【作者】 李刚；

【导师】 何敏；

【作者基本信息】 广西医科大学 ， 生物医学工程， 2010， 硕士

【摘要】 原发性肝癌(Primary hepatic carcinoma,PHC)是世界流行最广的恶性肿瘤之一,其在所有癌症中恶性程度高,治疗最困难,恶性发展速度最快,生存时间最短。2007年全世界的肝癌死亡例数为680 000,在全球肿瘤死因中位列第三。而我国由于肝炎和肝炎转肝硬化的病人众多,致使肝癌在我国的发病率和死亡率均高于世界平均水平。我国每年死于肝癌人数占全世界肝癌死亡人数的55%以上,目前尚缺乏有效的治疗药物。因此为降低其发病率和死亡率,诸多学者长期致力于防治肝癌的研究。细胞凋亡比例明显失调是肿瘤发生发展中的重要因素,如能诱导肿瘤细胞凋亡,使其凋亡的比例增加,则可能遏制肿瘤的发生发展。表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)是绿茶多酚提取物儿茶素(catechin)的主要成分,具有抑制肿瘤细胞增殖和诱导肿瘤细胞凋亡的作用。但是较高浓度的EGCG可直接导致细胞坏死,所以及早发现细胞的早期凋亡,控制EGCG的剂量,是减少药物对细胞损伤的最有效的方法。多数有关EGCG诱导肿瘤细胞凋亡的研究,是依据流式细胞术(FCM)的凋亡细胞峰(亚G1细胞峰)来做出评价的。然而,亚G1细胞群的出现并非是凋亡细胞的特异变化。因此,本研究建立了一种检测细胞早期凋亡的新方法—PSAB-VA∕EL法,来检测EGCG诱导人肝癌SMMC-7721细胞早期凋亡的发生。同时,运用SELDI-TOF-MS和MALDI-TOF/TOF蛋白质谱技术检测与EGCG诱导人肝癌SMMC-7721细胞凋亡相关的关键蛋白,探讨EGCG诱导人肝癌细胞早期凋亡的可能机制。第一章PSAB-VA∕EL法检测细胞早期凋亡方法的建立目的:建立一种经济实用且特异性高的细胞早期凋亡检测新方法,并与常用的Annexin V-FITC/PI法比较。方法:1、以不同质量浓度的EGCG作用Jurkat细胞,采用Annexin V-FITC/PI法检测Jurkat细胞凋亡率,并以此作为细胞凋亡模型;2、尝试用不同的标记有荧光染料的PSAB抗体和核酸荧光染料来检测此细胞凋亡模型。结果:1、经Annexin V-FITC/PI法检测,Jurkat细胞随着EGCG浓度的增加,正常细胞比例不断下降,而晚期凋亡细胞的百分比明显升高,同时早期凋亡细胞保持在10%左右;2、对各种标记有荧光染料的PSAB抗体和核酸荧光染料比较后,发现PSAB- VA和EL搭配使用,能较好地检测细胞凋亡。结论:1、PSAB-VA∕EL法和Annexin V-FITC/PI法在检测细胞凋亡各个阶段的百分比时,其结果的变化趋势一致;2、PSAB-VA∕EL法可能是一种比较灵敏的细胞早期凋亡检测方法。第二章EGCG诱导人肝癌SMMC-7721细胞早期凋亡目的:探讨表没食子儿茶素没食子酸酯(EGCG)诱导人肝癌SMMC-7721细胞早期凋亡的变化规律。方法:1、通过MTT法初步筛选EGCG诱导人肝癌SMMC-7721细胞凋亡的作用浓度;2、利用PSAB-VA∕EL法(第一章所述),通过流式细胞仪检测EGCG对SMMC-7721细胞增殖的抑制作用和早期凋亡的诱导效应。结果:EGCG诱导人肝癌SMMC-7721细胞早期凋亡的最佳作用剂量是100mg/L,早期凋亡率达48.16%。随着EGCG浓度增加(125~175mg/L),早期凋亡率明显下降,而晚期凋亡细胞的百分比明显升高。结论:EGCG有明显的诱导人肝癌SMMC-7721细胞早期凋亡的作用,且呈剂量依赖性,小剂量起诱导凋亡作用,而大剂量则对细胞株具有杀伤作用,直接导致细胞坏死。第三章EGCG诱导人肝癌SMMC-7721细胞凋亡后的相关蛋白变化及鉴定研究目的:利用蛋白质谱技术检测与EGCG诱导人肝癌SMMC-7721细胞凋亡相关的关键蛋白,探讨EGCG诱导人肝癌细胞早期凋亡的可能机制。方法:1、运用SELDI-TOF-MS技术筛选人肝癌血清和人肝癌SMMC-7721细胞裂解液中共同存在的蛋白;2、运用SELDI-TOF-MS技术检测EGCG诱导SMMC-7721细胞凋亡后的细胞裂解液中存在的蛋白;3、检测在肝癌血清、SMMC-7721细胞裂解液以及EGCG作用后的SMMC-7721细胞裂解液中,共有的关键蛋白;4、运用MALDI-TOF/TOF质谱技术平台,并结合iTRAQ蛋白质定量试剂的使用,对上述关键蛋白标志物进行鉴定。结果:1、人肝癌血清和人肝癌SMMC-7721细胞裂解液中共同存在的蛋白有23个;2、SMMC-7721细胞裂解液与EGCG诱导后的SMMC-7721细胞相比,有16个蛋白高表达,16个蛋白低表达;3、在肝癌血清、SMMC-7721细胞裂解液以及EGCG作用后的SMMC-7721细胞裂解液中,共有的关键蛋白为:4283.74 Da和8562.11 Da分子量蛋白,并以此作为EGCG诱导人肝癌SMMC-7721细胞凋亡后的关键蛋白标志物;4、MALDI-TOF/TOF质谱技术检测到的肝癌血清中,高表达的标志蛋白α-1-抗胰蛋白酶的酶解片段与关键蛋白4283.74 Da的分子量非常接近,而低表达的标志蛋白纤溶酶原的酶解片段与关键蛋白8562.11 Da的分子量非常接近。结论:1、运用SELDI-TOF-MS和MALDI-TOF/TOF蛋白质谱技术对肝癌血清和细胞裂解液的检测,有助于客观反映肝癌血清和细胞中低丰度蛋白的变化;2、EGCG诱导人肝癌SMMC-7721细胞凋亡的机制可能与α-1-抗胰蛋白酶(4283.74 Da)和纤溶酶原(8562.11 Da)这两个关键蛋白标志物有关。更多还原

【Abstract】 Primary hepatic carcinoma (PHC) is one of the most common malignant tumors in the world. It has some clinical features such as rapid development, high malignant degree, difficult therapy and less survival time. There were nearly 680 000 patients death of PHC in 2007 around the world. It was the third causes of cancer death in the world. In china, the liver cancer incidence and mortality rates were higher than the global due to the large numbers of hepatocirrhosis patients. The patients died from liver cancer in our country accounted 55% of the world population PHC deaths rate per year. And it has no specific drug therapy yet. Therefore, many researchers are dedicated to the research of prevention and cure liver cancer for a long time in order to decrease the incidence and mortality rate.The disproportion of apoptosis played an important role in the tumor development. The tumor development may be inhibited if apoptotic proportion increases of tumor cells being induced apoptosis. Epigallocatechin gallate (EGCG), was a major components of green tea polyphenols extractive catechin, which can inhibit tumor growth and induce cells apoptosis. But higher concentrations of EGCG can caused cell death, so the most effective way of reduce cell injury is by detecting in the early apoptosis of cell and controlling the EGCG dosage.Most studies on the EGCG induced tumor cell apoptosis were evaluation through apoptotic peak (sub G1 peak) measured by flow cytometry (FCM). But sub G1 peak is not specific change in apoptosis. Therefore, this study established a new early apoptosis detection method, PSAB-VA/EL method, to detect early apoptosis in human hepatocelluar carcinoma cell line SMMC-7721 by EGCG.At the same time, the key proteins of apoptosis in human hepatocelluar carcinoma cell line SMMC-7721 induced by EGCG were detected by SELDI-TOF-MS and MALDI-TOF/TOF. The possible mechanism of apoptosis inducing hepatocelluar carcinoma cell line SMMC-7721 by EGCG was investigated.Part OneEstablishment of a PSAB-VA/EL method for detected early apoptosis cellsObjective: To establish an economical, practical and high specificity method for detecting early apoptosis cells, then evaluated this method comparing with Annexin V-FITC/PI method.Methods: 1. The Jurkat cells was treated with different concentrations of EGCG, early apoptosis of Jurkat cells was measured by Annexin V-FITC/PI method, and took it as an apoptosis model; 2. Attempt several different of PSAB antibody and nucleic acid fluorescent to detect early apoptosis model. Results: 1. Jurkat cells were detected by Annexin V-FITC/PI method, with increasing the concentration of EGCG, the proportion of normal cells dropped consistently. While the percentage of apoptotic cells significantly increased, the early apoptotic cells remained at 10%; 2. After comparing PSAB antibody and nucleic acid fluorescent dye, founded that the collocation of PSAB antibody with EL may be used for detecting apoptosis better.Conclusion: 1. The change trends of results were consistent both in SAB-VA/EL and Annexin V-FITC/PI method which detected apoptosis in the percentage of each stage; 2. The PSAB-VA/EL may be a sensitive method used for detecting early apoptotic cells.Part Two The early apoptosis in human hepatocelluar carcinoma cell line SMMC-7721 induced by EGCGObjective: To investigate the changes of apoptosis in hepatocelluar carcinoma cell line SMMC-7721 induced by EGCG.Methods: 1. MTT assay was used to detect the effect concentration of apoptosis of SMMC-7721 cells induced by EGCG; 2. The growth inhibition and early apoptosis of SMMC-7721 cells was measured by flow cytometry using PSAB-VA/EL method (Described in Part One).Results: The best dose of EGCG effecting on induction of early apoptosis in SMMC-7721 cells was 100mg/L, and the early apoptosis rate was up to 48.16%. With increasing the concentration of EGCG (125~175mg/L), early apoptosis was significantly decreased, while the percentage of apoptotic cells was significantly increase.Conclusion: EGCG significantly induced early apoptosis in human hepatocelluar carcinoma SMMC-7721 cells, in a dose-dependent manner.Low dose could induce apoptosis, while large dose could kill cells and cause cell death. Part Three Alteration of proteomic expression in SMMC-7721 cells after induced by EGCG and identifyObjective: The key proteins of apoptosis in human hepatocelluar carcinoma cell line SMMC-7721 induced by EGCG were detected by protein mass spectrometry. To investigate the possible mechanism of apoptosis of hepatocelluar carcinoma cell line SMMC-7721 induced by EGCG.Methods: 1. Proteins coexisting in human hepatocellular carcinoma serum and human hepatocelluar carcinoma SMMC-7721 cells lysate were detected by SELDI-TOF-MS; 2. Proteins existing in human hepatocelluar carcinoma SMMC-7721 cells lysate treated by EGCG was detected by SELDI-TOF-MS; 3. Detection of the key proteins coexisting in human hepatocellular carcinoma serum, SMMC-7721 cells lysate and SMMC-7721 cells lysate treated by EGCG; 4. All the proteins mentioned above were detected by using iTRAQ labeling coupled with MALDI-TOF/TOF.Results: 1. there were 23 proteins coexisting in human hepatocellular carcinoma serum and human hepatocelluar carcinoma SMMC-7721 cells lysate; 2. Comprised with SMMC-7721 cells lysate treated by EGCG, 16 proteins were up-regulated expression and 16 proteins were down-regulated expression in SMMC-7721 cells lysate; 3. Among those proteins, the m/z value of key proteins were 4283.74 Da and 8562.11 Da; 4. The m/z value of highly expressedα-1-Antitrypsin enzymolysis fragment was very close to key proteins 4283.74 Da; The m/z value of low expressed Plasminogen enzymolysis fragment was very close to key proteins8562.11 Da.Conclusion: 1. By detection of human hepatocellular carcinoma serum and SMMC-7721 cells lysate with SELDI-TOF-MS and MALDI-TOF/TOF is helpful to reflect the alteration of low-abundance proteins objectively; 2. The possible mechanism of apoptosis inducing hepatocelluar carcinoma cell line SMMC-7721 by EGCG is concerned with the key proteins ofα-1-Antitrypsin and Plasminogen.更多还原