosarcoma,OSA)是最常见的骨组织原发恶性肿瘤，好发于儿童及青少年。其临床生物学特征为肿瘤恶性程度极高，生长迅速，早期发生转移。传统治疗方法（手术切除或截肢加辅助化疗）5年存活率仅为20%—50%。近年来，由于化疗药物的发展、及时诊断和严格按照外科分期选择手术，疗效有所提高，但总体治疗结果仍不理想，尤其是伴有远处转移者。骨肉瘤的治疗一直是临床研究的重点与难点，对骨肉瘤进行早期诊断，寻找特异、高效、副作用小的治疗手段即成为骨科领域亟待解决的重要课题。因此，在基因和蛋白质水平了解骨肉瘤的发生、发展具有极其重要意义。
近年来对相关癌基因（ras,myc,fos,bcl-2,met等）和抑癌基因（Rb、P53、P21等）进行了结构分析和蛋白表达研究，发现骨肉瘤可出现多个基因的异常，不同患者间基因的异常存在差别。得出了骨肉瘤细胞普遍存在染色体、癌基因和抑癌基因的异常，骨肉瘤的发生与多种癌基因和抑癌基因变异有关的结论。同时也说明癌基因及抑癌基因异常在骨肉瘤的发生中起着重要作用，骨肉瘤的发生是多种因素共同作用的结果，需要研究的不仅是一个或几个，而是整个基因组在从正常到癌变及其进展过程的各个阶段中成百上千个基因和其表达的蛋白质的动态变化。只有全面了解基因表达水平，才能理解基因型和表型的相互关系。
以往对骨肉瘤的研究方法通常是以个别基因或个别蛋白质为研究对象，往往是静态的、局部的，而面对复杂的、动态多基因相互作用肿瘤性疾病时，这些方法就表现出很大的局限性。
人类基因组计划(human genome project，HGP)的快速、大规模推进，上千种基因序列的相继问世，使人类对生命本质的认
识达到了一个空前的广度和深度。但人类基因组序列图谱初稿的公布，在揭示基因组的精细结构的同时，也凸现出基因数量有限性和基因结构的相对稳定性，这与生命现象的复杂性和多变性之间存在着巨大反差。这种反差使人们认识到：基因只是遗传信息的载体。要研究生命现象，阐述生命活动的规律，只了解基因组的结构是远远不够的，基因组研究已从结构基因组学转向功能基因组学，研究重心也已从揭示生命的所有遗传信息转移到在整体水平上对基因功能的认识。面对分析数以百万记的基因的复杂表达方式，确定细胞生命中错综复杂的调节表达信号的艰巨任务，传统的Northern Blot或点杂交的方法，以及电泳为基础的基因表达、序列测定、突变和多态性分析等一次只能研究少量蛋白质和基因的方法显然不能适应发展的要求。
伴随着后基因时代的到来，基因芯片(gene chip)和蛋白质组(Proteome)技术越来越受到国内外医学科学工作者的密切关注，并以其特有的思维方法和技术手段在解决生物学重大问题上开始显示出强大威力。
基因芯片是微列阵技术(microarray technology)的一个分支，含有大量生物信息的固相基质称为微列阵(microarray)，又称生物芯片(biochip)。根据储存的生物信息的类型又可分为组织微列阵(tissue microarray)，寡核苷酸微列阵(oligonucleotide microarray又称DNA微列阵)或DNA芯片(DNA microarray)，cDNA微列阵(cDNA microarray又称cDNA芯片)等。DNA微列阵和cDNA微列阵一起又称基因芯片。基本原理是分子杂交。将大量靶基因用点样仪有序地、高密度地点在玻璃、硅等载体上制作而成的。将待测样品用荧光染料标记制备成探针与芯片杂交，杂交信号用激光扫描仪检测，计算机分析检测结果，可获得类似于传统的点杂交的分子杂交数据，比较各组间靶基因表达谱的差异，以达到快速、高效、高通量及平行性地分析生物信息的目的。
蛋白质组的定义为一个有机体的基因组所表达的全部蛋白质。它是一个在空间和时间上动态变化着的整体。狭义上讲，蛋
白质组是指不同条件下细胞内蛋白质的变化，比如正常细胞和异常细胞之间，细胞用药和不用药之间的蛋白质表达谱的差别，这在疾病研究和药物筛选上很有意义。当前肿瘤蛋白质组学研究常见的思路是鉴定和定量分析某一特定组织内肿瘤细胞与其正常起源细胞之间或肿瘤细胞在药物处理前后差异表达的蛋白质，并以此作为肿瘤早期检测和治疗的生物标志物，同时建立相应肿瘤组织或细胞的蛋白质数据库。采用的方法主要是双向电泳(Two-dimensional gel electrophoresis，2-DE)、质谱(MS)和生物信息学(bioinformatics)分析等。
上述两种新技术给肿瘤学研究带来了全新的概念。国内外尚未见将其应用于骨肉瘤研究的报告。而其在骨肉瘤研究中的应用将极大地促进对骨肉瘤的发病机制、诊断、治疗和新药开发的研究，具有重要的理论和实际意义。
本文运用基因芯片及蛋白质组技术研究骨肉瘤组织与正常骨组织中蛋白质及基因的表达差异。
骨肉瘤标本（实验组）取自我院骨科新鲜手术切除的肿瘤组织,病理切片证实为成骨肉瘤。正常组织（对照组）为同一手术（截肢术）中切取的肿瘤远隔部位的骨膜和骨组织，病理切片证实为正常骨膜和骨组织。
骨肉瘤的形态学及免疫组化检查部分中骨肉瘤组织切片HE染色结果证明所采集的标本为低分化型、骨母细胞型成骨肉瘤组织，病理特征较为典型，为下一步实验的顺利进行奠定了良好的基础?
Osteosarcoma (OSA) is the most common primary malignant tumor of bone with highest incidence. It often occurs in juvenile and young people, and more frequently in male. Its pathological characteristics are as follow: sarcoma cells producing plenty of osteoid tissues, early distant metastasis, and the leading cause of mortality is pulmonary metastases. Traditional therapy mainly includes amputation of extremity, but the survival rate within 5 years is only 5~15% under pure surgery method; Nevertheless the patients suffering from pulmonary metastases will die within 6 months generally. Recently, owing to chemotherapy, prompt diagnosis, and operation performed strictly according to the surgery stage, the curative effect has improved to some extent. But the result is still unsatisfied in total, especially to those who accompanied with remote metastases. The researches of OSA are always the emphasis and difficulty in clinical and fundamental studies, therefore the early diagnosis of OSA and searching a kind of therapy which is specific, high effective and fewer side effects has become an important issue. So, it has a very important meaning to understand the formation and development of osteosarcoma in the genetic and protein level.
In recent years, structural analyse and protein expression studies on protooncogene(pro-onc) (ras, myc, fos, bcl-2, met, et al.) and antioncogene(anti-onc) (Rb, P53, P21, et al.) showed that multi-abnormity of genes may occue
in osteosarcoma, and different patients have different genetic abnormity. The ubiquity abnormities of chromosome, protooncogene and antioncogene exist in osteosarcoma cells illuminated that the formation of osteosarcoma related to many protooncogene and antioncogene mutations. Since the formation of osteosarcoma is related to multi-factors, the research should not limited on one or a few genes and proteins, but thousands of genes and proteins that kept changing in every phase of the mutation process of the whole genome.
Traditional ways of gene and protein study focus on certain gene and protein, and are static and partial. When the method face complicate, dynamic, multi-gene interaction tumor diseases, the incapable of these ways showed.
The rapid advance of human genome project (HGP) showed us thousands of gene sequence that we do not know before. The publish of the first draft of human genome atlas showed us the delicacy of human genome structure, but also raise the problem of comparatively fewer of the gene number contrast to delicacy and levity of life phenomenal. This contrast lead us to realize that gene is only the carrier of heritage information. It is far not enough to expatiate life activity rules with only understanding of the structure of genome. The research of genome has shifted from structural to functional, and the research barycenter has shifted from disclosing heritage information to understanding of the function of gene in an integrated level. Facing millions of gene expression manners and intricate adjustment of expression signals in cell, traditional research methods such as Northern Blot, dot cross bred and electrophoresis based gene expression and sequence
analysis etc. which focusing one or a few proteins or gene, are no longer suit the research needs.
As the imminent of post-genetic era, gene chip and proteome technologies are catching more and more attention of the researchers’ in recent years, and they have shown great power in solving important biological problems.
Microarray technology, namely biochip, includes tissue microarray, DNA microarray, cDNA microarray and so on. On the other hand, DNA microarray and cDNA microarray are also named together as gene chip, whose basic principle is molecular hybridization. It makes gene analysis possible of which is large scale, parallel, small size, and automation. Simultaneously, the result of the research is more objective, more precise. These important characteristics have brought a series of achievements, which can't be attained before. Main applications
近年来对相关癌基因（ras,myc,fos,bcl-2,met等）和抑癌基因（Rb、P53、P21等）进行了结构分析和蛋白表达研究，发现骨肉瘤可出现多个基因的异常，不同患者间基因的异常存在差别。得出了骨肉瘤细胞普遍存在染色体、癌基因和抑癌基因的异常，骨肉瘤的发生与多种癌基因和抑癌基因变异有关的结论。同时也说明癌基因及抑癌基因异常在骨肉瘤的发生中起着重要作用，骨肉瘤的发生是多种因素共同作用的结果，需要研究的不仅是一个或几个，而是整个基因组在从正常到癌变及其进展过程的各个阶段中成百上千个基因和其表达的蛋白质的动态变化。只有全面了解基因表达水平，才能理解基因型和表型的相互关系。
以往对骨肉瘤的研究方法通常是以个别基因或个别蛋白质为研究对象，往往是静态的、局部的，而面对复杂的、动态多基因相互作用肿瘤性疾病时，这些方法就表现出很大的局限性。
人类基因组计划(human genome project，HGP)的快速、大规模推进，上千种基因序列的相继问世，使人类对生命本质的认
识达到了一个空前的广度和深度。但人类基因组序列图谱初稿的公布，在揭示基因组的精细结构的同时，也凸现出基因数量有限性和基因结构的相对稳定性，这与生命现象的复杂性和多变性之间存在着巨大反差。这种反差使人们认识到：基因只是遗传信息的载体。要研究生命现象，阐述生命活动的规律，只了解基因组的结构是远远不够的，基因组研究已从结构基因组学转向功能基因组学，研究重心也已从揭示生命的所有遗传信息转移到在整体水平上对基因功能的认识。面对分析数以百万记的基因的复杂表达方式，确定细胞生命中错综复杂的调节表达信号的艰巨任务，传统的Northern Blot或点杂交的方法，以及电泳为基础的基因表达、序列测定、突变和多态性分析等一次只能研究少量蛋白质和基因的方法显然不能适应发展的要求。
伴随着后基因时代的到来，基因芯片(gene chip)和蛋白质组(Proteome)技术越来越受到国内外医学科学工作者的密切关注，并以其特有的思维方法和技术手段在解决生物学重大问题上开始显示出强大威力。
基因芯片是微列阵技术(microarray technology)的一个分支，含有大量生物信息的固相基质称为微列阵(microarray)，又称生物芯片(biochip)。根据储存的生物信息的类型又可分为组织微列阵(tissue microarray)，寡核苷酸微列阵(oligonucleotide microarray又称DNA微列阵)或DNA芯片(DNA microarray)，cDNA微列阵(cDNA microarray又称cDNA芯片)等。DNA微列阵和cDNA微列阵一起又称基因芯片。基本原理是分子杂交。将大量靶基因用点样仪有序地、高密度地点在玻璃、硅等载体上制作而成的。将待测样品用荧光染料标记制备成探针与芯片杂交，杂交信号用激光扫描仪检测，计算机分析检测结果，可获得类似于传统的点杂交的分子杂交数据，比较各组间靶基因表达谱的差异，以达到快速、高效、高通量及平行性地分析生物信息的目的。
蛋白质组的定义为一个有机体的基因组所表达的全部蛋白质。它是一个在空间和时间上动态变化着的整体。狭义上讲，蛋
白质组是指不同条件下细胞内蛋白质的变化，比如正常细胞和异常细胞之间，细胞用药和不用药之间的蛋白质表达谱的差别，这在疾病研究和药物筛选上很有意义。当前肿瘤蛋白质组学研究常见的思路是鉴定和定量分析某一特定组织内肿瘤细胞与其正常起源细胞之间或肿瘤细胞在药物处理前后差异表达的蛋白质，并以此作为肿瘤早期检测和治疗的生物标志物，同时建立相应肿瘤组织或细胞的蛋白质数据库。采用的方法主要是双向电泳(Two-dimensional gel electrophoresis，2-DE)、质谱(MS)和生物信息学(bioinformatics)分析等。
上述两种新技术给肿瘤学研究带来了全新的概念。国内外尚未见将其应用于骨肉瘤研究的报告。而其在骨肉瘤研究中的应用将极大地促进对骨肉瘤的发病机制、诊断、治疗和新药开发的研究，具有重要的理论和实际意义。
本文运用基因芯片及蛋白质组技术研究骨肉瘤组织与正常骨组织中蛋白质及基因的表达差异。
骨肉瘤标本（实验组）取自我院骨科新鲜手术切除的肿瘤组织,病理切片证实为成骨肉瘤。正常组织（对照组）为同一手术（截肢术）中切取的肿瘤远隔部位的骨膜和骨组织，病理切片证实为正常骨膜和骨组织。
骨肉瘤的形态学及免疫组化检查部分中骨肉瘤组织切片HE染色结果证明所采集的标本为低分化型、骨母细胞型成骨肉瘤组织，病理特征较为典型，为下一步实验的顺利进行奠定了良好的基础?
Osteosarcoma (OSA) is the most common primary malignant tumor of bone with highest incidence. It often occurs in juvenile and young people, and more frequently in male. Its pathological characteristics are as follow: sarcoma cells producing plenty of osteoid tissues, early distant metastasis, and the leading cause of mortality is pulmonary metastases. Traditional therapy mainly includes amputation of extremity, but the survival rate within 5 years is only 5~15% under pure surgery method; Nevertheless the patients suffering from pulmonary metastases will die within 6 months generally. Recently, owing to chemotherapy, prompt diagnosis, and operation performed strictly according to the surgery stage, the curative effect has improved to some extent. But the result is still unsatisfied in total, especially to those who accompanied with remote metastases. The researches of OSA are always the emphasis and difficulty in clinical and fundamental studies, therefore the early diagnosis of OSA and searching a kind of therapy which is specific, high effective and fewer side effects has become an important issue. So, it has a very important meaning to understand the formation and development of osteosarcoma in the genetic and protein level.
In recent years, structural analyse and protein expression studies on protooncogene(pro-onc) (ras, myc, fos, bcl-2, met, et al.) and antioncogene(anti-onc) (Rb, P53, P21, et al.) showed that multi-abnormity of genes may occue
in osteosarcoma, and different patients have different genetic abnormity. The ubiquity abnormities of chromosome, protooncogene and antioncogene exist in osteosarcoma cells illuminated that the formation of osteosarcoma related to many protooncogene and antioncogene mutations. Since the formation of osteosarcoma is related to multi-factors, the research should not limited on one or a few genes and proteins, but thousands of genes and proteins that kept changing in every phase of the mutation process of the whole genome.
Traditional ways of gene and protein study focus on certain gene and protein, and are static and partial. When the method face complicate, dynamic, multi-gene interaction tumor diseases, the incapable of these ways showed.
The rapid advance of human genome project (HGP) showed us thousands of gene sequence that we do not know before. The publish of the first draft of human genome atlas showed us the delicacy of human genome structure, but also raise the problem of comparatively fewer of the gene number contrast to delicacy and levity of life phenomenal. This contrast lead us to realize that gene is only the carrier of heritage information. It is far not enough to expatiate life activity rules with only understanding of the structure of genome. The research of genome has shifted from structural to functional, and the research barycenter has shifted from disclosing heritage information to understanding of the function of gene in an integrated level. Facing millions of gene expression manners and intricate adjustment of expression signals in cell, traditional research methods such as Northern Blot, dot cross bred and electrophoresis based gene expression and sequence
analysis etc. which focusing one or a few proteins or gene, are no longer suit the research needs.
As the imminent of post-genetic era, gene chip and proteome technologies are catching more and more attention of the researchers’ in recent years, and they have shown great power in solving important biological problems.
Microarray technology, namely biochip, includes tissue microarray, DNA microarray, cDNA microarray and so on. On the other hand, DNA microarray and cDNA microarray are also named together as gene chip, whose basic principle is molecular hybridization. It makes gene analysis possible of which is large scale, parallel, small size, and automation. Simultaneously, the result of the research is more objective, more precise. These important characteristics have brought a series of achievements, which can't be attained before. Main applications
引文
第一部分 综述
综述一 蛋白质组学的概念及研究现状
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综述二 蛋白质组学研究的技术方法与发展现状
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综述五 肿瘤癌基因、抑癌基因、凋亡相关基因的研究现状及基因芯片技术的应用前景
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第二部分 人骨肉瘤标本的形态学及免疫组化检查
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第三部分 人骨肉瘤癌基因、抑癌基因及凋亡相关基因表达差异的基因芯片研究
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第四部分 人骨肉瘤相关蛋白质组分析
实验一 双向凝胶电泳和图像分析技术的建立及优化
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实验二 差异蛋白质的质谱鉴定和数据库检索
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综述一 蛋白质组学的概念及研究现状
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综述二 蛋白质组学研究的技术方法与发展现状
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综述三 肿瘤蛋白质组学的研究概况
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综述五 肿瘤癌基因、抑癌基因、凋亡相关基因的研究现状及基因芯片技术的应用前景
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第二部分 人骨肉瘤标本的形态学及免疫组化检查
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第三部分 人骨肉瘤癌基因、抑癌基因及凋亡相关基因表达差异的基因芯片研究
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第四部分 人骨肉瘤相关蛋白质组分析
实验一 双向凝胶电泳和图像分析技术的建立及优化
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实验二 差异蛋白质的质谱鉴定和数据库检索
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