肝癌生物标本库的建立及应用
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摘要
肝细胞肝癌(Hepatocellular Carcinoma, HCC)简称肝癌,世界卫生组织数据库显示,全球每年新增约60万肝癌患者,为第三号癌症杀手。我国肝癌年发病人数约35万,有30万人死于此病,发病率和死亡率均占癌症第二位。
目前,以肝切除术为代表的外科治疗仍是肝癌首选治疗方法。近年来逐渐开展新的治疗方法为肝癌的治疗带来了多种选择,并大大改善了病人的预后。基因组学和蛋白质组学等高通量技术平台的迅猛进展,外源基因转导和表达技术的提高,细胞靶向与表位靶向载体的构建,生物信息数据库的日趋完备,肝癌的分子靶向治疗成为一种具有广泛临床应用前景的综合疗法。
虽然近年来的研究已经取得了一定的进展,但是依然存在着一些不尽人意的现象,如研究的重现性较差,研究周期长,进程较慢等。标本库的建立能有效地避免离体肿瘤组织因缺血在短期内失去其相应的生物学活性,如基因的降解、蛋白的变性等,利用同一标本库的标本进行相关研究能有效地提高研究结果的重现性。
肿瘤研究的最终目的是治疗人类的肿瘤,因此对肿瘤的研究模型也必须从体外实验和动物模型的研究逐渐转向人体肿瘤组织标本的研究以及临床研究。二十世纪末期以来,国际上先后建立了单病种和多病种的肿瘤组织库,并逐渐规范化,形成了标准化的操作体系。如欧洲不少国家的医疗机构拥有了自己的标准化的冰冻肿瘤组织标本库。近年国内某些研究机构也建立了肿瘤库,但多数肿瘤标本库都缺乏标准化、规范化的操作体系,所收集的肿瘤标本不能够为临床和基础研究所应用,或者在应用中受到很大的限制,因此标准化肿瘤标本库的建立具有重大的现实意义。
端粒是真核细胞染色体末端的一个特化结构,由一定长度高度保守的串联重复DNA序列构成,是稳定染色体的关键结构。端粒长度的变短与肿瘤发病密切相关,如:端粒缩短导致乳腺癌、前列腺癌、肺癌和膀胱癌等肿瘤的发生率显著增加。利用临床组织样本进行的研究表明:与临近的非肝癌组织和肝硬化组织相比,肝癌组织的平均端粒长度显著缩短,在肝癌发病中扮演了极其重要的角色。
因此,在我国西部建立一个标准化的肝癌标本库,对于肝癌的研究和临床治疗势在必行,本研究拟建立一个标准化的、规模化的肝癌标本库,并初步利用此样本库来进行肝癌标本平均端粒长度与肝癌发病风险关系的评估。
本研究分四个部分:
第一部分肝癌生物标本的规范化收集
目的:探讨建立肝癌生物标本库的标准化程序,研究并制定规范化的肝癌生物标本采集、处理、保存操作流程,建立功能完善,信息丰富的肝癌生物标本库。
方法:借鉴国内外标本库建立的标准化程序,制定规范化的肝癌生物标本收集操作流程体系,收集来自3家临床医院的肝癌、慢性肝疾病(Chronic liver disease,CLD)和对照患者的组织标本和血液标本,采用收集—反馈—修改的模式对标本收集的操作流程不断完善,并健全各种管理和质量控制措施,完成各种表格登记。
结果:通过制定标准化的标本收集流程,收集到了来自3家医院的604例肝癌组织和血液标本。并随机抽取20份DNA进行检测,证明所收集的标本具有较高的质量。
结论:规范化操作流程的建立有利于提高标本收集的效率,提高标本库的质量,具有很好的可操作性。
第二部分肝癌标本临床资料的信息化管理
目的:建立肝癌标本临床资料的信息化管理系统,对所收集的肝癌标本以及相关的临床资料信息进行系统化管理。
方法:利用标准化的资料表格,通过系统的临床信息采集,不断完善所收集的肝癌生物标本的相关临床信息。建立基于EpiData软件的数据库管理系统,通过简单便捷的数据录入、查询、分析界面,对肝癌标本进行系统化的管理。
结果:通过604份肝癌标本信息的录入和初步分析,检验了对收集肝癌标本的管理系统。
结论:通过建立标准化的肝癌标本临床信息管理系统,能够有效提高标本管理的准确性和可操作性。
第三部分肝癌标本平均端粒长度用于肝癌发病风险的评估
目的:对肝癌标本的平均端粒长度进行检测,探讨其与肝癌发病风险之间的联系。
方法:采用实时定量PCR的方法,检测肝癌标本及正常对照标本的全部染色体平均端粒长度,通过评估肝癌的发病风险和全部染色体端粒长度的相关性,进而确定二者的关系。
结果:通过对33例标本的检测,发现肝癌标本的平均端粒长度较正常对照组显著缩短。
结论:肝癌的发病风险与标本中平均端粒长度有关。
Hepatocellular carcinoma(HCC), the WHO data show that about 600,000 patients with HCC increase yearly, HCC became the third leading cause of cancer. In China HCC cause 350,000 patients and 300,000 deaths every year. It is the second cancer for its incidence and mortality.
Currently, with liver resection as the representative of the surgical treatment is still the standard therapy for HCC. In recent years, developing new therapies for the treatment of HCC brought a variety of options, and improved the prognosis of the patient significantly. With the genomics and proteomics technology platform for high-throughput progress rapidly, exogenous gene transduction and expression technology advances, complete of bio-information database and cell targeting and site targeting vector array. Molecular targeted treatment of liver cancer become a combination therapy for clinical.
Although, recent studies have made some progress, there remain a number of undesirable phenomena, such as poor reproducibility, the long cycle, slow process, etc. The establishment of bio-bank can avoid these problems effectively. Since the late twentieth century, it has established tumor tissue banks for single disease or multi disease. The operating systems were normalized and standardized. American and European medical institutions have already established their own standardized frozen tumor tissue banks. In recent years, some Chinese research institutions have also established tumor banks, but most of there were lack of normalized and standardized operating systems. The collection of tumor specimens can not be applied to clinical and basic research, Or have limitations in the application. Therefore, the establishment of a standardized tumor bio-bank has great practical significance.
Telomeres are the specialized ends of eukaryotic chromosome. It formed by highly conservative repeat DNA with certain length, is the key structure to stabilizing the chromosome. Telomere shortening is closely related with cancer incidence, such as breast, prostate, lung and bladder cancer. The clinical studies used of tissue samples have shown that tissue of HCC have significantly reduced average telomere length, compare to the adjacent non-HCC tissue, and hepatic cirrhosis tissue. Telomere shortening plays an important role in HCC incidence.
Thus, in Western China, the establishment of a standardized HCC bio-bank is imperative for cancer research and clinical therapy. This study is intended to establish a standardized, large-scale HCC bio-bank,and preliminary use this resources to assess the average telomere length and HCC incidence. This study is divided into three parts:
Part I The standardized collection of HCC biological specimens
Aim: To explore the standardized procedures for establishment of HCC bio-bank, development of a standardized HCC bio-specimens collection, processing and preservation operation process. Establish a functional, information-rich HCC bio-bank. Methods: Reference standardized procedures to establish bio-banks, to develop and standardize the operational processes system in HCC bio-sample collection. Collect liver tissue and blood samples from 3 clinical hospitals. Using of the collection - Feedback - modify mode to improve the specimens collection operational processes, strengthen management and quality control measures, complete the registration of forms. Results: Through the development of standardized sample collection procedures, collected 604 cases of HCC、CLD and control tissues and blood samples from the 3 hospitals. And 20 were randomly selected for DNA testing, the results prove that the collection of specimens have a high quality. Conclusion: The establishment of standardized operational processes is helpful to improve the efficiency of sample collection and quality.
Part II The Information management of liver specimens’clinical data
Aim: To establish the information management system to conduct a systematic management of clinical information of collected specimens. Methods:Using the standardized basic personal information and medical records summary table, through the systemic collection of clinical information, complete the clinical information of collected biological specimens. To establish a management system based EpiData software. Through a simple and convenient interface of data entry, query and analysis, manage the HCC specimens systematic. Results: Through collection and entry of 604 samples’information established and improved the HCC specimens’information management system. Conclusion: Through the establishment of a standardized clinical information management system, to effectively improve the accuracy and operability of specimen management have been enabled.
Part III The assessment of average telomere length and the risk of HCC
Aim: Testing the average telomere length of liver specimens, explore its relationship with the risk of HCC. Methods: Using the Real-time quantitative PCR reaction, to detect the average telomere length of HCC and non-HCC specimens. Assess the correlation between HCC risk and telomere length. Results: Through the testing of 33 cases of HCC, CLD and control samples, found that the average telomere length of HCC specimens are significantly shorter than control. Conclusion: The risk of HCC is related in the average telomere length of the specimen.
目前,以肝切除术为代表的外科治疗仍是肝癌首选治疗方法。近年来逐渐开展新的治疗方法为肝癌的治疗带来了多种选择,并大大改善了病人的预后。基因组学和蛋白质组学等高通量技术平台的迅猛进展,外源基因转导和表达技术的提高,细胞靶向与表位靶向载体的构建,生物信息数据库的日趋完备,肝癌的分子靶向治疗成为一种具有广泛临床应用前景的综合疗法。
虽然近年来的研究已经取得了一定的进展,但是依然存在着一些不尽人意的现象,如研究的重现性较差,研究周期长,进程较慢等。标本库的建立能有效地避免离体肿瘤组织因缺血在短期内失去其相应的生物学活性,如基因的降解、蛋白的变性等,利用同一标本库的标本进行相关研究能有效地提高研究结果的重现性。
肿瘤研究的最终目的是治疗人类的肿瘤,因此对肿瘤的研究模型也必须从体外实验和动物模型的研究逐渐转向人体肿瘤组织标本的研究以及临床研究。二十世纪末期以来,国际上先后建立了单病种和多病种的肿瘤组织库,并逐渐规范化,形成了标准化的操作体系。如欧洲不少国家的医疗机构拥有了自己的标准化的冰冻肿瘤组织标本库。近年国内某些研究机构也建立了肿瘤库,但多数肿瘤标本库都缺乏标准化、规范化的操作体系,所收集的肿瘤标本不能够为临床和基础研究所应用,或者在应用中受到很大的限制,因此标准化肿瘤标本库的建立具有重大的现实意义。
端粒是真核细胞染色体末端的一个特化结构,由一定长度高度保守的串联重复DNA序列构成,是稳定染色体的关键结构。端粒长度的变短与肿瘤发病密切相关,如:端粒缩短导致乳腺癌、前列腺癌、肺癌和膀胱癌等肿瘤的发生率显著增加。利用临床组织样本进行的研究表明:与临近的非肝癌组织和肝硬化组织相比,肝癌组织的平均端粒长度显著缩短,在肝癌发病中扮演了极其重要的角色。
因此,在我国西部建立一个标准化的肝癌标本库,对于肝癌的研究和临床治疗势在必行,本研究拟建立一个标准化的、规模化的肝癌标本库,并初步利用此样本库来进行肝癌标本平均端粒长度与肝癌发病风险关系的评估。
本研究分四个部分:
第一部分肝癌生物标本的规范化收集
目的:探讨建立肝癌生物标本库的标准化程序,研究并制定规范化的肝癌生物标本采集、处理、保存操作流程,建立功能完善,信息丰富的肝癌生物标本库。
方法:借鉴国内外标本库建立的标准化程序,制定规范化的肝癌生物标本收集操作流程体系,收集来自3家临床医院的肝癌、慢性肝疾病(Chronic liver disease,CLD)和对照患者的组织标本和血液标本,采用收集—反馈—修改的模式对标本收集的操作流程不断完善,并健全各种管理和质量控制措施,完成各种表格登记。
结果:通过制定标准化的标本收集流程,收集到了来自3家医院的604例肝癌组织和血液标本。并随机抽取20份DNA进行检测,证明所收集的标本具有较高的质量。
结论:规范化操作流程的建立有利于提高标本收集的效率,提高标本库的质量,具有很好的可操作性。
第二部分肝癌标本临床资料的信息化管理
目的:建立肝癌标本临床资料的信息化管理系统,对所收集的肝癌标本以及相关的临床资料信息进行系统化管理。
方法:利用标准化的资料表格,通过系统的临床信息采集,不断完善所收集的肝癌生物标本的相关临床信息。建立基于EpiData软件的数据库管理系统,通过简单便捷的数据录入、查询、分析界面,对肝癌标本进行系统化的管理。
结果:通过604份肝癌标本信息的录入和初步分析,检验了对收集肝癌标本的管理系统。
结论:通过建立标准化的肝癌标本临床信息管理系统,能够有效提高标本管理的准确性和可操作性。
第三部分肝癌标本平均端粒长度用于肝癌发病风险的评估
目的:对肝癌标本的平均端粒长度进行检测,探讨其与肝癌发病风险之间的联系。
方法:采用实时定量PCR的方法,检测肝癌标本及正常对照标本的全部染色体平均端粒长度,通过评估肝癌的发病风险和全部染色体端粒长度的相关性,进而确定二者的关系。
结果:通过对33例标本的检测,发现肝癌标本的平均端粒长度较正常对照组显著缩短。
结论:肝癌的发病风险与标本中平均端粒长度有关。
Hepatocellular carcinoma(HCC), the WHO data show that about 600,000 patients with HCC increase yearly, HCC became the third leading cause of cancer. In China HCC cause 350,000 patients and 300,000 deaths every year. It is the second cancer for its incidence and mortality.
Currently, with liver resection as the representative of the surgical treatment is still the standard therapy for HCC. In recent years, developing new therapies for the treatment of HCC brought a variety of options, and improved the prognosis of the patient significantly. With the genomics and proteomics technology platform for high-throughput progress rapidly, exogenous gene transduction and expression technology advances, complete of bio-information database and cell targeting and site targeting vector array. Molecular targeted treatment of liver cancer become a combination therapy for clinical.
Although, recent studies have made some progress, there remain a number of undesirable phenomena, such as poor reproducibility, the long cycle, slow process, etc. The establishment of bio-bank can avoid these problems effectively. Since the late twentieth century, it has established tumor tissue banks for single disease or multi disease. The operating systems were normalized and standardized. American and European medical institutions have already established their own standardized frozen tumor tissue banks. In recent years, some Chinese research institutions have also established tumor banks, but most of there were lack of normalized and standardized operating systems. The collection of tumor specimens can not be applied to clinical and basic research, Or have limitations in the application. Therefore, the establishment of a standardized tumor bio-bank has great practical significance.
Telomeres are the specialized ends of eukaryotic chromosome. It formed by highly conservative repeat DNA with certain length, is the key structure to stabilizing the chromosome. Telomere shortening is closely related with cancer incidence, such as breast, prostate, lung and bladder cancer. The clinical studies used of tissue samples have shown that tissue of HCC have significantly reduced average telomere length, compare to the adjacent non-HCC tissue, and hepatic cirrhosis tissue. Telomere shortening plays an important role in HCC incidence.
Thus, in Western China, the establishment of a standardized HCC bio-bank is imperative for cancer research and clinical therapy. This study is intended to establish a standardized, large-scale HCC bio-bank,and preliminary use this resources to assess the average telomere length and HCC incidence. This study is divided into three parts:
Part I The standardized collection of HCC biological specimens
Aim: To explore the standardized procedures for establishment of HCC bio-bank, development of a standardized HCC bio-specimens collection, processing and preservation operation process. Establish a functional, information-rich HCC bio-bank. Methods: Reference standardized procedures to establish bio-banks, to develop and standardize the operational processes system in HCC bio-sample collection. Collect liver tissue and blood samples from 3 clinical hospitals. Using of the collection - Feedback - modify mode to improve the specimens collection operational processes, strengthen management and quality control measures, complete the registration of forms. Results: Through the development of standardized sample collection procedures, collected 604 cases of HCC、CLD and control tissues and blood samples from the 3 hospitals. And 20 were randomly selected for DNA testing, the results prove that the collection of specimens have a high quality. Conclusion: The establishment of standardized operational processes is helpful to improve the efficiency of sample collection and quality.
Part II The Information management of liver specimens’clinical data
Aim: To establish the information management system to conduct a systematic management of clinical information of collected specimens. Methods:Using the standardized basic personal information and medical records summary table, through the systemic collection of clinical information, complete the clinical information of collected biological specimens. To establish a management system based EpiData software. Through a simple and convenient interface of data entry, query and analysis, manage the HCC specimens systematic. Results: Through collection and entry of 604 samples’information established and improved the HCC specimens’information management system. Conclusion: Through the establishment of a standardized clinical information management system, to effectively improve the accuracy and operability of specimen management have been enabled.
Part III The assessment of average telomere length and the risk of HCC
Aim: Testing the average telomere length of liver specimens, explore its relationship with the risk of HCC. Methods: Using the Real-time quantitative PCR reaction, to detect the average telomere length of HCC and non-HCC specimens. Assess the correlation between HCC risk and telomere length. Results: Through the testing of 33 cases of HCC, CLD and control samples, found that the average telomere length of HCC specimens are significantly shorter than control. Conclusion: The risk of HCC is related in the average telomere length of the specimen.
引文
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