基因芯片联合组织芯片探讨原发性肝癌发生发展的分子机制
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摘要
制全貌，为临床诊治提供新的潜在靶点。
材料与方法：1、收集10例施行正位肝移植的HCC患者的癌、癌旁和正常肝组织，应用H&E染色确认相关组织的病理类型，应用Trizol一步法抽提配对组织的总RNA；2、应用1％琼脂糖凝胶电泳和芯片实验室(Lab-on-chip)进行RNA质量检测；3、按QIAGENRNeasy(?) Mini Kit说明，进行总RNA纯化、逆转录cRNA合成、荧光标记和纯化；4、将癌组织和正常肝组织、癌组织和癌旁肝组织的cRNA探针分别与Agilent寡核苷酸芯片(21，073探针)进行杂交，洗涤后应用Aailent Scanner获取图像，应用Feature Extraction软件进行定量分析处理，应用cluster分析软件进行聚类分析，通过Treeview软件以树状图形式显示；5、挑选明显差异表达的代表基因4个，其中包括上调和下调基因各2个，以β-actin为内对照基因，由上海生工生物公司设计合成目的基因引物前导链和后随链，按Invitrogen RT-PCR kit说明进行SYBR Green Ⅰ染料掺入的荧光real time RT-PCR绝对定量验证分析；6、挑选差异表达的代表基因6个，其中包括5个上调基因和1个下调基因，在高通量肝癌特异性组织芯片(产品批号：OD-CT-DgLiv01-001)上应用S-P法验证代表基因在蛋白水平的表达，采用SPSS10.0软件包分析相关基因的表达与HCC临床病理参数之间以及基因相互之间的相关性。
结果：1、H&E染色结果显示癌、癌旁以及正常肝组织呈现典型的组织病理变化，1％琼脂糖凝胶电泳和Lab-on-chip电泳结果均提示提示配对组织总RNA的质量高，无降解现象。2、高通量Agilent寡核苷酸芯片扫描发现在癌和癌旁组织的差异基因表达谱中，2倍上调的的差异表达基因(differentially expressed genes，DEGs)共1295个，而癌与正常肝组织的数目为1074个，综合两者的共同DEGs，共420个；相应的，在癌和癌旁组织2倍下调的DEGs共1320个，而癌与正常肝组织的数目为1107个，共同下调的DEGs为552个。3、Cluster聚类分析显示HCC的发生发展过程涉及癌基因与抑癌基因、编码离子通道和蛋白转运相关的基因、编码细胞周期相关蛋白的基因、与细胞应激相关的基因、细胞骨架和运动相关的基因、细胞凋亡相关的基因、调节DNA合成、修复和重排相关的基因、细胞因子受体相关基因、免疫蛋白相关基因、细胞代谢相关基因、信号转导相关基因、调节转录的基因、与生长发育相关的基因等，尚有部分基因不能分类或为表达序列标签(expression sequence tags，ESTs)。4、差异基因表达谱中，包含12个5倍以上上调表达的基因，分别是CTHRC1、UCHL1、PPP1R9A、CTSL2、POSTN、NQO1、DKK1、AGR2、MMP-12、SULT1C1、LAPTM4B和PEG10；7个10倍以上下调表达的基因，分别是3个细胞色素P450基因(CYP2C9、CYP2A7和CYP2C8)、
Background and objective: Hepatocellular carcinoma(HCC), an aggressive malignancy with poor prognosis and one of the most common tumors in human beings, has become a leading cause of cancer-related death in adults from Asia and sub-Saharan-Africa. Although advances have been achieved in the diagnosis and treatment of HCC, the prognosis of patients with HCC remains dismal. The 5-year survival rate is less than 25% in the subgroups of patients who have the most favorable characteristics and are eligible for surgical resection. The poor prognosis of HCC has been associated with recurrence and metastasis. Therefore, a better understanding of molecular mechanisms involved in HCC development and metastasis should be investigated urgently.
HCC is associated with multiple risk factors and is believed to arise from preneoplastic lesions, usually in the background of cirrhosis. Extensive studies on HCC and its precursors have demonstrated complex and heterogeneous genetic or chromosomal abnormalities along the way from preneoplastic lesions to HCCs. These genetic abnormalities include loss of heterozygosity, microsatellite instability, gene alterations, and aberrant global gene expression profiles. Although some genetic alterations involving the p53 family, Rb family, and Wnt pathways are particularly important in the development of HCCs, the molecular pathogenesis of HCC differs with etiology in some extent. Recent studies using DNA microarray technique have identified some unique gene expression profiles in hepatitis B virus (HBV)- and hepatitis C virus (HCV)- associated HCCs. Gene expression profiling also allows people to distinguish HCCs from normal tissue or preneoplastic lesions and to evaluate metastatic or recurrent potentials. These unique genes or gene products associated with malignant transformation and recurrent or metastatic potentials may serve as molecular markers for early diagnosis, prediction of prognosis, and responsiveness to therapy. To date, information that has accumulated for the past several decades is still incomplete, and we still are faced with a great challenge in deciphering the molecular mechanisms of HCCs.
The development of microarray technologies, which allow us to undertake parallel analyses of many genes, has led to a new era in medical science. New genomic high-throughput technologies, such as DNA microarrays, may facilitate considerably the molecular profiling of
材料与方法：1、收集10例施行正位肝移植的HCC患者的癌、癌旁和正常肝组织，应用H&E染色确认相关组织的病理类型，应用Trizol一步法抽提配对组织的总RNA；2、应用1％琼脂糖凝胶电泳和芯片实验室(Lab-on-chip)进行RNA质量检测；3、按QIAGENRNeasy(?) Mini Kit说明，进行总RNA纯化、逆转录cRNA合成、荧光标记和纯化；4、将癌组织和正常肝组织、癌组织和癌旁肝组织的cRNA探针分别与Agilent寡核苷酸芯片(21，073探针)进行杂交，洗涤后应用Aailent Scanner获取图像，应用Feature Extraction软件进行定量分析处理，应用cluster分析软件进行聚类分析，通过Treeview软件以树状图形式显示；5、挑选明显差异表达的代表基因4个，其中包括上调和下调基因各2个，以β-actin为内对照基因，由上海生工生物公司设计合成目的基因引物前导链和后随链，按Invitrogen RT-PCR kit说明进行SYBR Green Ⅰ染料掺入的荧光real time RT-PCR绝对定量验证分析；6、挑选差异表达的代表基因6个，其中包括5个上调基因和1个下调基因，在高通量肝癌特异性组织芯片(产品批号：OD-CT-DgLiv01-001)上应用S-P法验证代表基因在蛋白水平的表达，采用SPSS10.0软件包分析相关基因的表达与HCC临床病理参数之间以及基因相互之间的相关性。
结果：1、H&E染色结果显示癌、癌旁以及正常肝组织呈现典型的组织病理变化，1％琼脂糖凝胶电泳和Lab-on-chip电泳结果均提示提示配对组织总RNA的质量高，无降解现象。2、高通量Agilent寡核苷酸芯片扫描发现在癌和癌旁组织的差异基因表达谱中，2倍上调的的差异表达基因(differentially expressed genes，DEGs)共1295个，而癌与正常肝组织的数目为1074个，综合两者的共同DEGs，共420个；相应的，在癌和癌旁组织2倍下调的DEGs共1320个，而癌与正常肝组织的数目为1107个，共同下调的DEGs为552个。3、Cluster聚类分析显示HCC的发生发展过程涉及癌基因与抑癌基因、编码离子通道和蛋白转运相关的基因、编码细胞周期相关蛋白的基因、与细胞应激相关的基因、细胞骨架和运动相关的基因、细胞凋亡相关的基因、调节DNA合成、修复和重排相关的基因、细胞因子受体相关基因、免疫蛋白相关基因、细胞代谢相关基因、信号转导相关基因、调节转录的基因、与生长发育相关的基因等，尚有部分基因不能分类或为表达序列标签(expression sequence tags，ESTs)。4、差异基因表达谱中，包含12个5倍以上上调表达的基因，分别是CTHRC1、UCHL1、PPP1R9A、CTSL2、POSTN、NQO1、DKK1、AGR2、MMP-12、SULT1C1、LAPTM4B和PEG10；7个10倍以上下调表达的基因，分别是3个细胞色素P450基因(CYP2C9、CYP2A7和CYP2C8)、
Background and objective: Hepatocellular carcinoma(HCC), an aggressive malignancy with poor prognosis and one of the most common tumors in human beings, has become a leading cause of cancer-related death in adults from Asia and sub-Saharan-Africa. Although advances have been achieved in the diagnosis and treatment of HCC, the prognosis of patients with HCC remains dismal. The 5-year survival rate is less than 25% in the subgroups of patients who have the most favorable characteristics and are eligible for surgical resection. The poor prognosis of HCC has been associated with recurrence and metastasis. Therefore, a better understanding of molecular mechanisms involved in HCC development and metastasis should be investigated urgently.
HCC is associated with multiple risk factors and is believed to arise from preneoplastic lesions, usually in the background of cirrhosis. Extensive studies on HCC and its precursors have demonstrated complex and heterogeneous genetic or chromosomal abnormalities along the way from preneoplastic lesions to HCCs. These genetic abnormalities include loss of heterozygosity, microsatellite instability, gene alterations, and aberrant global gene expression profiles. Although some genetic alterations involving the p53 family, Rb family, and Wnt pathways are particularly important in the development of HCCs, the molecular pathogenesis of HCC differs with etiology in some extent. Recent studies using DNA microarray technique have identified some unique gene expression profiles in hepatitis B virus (HBV)- and hepatitis C virus (HCV)- associated HCCs. Gene expression profiling also allows people to distinguish HCCs from normal tissue or preneoplastic lesions and to evaluate metastatic or recurrent potentials. These unique genes or gene products associated with malignant transformation and recurrent or metastatic potentials may serve as molecular markers for early diagnosis, prediction of prognosis, and responsiveness to therapy. To date, information that has accumulated for the past several decades is still incomplete, and we still are faced with a great challenge in deciphering the molecular mechanisms of HCCs.
The development of microarray technologies, which allow us to undertake parallel analyses of many genes, has led to a new era in medical science. New genomic high-throughput technologies, such as DNA microarrays, may facilitate considerably the molecular profiling of
引文
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