【作者】 罗速；

【导师】 张今；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 肝癌是我国高发恶性肿瘤，位于恶性肿瘤死亡率第二位。它具有早期不易发现，难以早期诊断，防治困难，生存率低的特点。肝癌已成为我国医学研究的重要课题，更是分子生物学及其技术应用的热点领域之一。恶性实体瘤的发生发展本质就是细胞生长失控，其根本原因在于肿瘤相关基因或特异基因的异常，使细胞的生长与调控的有关基因结构与功能变化，代谢改变。肝癌的发生有其独特病因，发展也有其特殊规律，进行分子水平指标的研究，才能解决肝癌早期诊断的问题，为临床赢得更多治疗时间和空间，提高生存率。本研究从基因及其表达产物水平上，对肝癌标志性检测指标抑癌基因P16、癌相关基因IGF-Ⅱ和AFP进行单项与联合检测的实验研究，以期阐明肝肿瘤标志物与肝癌早期诊断的分子机理，建立肝肿瘤标志物联合检测体系，提高肝癌阳性检出率，为肝癌早期诊断提供理论依据和临床实验诊断标准。本研究建立生物发光免疫分析技术检测血清AFP作为肝癌早期诊断的公认标志物。利用海洋明亮发光菌502提取细菌荧光素酶，通过Sepharose-4B将细菌荧光素酶和由G6PDH标记的抗人AFP抗体，以及其二抗等生物发光体系组成成分固相化，提高细菌荧光素酶的催化活性和抗原抗体免疫反应活性。并应用生物发光免疫技术进行肝癌特异性癌胚蛋白AFP含量的检测。显示肝癌阳性检出率达到90%以上，证明生物发光免疫分析技术测定AFP的敏感性好，是唯一能够与目前测定AFP最敏感技术放射免疫法相媲美的技术，解决了放射免疫测定法存在的放射污染问题，安全简便，微量快速。尤其适用于肝癌患者血清AFP持续低浓度升高的小肝癌的早期诊断。表明AFP作为肝癌早期诊断的特异性标志物仅是肝癌发生发展特定阶段的产物，反映了肝癌发展过程中某阶<WP=126>段的规律性，与肝癌发生发展密切相关，具有应用于健康人群肝癌普查的实用性，提供肝癌早期诊断的临床实验诊断新手段。本研究利用PCR-RFLP方法，探讨IGF-Ⅱ基因P3启动子DNA多态性改变作为肝癌早期诊断的特征性标志物的可能性，以及IGF-Ⅱ基因P3启动子DNA多态性改变与肝癌发生发展的相关性。使用BstE-Ⅱ、EcoR-Ⅰ、Sac-Ⅱ、BgI-Ⅱ和Ava-Ⅱ酶切的结果显示EcoR-Ⅰ、Sac-Ⅱ、BgI-Ⅱ和Ava-Ⅱ酶切位点在肝癌肝细胞癌变过程中可能未有碱基的变异，暗示这些基因位点可能与肝癌发生发展关系不大。只有BstE-Ⅱ酶切位点处出现阳性结果，其阳性检出率达到63.33%，与良性肝脏疾患比较有显著差异，说明IGF-Ⅱ基因P3启动子DNA多态性改变与肝癌发生发展有极大相关性，提示肝癌早期时IGF-Ⅱ基因P3启动子DNA的异常可能是先于其他指标出现。IGF-Ⅱ基因P3启动子与癌胚蛋白AFP不同，可在癌前状态的肝组织中异常表达，特异性较强，具有早于其他指标出现在肝癌组织中的特点。因此，IGF-Ⅱ基因P3启动子DNA多态性改变作为肝癌早期诊断新的特异标志物具备实验依据，也符合肝癌致病机制相关癌基因特殊作用规律。依据IGF-Ⅱ基因P3启动子DNA多态性变化与肝癌组织的密切相关性特点有望作为基因治疗的靶基因。IGF-Ⅱ基因P3启动子DNA多态性改变检测假阳性率较低，可望成为肝癌发生、发展及疗效的监测新标志物，具有肝癌早期诊断的临床应用价值。本研究应用MSP方法检测血浆P16基因甲基化的变化，分析其与肝癌发生发展之间关系，确证P16基因甲基化是否是肝癌早期或较早期出现的特异性抑癌基因突变者，能否成为另一个新基因诊断指标。实验表明P16基因甲基化的出现是肝癌细胞癌变前或癌变的特征性基因突变，也发现P16基因甲基化是迄今唯一与肝癌病理分化程度呈负相关性的肝肿瘤标志物，肝癌病理分化程度越高，恶性程度越低，血浆P16基因甲<WP=127>基化就越强，阳性检出率就增多。并且，研究还发现，血浆P16基因甲基化无假阳性的结果，说明P16基因甲基化对其他良性肝脏疾患与肝癌的鉴别诊断具有极大的特异性和重大的临床意义，这也是血浆P16基因甲基化指标不同于其他临床肝肿瘤标志物最大特点之一。也在实验中得到证实，血浆P16基因甲基化不与年龄、性别相关，也与肝癌分期无关，在肝癌各期均可发生，提示可能是肝癌早期或较早期诊断有意义的指标。P16基因甲基化血浆测定的MSP方法操作方便，特异性强，尤其适用于肝癌高危人群的筛查，是肝癌早期诊断较为理想的新标志物。本研究在分别探讨AFP、P16基因甲基化和IGF-Ⅱ基因P3启动子DNA多态性各项指标与肝癌发生发展的关系及其作为肝肿瘤标志物的敏感性和特异性的基础上，更进一步地探讨三种肝肿瘤标志物互补性对肝癌早期诊断的临床意义。设计二种和三种肝肿瘤标志物联合检测。然后，选择更好地肝肿瘤标志物联合检测的方式，寻找提高肝癌早期诊断的阳性检出率和特异性的理想途径。研究结果显示三种肝肿瘤标志物联合检测肝癌早期诊断的阳性率和特异性为最佳（分别为96.7%、100%），二种肝肿瘤标志物联合检测以血清AFP和血浆P16基因甲基化联合检测效果最好，接近三种肝肿瘤标志物联合检测的阳性率和特异性（分别为93.3%和100%）。因IGF-Ⅱ基因P3启动子DNA多态性的检测肝组织取材困难的缘故，AFP和P16基因甲基化二种肝肿瘤标志物的组合是基因诊断血清肝肿瘤标志物最敏感特异、便捷易行的联更多还原

【Abstract】 Liver carcinoma is one of most common malignant tumor in China and its death rate is in the second of all malignant tumors. It has such characteristics as not easy to find out and diagnose in its early period, difficult to prevent and cure and low survival rate etc. Liver carcinoma has become one of the most important subject in medical research of China and one of most hotspots in the field of molecule biology.The essential of the development of malignant entity tumor is to lose control of cell growth, the basic reason about it is the abnormity of genes related cancer and special genes, which make the structure and function of these gene changed and lead to metabolizability changed. For the occurrence and development of liver cancer has its particular reason and rule, only the research on molecule level can resolve the problem of its early diagnosis, which will benefit to the clinic for more time and space and higher living rate. This paper is focused on study of the marked test indexes of liver cancer such as a inhibitory gene P16, IGF-II gene related cancer and AFP in a way or a combined way in order to make out the mechanism between marked substances and early diagnosis, to set up a combined test system based on marked substances, to improve the rate of positive test and to provide theoretical basis and clinic standard for early diagnosis of liver carcinoma.The paper proposes bioluminescence immunoassay technology (BLIAT) to test AFP in the serum as a proverbial marked substance for the early diagnosis of liver carcinoma. A biological luminous system consisted of the bacterial luciferase distilled with oceanic photobacterium phosphoreum 502, AFP antibody marked by G6PDH and its anti - antibody is fixed in a solid state through Sepharose – 4B, which will improve the catalytic activity of bacterial luciferase and <WP=129>immunoreaction activity of antigen and antibody. The concentration of AFP in serum of liver carcinoma is examined through BLIAT and the result shows that the positive examination rate is over 90%, which shows that BLIAT has good sensitivity to test AFP. BLIAT has such characteristics as safe, easy, minim and quick ect., it is the only technology with good quality compared favourably with the irradiation immunity method, which has irradiation pollution. It is especially suitable for the carcinoma patient with30%~40% AFP keeping increasing in low concentration and early diagnosis for small liver carcinoma. AFP to be a idiosyncratic marked substance in early diagnosis of liver carcinoma is only the production in the particular period and reflects the regularity in one phase, it has relationship with the occurrence and development of liver carcinoma, has practicability for general survey of liver carcinoma and provide a new means to early diagnosis.The paper discusses the possibility of DNA polymorphism of P3 promoter of IGF-II(insulin like growth factor II) gene to be a idiosyncratic marked substance for early diagnosis of liver carcinoma and relationship between DNA polymorphism change of P3 promoter DNA of IGF-II gene and the occurrence and development of liver carcinoma with the PCR-RFLP method. The enzyme slices of BstEII, EcoRI, Sac II, BglII and AvaII show that there are no the variation of alkaline radical in the points of enzyme slices of EcoRI, Sac II, BglII and AvaII, this shows that they have no relationship with carcinoma, whereas a positive result appeared on the point of BstEII enzyme slice, its positive test rate is 63.3%, which is totally different from the benign tumor, that accounts for DNA polymorphism change of P3 promoter of IGF-II gene has great relationship with the occurrence and development of liver cancer and abnormity DNA of P3 promoter of IGF-II gene maybe first appear than other index. <WP=130>P3 promoter DNA of IGF-II gene is different from AFP in the embryo protein of liver carcinoma and can be expressed before the occurrence of liver carcinoma, so its idiosyncrasy is strong. Therefore DNA polymorphism change of P3 promoter of IGF-II gene to be a idiosyncrat更多还原