【作者】 刘志强；

【导师】 梁廷波；

【作者基本信息】 浙江大学 ， 外科学， 2007， 硕士

【摘要】 背景真核细胞的基因组DNA在细胞核里是以染色质的形式存在的，染色质的基本单位是核小体，由核心组蛋白（H2A、H2B、H3和H4）、H1及DNA组成。在细胞生命活动的选择性基因沉默或基因表达过程中，包裹于染色质中的基因组DNA序列一般不发生改变，但细胞核内的染色质结构可以发生高度动态变化，使一些特定基因组区域的转录活性呈现相应的改变。这种影响基因转录活性但不涉及DNA序列改变的基因表达调控方式称为表观遗传调控。长期以来，肿瘤的形成一直被认为是基因突变的结果。然而，越来越多的证据表明，表观遗传调控在肿瘤的发生发展中同样至关重要。表观遗传调控主要包括DNA甲基化修饰和组蛋白修饰，这些修饰改变染色质结构，导致基因转录调节发生变化，从而导致细胞增殖失常而致癌。组蛋白的修饰比DNA甲基化复杂得多，不同组蛋白（组蛋白H3和H4）的不同氨基酸可以发生不同类型的修饰，包括乙酰化、甲基化、磷酸化、泛素化、糖基化、羧基化等，其中以乙酰化尤为重要。组蛋白乙酰化修饰是可逆的动态过程，受组蛋白乙酰基转移酶（histone acetyltraasferases，HATs）和组蛋白去乙酰化酶（histone deacetylases，HDACs）调控，前者使组蛋白高乙酰化，后者则相反，使组蛋白去乙酰化。许多研究已经证实了组蛋白乙酰化失衡（高／低乙酰化）与肿瘤发生密切相关，一方面，人们发现组蛋白乙酰化和去乙酰化的变化与细胞的形态变化有关；另一方面，催化组蛋白乙酰化的HATs或催化组蛋白去乙酰化的HDACs可与一些癌基因和抑癌基因相互作用，从而影响细胞分化和细胞增殖。既往在癌细胞中发现组蛋白翻译后修饰变化主要是在基因的一些启动子，事实上，组蛋白的修饰，如赖氨酸残基上的乙酰化修饰，可发生在染色质的编码区和非启动子序列上，这种修饰被称为组蛋白的整体乙酰化修饰，不仅影响基因转录，也决定了细胞或有机体内组蛋白的整体乙酰化状态。肝癌是世界上第五大最常见的恶性肿瘤，每年有50万以上的新发病例，其中我国占50％，位居我国癌症死亡原因的第二位。随着人们对表观遗传调控机制在肿瘤发生中的重要作用的深入认识，肝癌的表观遗传学研究逐渐成为热点，已有研究发现DNA甲基化异常和组蛋白乙酰化失衡在肝癌的发生中起了重要的作用。然而，迄今为止，关于组蛋白乙酰化失衡在肝癌中作用的认识主要来自一些应用不同的化学物质改变细胞乙酰化状态的体外研究，并且不同的研究结果不相一致。有些研究认为组蛋白去乙酰化酶抑制剂（如曲古抑菌素A、二烯丙基二硫等）使组蛋白高乙酰化，从而可以诱导肝癌细胞凋亡、阻滞细胞周期、抑制细胞增殖等；相反，另一些研究则认为使组蛋白低乙酰化的化合物如姜黄素（Cur），镍离子和1，10-二氮杂菲铜(1，10-phenanthroline-Cu²⁺)等可促进肝癌细胞凋亡。互相矛盾的体外实验结果，促使我们有必要进一步了解肝癌组织中组蛋白的整体乙酰化水平。目前，调整组蛋白乙酰化状态已经成为药物治疗肿瘤的重要靶点，了解肝癌组织的组蛋白整体乙酰化状态将为该类抗癌药物的研制以及临床应用提供依据。材料和方法1．病例资料病例为因原发性肝脏肿瘤在浙江大学医学院附属第一医院肝胆胰外科行肝切除或肝移植手术、术后病理确诊为肝细胞性肝癌的癌及癌旁组织标本各82例；查阅病历，收集包括患者年龄、性别、乙型肝炎病史、有无肝硬化基础、血甲胎蛋白水平、肿瘤数目（单发或多发）、肿瘤大小、肿瘤有无包膜、有无门静脉癌拴、肿瘤TNM分期、以及组织学分级等在内的临床资料。2．构建组织芯片，应用抗乙酰化组蛋白H3（Lys 9）、H4（Lys 8）的多克隆抗体采用免疫组织化学染色法检测肝癌及癌旁组织中的乙酰化组蛋白H3（Lys 9）、H4（Lys 8）的蛋白表达。3．免疫组化染色结果判断细胞核呈棕色的细胞是阳性细胞；结合细胞核着色强度和着色细胞百分比的积分之和判断免疫染色结果，着色强度积分为：不着色为0，轻度着色为1，中度着色为2，强着色为3；着色百分比积分为：无着色细胞为0，着色细胞数百分比＜25％为1，25％～50％为2，50％～75％为3，＞75％为4。若两种积分之和＞2分为阳性，≤2分为阴性。结果阴性的有可能在细胞的某些基因位点存在组蛋白乙酰化，但是其乙酰化组蛋白表达量低于检测水平，即提示整体组蛋白乙酰化水平低，结果阳性提示整体组蛋白乙酰化水平高。4．比较肝癌及癌旁组织整体组蛋白H3、H4的乙酰化水平。5．分析肝癌的组蛋白H3、H4乙酰化水平和各种临床病理特征之间的关系。6．统计分析应用SPSS 13.0进行统计分析，统计学方法采用卡方检验、柯尔莫诺夫-斯米尔诺夫检验。所有P值均为双向检验值，P＜0.05为统计学差异有显著性。结果1．组织芯片构建理想，所取组织符合研究需要。2．肝癌中乙酰化组蛋白H3、H4的蛋白表达阳性率分别为68.3％（56／82）和81.7％（67／82），和其相应的癌旁组织的阳性率31.7％（26／82）及45.1％（37／82）相比明显增高（P＜0.05）。3．乙酰化组蛋白H3、H4的蛋白表达水平与肝癌临床病理特征包括患者年龄、性别、乙型肝炎病史、有无肝硬化基础、血甲胎蛋白水平、肿瘤数目（单发或多发）、肿瘤大小、肿瘤有无包膜、有无门静脉癌拴、肿瘤TNM分期、以及组织学分级等之间均无明显相关（P＞0.05）。结论肝癌组织中组蛋白的整体乙酰化程度明显高于癌旁组织，这一结果提示组蛋白H3、H4的整体高乙酰化与肝癌的发生有关，但其内在机制尚未明确，需要深入研究。以调整组蛋白乙酰化失衡为治疗靶点的抗癌药物在肝癌中的确切作用机制、疗效和应用前景有待进一步研究和评估。更多还原

【Abstract】 BackgroundDNA in the eukaryotic genome exists in nucleus as chromatin. Nucleosome, which is formed by core histone protein （ H2A, H2B, H3 and H4 ）, H1 and DNA, is the basic unit of eukaryotic chromatin. In the process of gene expression or selective gene silencing, dynamic and large-scale changes occur on the structure of chromatin while the DNA that packaged into chromatin rarely changes in sequence. The changes influence the transcriptional activity at some certain regions of genome. Regulation of gene expression by influencing transcriptional activity that is not accompanied with changes in DNA sequence is defined as epigenetics. It is believed for a long time that gene mutations promote oncogenesis. However, more and more evidences tell us that epigenetics is also important in the initiation and progression of tumor. Epigenetics includes DNA methylation and histone modification. These modifications can change the structure of chromatin and modify transcription. The disturbance of transcription leads to hyperplasy, even causes cancer.Histone modification is more complicated than the DNA methylation, and different histone （histone H3/H4） have different types of modifications, including acetylation, methylation, phosphorylation, ubiquitination, glycosylation, carboxylation and so on. Among these modifications acetylation is the most important one. Histone acetylation is a dynamic, reversible process which is regulated by histone acetyltransferases （HATs） and histone deacetylases （HDACs）. HATs cause hyperacetylation while HDACs have the opposite effect. Many researches have confirmed that disequilibrium （hyperacetylation/hypoacetylation） of histone acetylation has a close relationship with initiation of tumor. Histone acetylation or deacetylation can change the morphous of cells; On the other hand, HATs and HDACs can influence the differentiation and generation of cells by interacting with oncogenes and anti-oncogenes. In addition, researches have found that post-translational modifications of histone occur mainly on the promoters of genes. In fact, other than being targeted to promoters, modifications of histones, such as acetylation of lysine, also occur over large regions of chromatin including coding regions and non-promoter sequences, which are referred to as global histone modifications. The modification can influence genetic transcription and also determine the global acetylation state of histones in cell and organism.Hepatocellular carcinoma （HCC） is the fifth most common malignant tumor in the world. There are more than 500 thousand new cases of HCC each year and 50% cases of these patients are Chinese. HCC is the second leading cause of cancer deaths in our country. Accompanied with the cognition on the importance of epigenetics in the initiation of tumor, epigenetics has become the hot spot in the HCC’s research. Some researches have demonstrated that DNA methylation and disturbance of hisone acetylation play an important role in the initiation of HCC. However, up to now, the role of disturbance of hisone acetylation has been studiedonly in vitro by using different chemicals which can change the state of acetylation, and different researches displayed discrepancy results. Some researches reveal that some chemical materials （mainly histone deacetylase inhibitor such as Trichostatin A, diallyl disulfide） which cause histone hyperacetylation can induce apoptosis, cell cycle arrest, antiproliferative effect in HCC. Conversely, some researches exhibit that hypoacetylation (caused by Curcumin, Ni²⁺ and 1, 10-phenanthroline-Cu²⁺) promote the apoptosis of HCC. Contradictory results in vitro spur us to get knowledge about the global acetylation state of histone in HCC. Today, regulating the state of acetylation of histone becomes an important target on cancer treatment with drugs. The knowledge of global acetylation state of histone in HCC will provide evidence for the drug research and clinical application.Materials and Methods1. PatientsA total of 82 patients with primary HCC undergoing hepatectomy or liver transplantation in the Department of Hepatobiliary and Pancreatic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, were involved in the study. The diagnosis of HCC was confirmed by histopathological examination of each resected tissue. Samples of the HCC and para-cancerous tissue were collected. Collected clinical data such as age, gender, history of Viral Hepatitis Type B, background of cirrhosis, level of AFP, tumor numbers（solitary or mutiple）, tumor size, tunicary tumors, portal vein tumor thrombus , TNM grading, histopathological grading of the tumors.2. Microarray chip preparation and immunohistochemistry detectionImmunohistochemistry combined with tissue microarrays was employed to determine the expression of acetylation of histone on HCC tissue and para-canceroustissue. We analysed the levels of acetylated histone H3 （Lys 9） and H4 （Lys 8）, using highly specific antibodies （antibody to acetyl-histone H3, acetyl-histone H4）.3. Evaluation of stained resultsThe nuclei which are brown stained are regarded as positive cells; For evaluation of expression results a score corresponding to the sum of both staining intensity and percentage of positive cells was determined. Staining intensity: 0, negative; 1, weak; 2, intermediate; 3, strong. Percentage of positive cells: 0, negative ; 1, <25% positive cells ; 2, 25%-50% positive cells ; 3 , 50%-75% positive cells ; 4, >75% positive cells . A score greater than 2 was regarded as positive, while the score lower than or equal to 2 was regarded as negative. The unstained cells might still contain the acetylations at certain genomic loci but their levels are below the detection limits, which are considerably decreased in these cells. The positive results are regarded as a high level global acetylation of histone.4. To compare the level of global acetylation of histone H3/H4 in HCC tissue with that of para-cancerous tissue.5. To analyze whether the level of acetylation of histone H3/H4 correlated with clinical data.6. Statistical analysisx² test and kolmogorov-smirnov test were used for statistical analysis. All analyses were performed with the SPSS statistical package on a personal computer, Release 13.0. All probability values were two-sided, and P≤0.05 was considered significant.Results1. Tissue microarray was successfully constructed and the tissue was fit for the research.2. Among the 82 cases of HCC , the positive rate of acetylation of histone H3/H4 were 68.3% （56/82） 81.7% （67/82）, significantly higher than the para-cancerous tissue 31.7% （26/82）, 45.1% （37/82） （P<0.05）.3. Expression of the two kinds of proteins was not related to clinical characters, including age, gender, history of Viral Hepatitis Type B , background of cirrhosis , level of AFP, tumor numbers（solitary or mutiple）, tumor size, tunicary tumors, portal vein tumor thrombus, TNM grading, histopathological grading of the tumors（P>0.05） .ConclusionsGlobal acetylation of histone in HCC tissue is significantly higher man that in the para-cancerous tissue. The result revealed that global hyperacetylation of histone H3/H4 is related to the genesis of HCC. However, the mechanism is not clear yet and should be studied in the future. It needs further research and evaluation for the drug’s precise mechanisms, therapeutic effects and clinical applications on the treatment of HCC, which target on regulating the disturbance of acetylation of global histone.更多还原