肝肿瘤标志物与肝癌早期诊断的研究
|
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
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
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
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
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.
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
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
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
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
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
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.
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
引文
1. 汤钊猷. 临床肝癌学[M]，上海，上海科技教育出版社，2001.
2. Lee HY, Murata J. Cloning, Chromosomal localization and tissue expression of artotation from human carcinoma. Biochem Biophy Res Commun,1996, 218:714.
3. Peng S,Kong G,Rao PM, et al. The genetic charge of DNA in HCC patients. Hepatology,1997,21:35-36.
4. Crowe M.The pair effect of DNA in product of HCC. Gastroenterology, 1997,106:A356-A359.
5. Cains P.Rates of P16 (CDKN2) mutations in primary tumors with Op loss. Science, 1994, 264:415-417.
6. 中国抗癌协会肝癌专业委员会，原发性肝癌诊断标准。中华肝脏杂志，2000,8（3）：135.
7. 袁爱力，刘为纹，晋化源等. 原发性肝癌、良性肝病及胚胎组织中甲胎蛋白异质体的研究。中华消化杂志，1988,8（1）：10-12.
8. 房殿春，罗元辉，刘为纹等。应用逆转录聚合酶链反应选择原发性肝癌患者外周血肿瘤细胞。中华内科杂志，1998,37：168-170.
9. Lemoine A, LeBricon T,Salvucci M, et al. Prospective evaluation of circulating hepatocyte by alpha-feto protein mRNA in human during live surgery. Am surg. 1997,226:43
10.沈鼎明，沈薇，左国庆等。血清肿瘤标志物在原发性肝癌诊断中的价值。中华消化杂志，1992,12：223-226.
11.孟宪镛。肝癌标志物的研究是早期诊断的关键。中华消化杂志。1993,13：251-252.
12.张柏和，吴孟超。抗人甲胎蛋白异质体单克隆抗体诊断肝细胞癌的临床应用价值。中华消化杂志，1992,12（3）：129-132.
13.Nomura F,Ishijma M,Kuwa K,etal.Serum des-gammacarbooxy prothrombin levels determined by a new generation of sensitive immunoassays in patients with small –sized hepatocellular cacinoma.Am J Gas troenteral,1999,94(3):650-654.
14.王斌，唐心利.利用细菌荧光素酶建立酶联免疫吸附试验检测抗-HBs的研究.医学研究通讯, 2003, 32（1）：9-12.
15.曾祝伦，唐波.肿瘤相关物质在肝病中的诊断价值.中国医学检验杂志，2003,4（2）：110-111.
16.黄建钊，沈文律，李波等.原发性肝细胞肝癌中P16基因的失活与P16蛋白表达的研究.华西医科大学学报,2000,31（3）：306-309.
17.Terouanne B,Nicolas JC,Crastes et al. Paulet A.Bioluminesceny
immunoassay for alpha –fetoprotein. Anal Biochem.1986 Apr, 154 (1); 132-7.
18.Wang IH ,LoYM,Yeo W,et al .Frequent P15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients .Clin Cancer Res ,2000 sep,6(9):3516-21.
19.杨冬华.加强人胰岛素样生长因子Ⅱ基因启动子与肝细胞癌变关系的研究.中华内科杂志,1999,38（2）：79-80.
20.J.F.Suo ,Liu YF,Zhang et al .Expression of insulin-like growth factor Ⅱin hepatitis B,cirrhosis and hepatocellular carcinoma :its relationship with hepatitis B virus angen expression. Hepatology, 1994, 20: 7880-7896.
21.Lix ,Cui H,et al .Expression levels of the insulin like growth factor Ⅱ(IGF-Ⅱ)in the human liver developmental relationships of the four promoters.J.Endo Chionolgy, 1996, 149: 117-124.
22.Ng IO,Lee,JM,et al. Expression of insulin-like growth factor Ⅱ mRNA in hepatocellular carcinoma.J.Gastroentral Hepatal, 1998, 13(2): 152-157.
23.Tetsurosohda,Kankatsa Yun,etal. Incrceased expression of insulin-like growth factor 2 in hepatocellular carcinoma is primary regulated at the transcriptional level. Lab. Invest, 1996, 75:307-311.
24.XuriLi,Zhang Nong.christin a ekstom disrupted IGF2 promoter control by silencing of promoter P1 in human hepatocellular carcinoma .Cancer Res,1997,57:2048-2054.
25.VuTh,Li T et al.Symmetric and asymmetric DNA methylation in human IGF2 H19 imprined region .Genomics,2000, 1:64(2): 132-143.
26.Li X,Gray SG,et al. Developmented dependent DNA methylation of the IGF2 and H19 promoters is correlated to the promoter activities in human liver development .Int J Dev. Bioal, 1998, 42(5)687-693.
27.Seo JH,Kin KW,Park.BC.Different Protein binding patterns in P3 promoter region of the human insulin-like growth factor Ⅱgene in the human liver cirrhosis and hepatocellular carcinoma tissues.J .Korean Men.Sci.1998,13(2):171-178.
28.Raizis AM,Eales MR,Reeve AE.Structural analysis of the human insulin-like growth factor ⅡP3 promoter.Biochem, 1993, 1(289):
139-149.
29.Kamb A,Gruis NA,Wearer-Feldans J,et al.A cell cycle regulator potentially involved in genesis of many tumur types. Science, 1994, 264:436-440.
30.Serrao M,Hamon GJ.A new regulation motif in cell-cycle control causing specific inhibition of cycle D/CDK4.Nature, 1993, 366: 704-707.
31.Marx J.New tumor suppressor may rival P53.Science,1991, 264:344-345.
32.Jin X,Nguyen D,Zhang WW. Cell cycle arrest and ihibition of tumor cell proliferation by the P16 INK4 gene mediated by an adenovirus vector. Cancer Res,1995,55:3250-3253.
33.Arap W,Nishikawa,Funai FB, et al. Replacement of the P16/CDKN2 gene suppresses human gilioma cell growth. Cancer Res, 1995, 55: 1451-1354.
34.Serrao M,Lee HW,Chin L, et al .Role of the P16 INK4a locas in tumor suppression and cell mortality .Cell,1996,85:27-37.
35.Hui AM,Shi YZ,Lix, et al .Loss of P16 INK4 protein alone and together with loss of retinoblastoma protein ,correlate with hepatocellular carcinoma progression .Cancer Lett, 2000, 154(1) :93-99.
36.Liu Jianyu,Li Bo, et al.P16 gene expression and methylation status of CpG island in human primary hepatocellular carcinoma. Journal of West China University of medical Science, 2002, 33(4): 540-543.
37.Huang Jian zhao ,Shen Wenlv,Li Bo, et al .A study on the inactivation of P16 gene and the expression of P16 protein in primary hepatocellular carcinoma.Journal of West China University of Medical Science ,2000,31(3):306.
38.Kita R,Nishida N,Fukuda Y et al. Infreguent alterations of the P16 INK4 gene in liver cancer.Int J Cancer,1996,67(2):176.
39.Matsuda Y,Ichida T,Mastuzawa J,et al .P16 INK4 is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology. 1999,116(2):394-400.
40.Willian H ,Ligget Jr,David Sidransky.Role of the P16 tumor suppressor gene in cancer. J Clininal Oncology, 1998, 16(3); 1197.
41.Ivy HN,Wong YM, Dennis LD, et al . Detection of aberrant P16 methylotion in the plasma and serum of liver cancer patients. Cancer
Res,1999,59(1):71-73.
42.Qin Y,Li B,Tan YS, et al .Polymorphism of P16 INK4a gene and rare mutation of P15 INK4a gene exon 2 in primary hepato carcinoma. Wolel Journal of Gastroenleralogy ,2000,6(3):411.
43.Beak MJ,Piao Z,Kim NG,et al . P16 is a major inactivation target in hepatocellular carcinoma .Cancer ,2000,89(1):60.
44.Lee TJ , Bae JJ, et al .Abrogation of the P16-Rb pathway in Korean hepatocellular carcinoma. Hepatogastroenterology, 2000, 47(36): 1663-1668.
45.Clen TC, Hsieh LL, Kuo TT, et al .P16 INK4 gene mutation and allelie loss of chromosome qp21-22 in Taiwanese hepatocellular carcinoma. Anticancer Res ,2000,20(3A):1621-1668.
46.Wang G, Huang Ch, Zhao Y, et al .Genetic aberrantion in primary hepatocellular carcinoma:correlation beween P15 gene mutation and loss of hetozygosity on chromasome 16q21-23,9p21-23,Cell Res. 2000, 10(4): 311-323.
47.Huang JianZhao ,Shen Wenliu ,Xia Suisheng ,et al.The status of P16 gene in primary hepatocellular carcinoma from a Chinese papulation.Chinese Journal of Cancer,2000,1:45-47.
48.Lee YY,Kang SH, Seo JY, et al. Alteration of P16 INK4a and P53 INK4a and P53INKB genes in gastric carcinomas. Cancer, 1997, 80: 1889-1896.
49.Lin YM,Chen CH,Huang GT, et al. Infrequent muation and no methylation of CDKN2A(P16/MTS1) and CDK2B(P15/MTS2) in heoatocullular carcinoma in Taiwan .Eur J Cancer, 1998, 34(11): 1789-1795.
50.Huang J,Shen W, Li B ,et al .Molecular and immunohistochemical study of the inactivation of the P16 gene in primary hepatocellular carcinoma. Chin Med J(EngⅠ), 2000, 113(10): 889-892.
51.Jin M,Piao Z,Kim NG, et al. P16 is a major inactivation target in hepatocellular carcinoma. Cancer ,2000,89(1):60-68.
52.Herman JG,Graff JR,Myohanen S, et al. Methylation specific PCR ,a novel PCR assay for methylation status of CpG is land .Proc Natl Acad,Sci USA.1996,93:9821-9826.
53.Liew G,LiMH,LoKW. High frequency of P16INK4 gene alteration in
hepatocellular carcinoma. Oncongene .1999,18:789-795
54.Singal R, Ginder GD, DNA methylation. Blood, 1999, 93(12): 4059-4070.
55.Ghoshal K,Majumder S,Li Z, et al .Suprression of metallothionein gene expression in a rat hepatoma because of promotor-specific DNA methylation .J Biological Chemistry .2000,275(1):539-547.
56.Robertson KD,Jones PA .DNA methylation past ,Present and future direction .Carcinogenesis.2000,219(3):461-467.
57.Tate PH ,Bird AP. Effects of DNA methylation on DNA binding proteins and gene expression.Curr Opin Genet Dev. 1993, 3(2): 226-231.
58.Kondo Y,Kanai Y, Saksmoto M, et al. Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis a comprehensive study of loss of heterozygosity and microsatellete instability at 39 loci and DNA hepermethylation on CpG insland in microdissected speciments from patients with hepatocellular carcinoma. Hepatology . 2000, 32(5): 970-979.
59.Pulling LC,Klinge DM,Belinshy SA.P16 INK4a and beta–catenin alteration in rat liver tumors individual by NNK. Carcinogenesis. 2002,30(3):561-568.
60.Stio Y, Kanai Y,Sakamoto M, et al .Expression of mRNA for DNA methytransferases and methyl-CpG-bind proteins and DNA methylation status on CpG island and pericentromeric satellite regions during human hepatocarinogenesis. Heoatology, 2002, 33(3): 561-568.
61.Park TJ, Kim HS,Byun KH, et al .Sequential changes in hepatocarcinogenesis induced by diethyl nitrosamine plus thioacetamide in fisher 344 rats ,induction of gankyrinexpression in liver fibrosis:pRb degradation in cirrhosis and methylation of P16 INK4a in hepatocellular carcinoma.Carciogenesis.2001,30(3):138-150.
62.Leon SA,Shapiro B,Sharoff DM, et al .Free DNA in the serum of cancer patients and effect of therapy .Cancer Res. 1997, 37(3): 646-650.
63.Zou HZ,Yu BM,Wang ZW, et al .Detection of aberrant P16 methylation in the serum of colorected cancer patients .Clin Cancer Res.2002,8:188-191.
64.Nunes DN,Kowalski LP,Simpson AJ, et al .Circulations tumor –derived DNA may permit the early diagnisis of head and reck sguamous cell .Int J Cancer .2001,92:214-219.
65.Jahr S,Hentze H,Englisch S, et al .DNA fragments in the blood plasma of
cancer patients quantitations and evidence for their origin from apoptotic and necrotic cell.Cancer Res. 2000,61(4):1659-1665.
66.Giacona MB,Ruben GC,Lczkowski KA, et al .Cell-free DNA in human blood plasma :length measurements in patients with pancreatic cancer and health control .Pancreas.1998,17:89
67.Steoun M,Maurice P,vasioukhin V, et al .The origin and mechanism of circulating DNA.Ann NY A cad Sci.2000,906:165.
68.Anker P,Struon M. Tumor-related alterations in circulating DNA, Poriential for diagnosis and detection of minimal residual disease .Leukemia,2001,15(2):289.
69.Suh SI,Pyun HY,Cho JW, et al .5-Aza-2 deoxycytidine leads to down regulation of aberrant P16 INK4a RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines.Cancer Letter. 2000, 160(1):81.
70.Chao Hong Xia,Sun Jianreng, Lu Jixin , et al .Slineal and experimental study on endometrial carcinoma .Chinese Journal of Obstetrics and Gynecology,2000,35(4):229.
2. Lee HY, Murata J. Cloning, Chromosomal localization and tissue expression of artotation from human carcinoma. Biochem Biophy Res Commun,1996, 218:714.
3. Peng S,Kong G,Rao PM, et al. The genetic charge of DNA in HCC patients. Hepatology,1997,21:35-36.
4. Crowe M.The pair effect of DNA in product of HCC. Gastroenterology, 1997,106:A356-A359.
5. Cains P.Rates of P16 (CDKN2) mutations in primary tumors with Op loss. Science, 1994, 264:415-417.
6. 中国抗癌协会肝癌专业委员会，原发性肝癌诊断标准。中华肝脏杂志，2000,8（3）：135.
7. 袁爱力，刘为纹，晋化源等. 原发性肝癌、良性肝病及胚胎组织中甲胎蛋白异质体的研究。中华消化杂志，1988,8（1）：10-12.
8. 房殿春，罗元辉，刘为纹等。应用逆转录聚合酶链反应选择原发性肝癌患者外周血肿瘤细胞。中华内科杂志，1998,37：168-170.
9. Lemoine A, LeBricon T,Salvucci M, et al. Prospective evaluation of circulating hepatocyte by alpha-feto protein mRNA in human during live surgery. Am surg. 1997,226:43
10.沈鼎明，沈薇，左国庆等。血清肿瘤标志物在原发性肝癌诊断中的价值。中华消化杂志，1992,12：223-226.
11.孟宪镛。肝癌标志物的研究是早期诊断的关键。中华消化杂志。1993,13：251-252.
12.张柏和，吴孟超。抗人甲胎蛋白异质体单克隆抗体诊断肝细胞癌的临床应用价值。中华消化杂志，1992,12（3）：129-132.
13.Nomura F,Ishijma M,Kuwa K,etal.Serum des-gammacarbooxy prothrombin levels determined by a new generation of sensitive immunoassays in patients with small –sized hepatocellular cacinoma.Am J Gas troenteral,1999,94(3):650-654.
14.王斌，唐心利.利用细菌荧光素酶建立酶联免疫吸附试验检测抗-HBs的研究.医学研究通讯, 2003, 32（1）：9-12.
15.曾祝伦，唐波.肿瘤相关物质在肝病中的诊断价值.中国医学检验杂志，2003,4（2）：110-111.
16.黄建钊，沈文律，李波等.原发性肝细胞肝癌中P16基因的失活与P16蛋白表达的研究.华西医科大学学报,2000,31（3）：306-309.
17.Terouanne B,Nicolas JC,Crastes et al. Paulet A.Bioluminesceny
immunoassay for alpha –fetoprotein. Anal Biochem.1986 Apr, 154 (1); 132-7.
18.Wang IH ,LoYM,Yeo W,et al .Frequent P15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients .Clin Cancer Res ,2000 sep,6(9):3516-21.
19.杨冬华.加强人胰岛素样生长因子Ⅱ基因启动子与肝细胞癌变关系的研究.中华内科杂志,1999,38（2）：79-80.
20.J.F.Suo ,Liu YF,Zhang et al .Expression of insulin-like growth factor Ⅱin hepatitis B,cirrhosis and hepatocellular carcinoma :its relationship with hepatitis B virus angen expression. Hepatology, 1994, 20: 7880-7896.
21.Lix ,Cui H,et al .Expression levels of the insulin like growth factor Ⅱ(IGF-Ⅱ)in the human liver developmental relationships of the four promoters.J.Endo Chionolgy, 1996, 149: 117-124.
22.Ng IO,Lee,JM,et al. Expression of insulin-like growth factor Ⅱ mRNA in hepatocellular carcinoma.J.Gastroentral Hepatal, 1998, 13(2): 152-157.
23.Tetsurosohda,Kankatsa Yun,etal. Incrceased expression of insulin-like growth factor 2 in hepatocellular carcinoma is primary regulated at the transcriptional level. Lab. Invest, 1996, 75:307-311.
24.XuriLi,Zhang Nong.christin a ekstom disrupted IGF2 promoter control by silencing of promoter P1 in human hepatocellular carcinoma .Cancer Res,1997,57:2048-2054.
25.VuTh,Li T et al.Symmetric and asymmetric DNA methylation in human IGF2 H19 imprined region .Genomics,2000, 1:64(2): 132-143.
26.Li X,Gray SG,et al. Developmented dependent DNA methylation of the IGF2 and H19 promoters is correlated to the promoter activities in human liver development .Int J Dev. Bioal, 1998, 42(5)687-693.
27.Seo JH,Kin KW,Park.BC.Different Protein binding patterns in P3 promoter region of the human insulin-like growth factor Ⅱgene in the human liver cirrhosis and hepatocellular carcinoma tissues.J .Korean Men.Sci.1998,13(2):171-178.
28.Raizis AM,Eales MR,Reeve AE.Structural analysis of the human insulin-like growth factor ⅡP3 promoter.Biochem, 1993, 1(289):
139-149.
29.Kamb A,Gruis NA,Wearer-Feldans J,et al.A cell cycle regulator potentially involved in genesis of many tumur types. Science, 1994, 264:436-440.
30.Serrao M,Hamon GJ.A new regulation motif in cell-cycle control causing specific inhibition of cycle D/CDK4.Nature, 1993, 366: 704-707.
31.Marx J.New tumor suppressor may rival P53.Science,1991, 264:344-345.
32.Jin X,Nguyen D,Zhang WW. Cell cycle arrest and ihibition of tumor cell proliferation by the P16 INK4 gene mediated by an adenovirus vector. Cancer Res,1995,55:3250-3253.
33.Arap W,Nishikawa,Funai FB, et al. Replacement of the P16/CDKN2 gene suppresses human gilioma cell growth. Cancer Res, 1995, 55: 1451-1354.
34.Serrao M,Lee HW,Chin L, et al .Role of the P16 INK4a locas in tumor suppression and cell mortality .Cell,1996,85:27-37.
35.Hui AM,Shi YZ,Lix, et al .Loss of P16 INK4 protein alone and together with loss of retinoblastoma protein ,correlate with hepatocellular carcinoma progression .Cancer Lett, 2000, 154(1) :93-99.
36.Liu Jianyu,Li Bo, et al.P16 gene expression and methylation status of CpG island in human primary hepatocellular carcinoma. Journal of West China University of medical Science, 2002, 33(4): 540-543.
37.Huang Jian zhao ,Shen Wenlv,Li Bo, et al .A study on the inactivation of P16 gene and the expression of P16 protein in primary hepatocellular carcinoma.Journal of West China University of Medical Science ,2000,31(3):306.
38.Kita R,Nishida N,Fukuda Y et al. Infreguent alterations of the P16 INK4 gene in liver cancer.Int J Cancer,1996,67(2):176.
39.Matsuda Y,Ichida T,Mastuzawa J,et al .P16 INK4 is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology. 1999,116(2):394-400.
40.Willian H ,Ligget Jr,David Sidransky.Role of the P16 tumor suppressor gene in cancer. J Clininal Oncology, 1998, 16(3); 1197.
41.Ivy HN,Wong YM, Dennis LD, et al . Detection of aberrant P16 methylotion in the plasma and serum of liver cancer patients. Cancer
Res,1999,59(1):71-73.
42.Qin Y,Li B,Tan YS, et al .Polymorphism of P16 INK4a gene and rare mutation of P15 INK4a gene exon 2 in primary hepato carcinoma. Wolel Journal of Gastroenleralogy ,2000,6(3):411.
43.Beak MJ,Piao Z,Kim NG,et al . P16 is a major inactivation target in hepatocellular carcinoma .Cancer ,2000,89(1):60.
44.Lee TJ , Bae JJ, et al .Abrogation of the P16-Rb pathway in Korean hepatocellular carcinoma. Hepatogastroenterology, 2000, 47(36): 1663-1668.
45.Clen TC, Hsieh LL, Kuo TT, et al .P16 INK4 gene mutation and allelie loss of chromosome qp21-22 in Taiwanese hepatocellular carcinoma. Anticancer Res ,2000,20(3A):1621-1668.
46.Wang G, Huang Ch, Zhao Y, et al .Genetic aberrantion in primary hepatocellular carcinoma:correlation beween P15 gene mutation and loss of hetozygosity on chromasome 16q21-23,9p21-23,Cell Res. 2000, 10(4): 311-323.
47.Huang JianZhao ,Shen Wenliu ,Xia Suisheng ,et al.The status of P16 gene in primary hepatocellular carcinoma from a Chinese papulation.Chinese Journal of Cancer,2000,1:45-47.
48.Lee YY,Kang SH, Seo JY, et al. Alteration of P16 INK4a and P53 INK4a and P53INKB genes in gastric carcinomas. Cancer, 1997, 80: 1889-1896.
49.Lin YM,Chen CH,Huang GT, et al. Infrequent muation and no methylation of CDKN2A(P16/MTS1) and CDK2B(P15/MTS2) in heoatocullular carcinoma in Taiwan .Eur J Cancer, 1998, 34(11): 1789-1795.
50.Huang J,Shen W, Li B ,et al .Molecular and immunohistochemical study of the inactivation of the P16 gene in primary hepatocellular carcinoma. Chin Med J(EngⅠ), 2000, 113(10): 889-892.
51.Jin M,Piao Z,Kim NG, et al. P16 is a major inactivation target in hepatocellular carcinoma. Cancer ,2000,89(1):60-68.
52.Herman JG,Graff JR,Myohanen S, et al. Methylation specific PCR ,a novel PCR assay for methylation status of CpG is land .Proc Natl Acad,Sci USA.1996,93:9821-9826.
53.Liew G,LiMH,LoKW. High frequency of P16INK4 gene alteration in
hepatocellular carcinoma. Oncongene .1999,18:789-795
54.Singal R, Ginder GD, DNA methylation. Blood, 1999, 93(12): 4059-4070.
55.Ghoshal K,Majumder S,Li Z, et al .Suprression of metallothionein gene expression in a rat hepatoma because of promotor-specific DNA methylation .J Biological Chemistry .2000,275(1):539-547.
56.Robertson KD,Jones PA .DNA methylation past ,Present and future direction .Carcinogenesis.2000,219(3):461-467.
57.Tate PH ,Bird AP. Effects of DNA methylation on DNA binding proteins and gene expression.Curr Opin Genet Dev. 1993, 3(2): 226-231.
58.Kondo Y,Kanai Y, Saksmoto M, et al. Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis a comprehensive study of loss of heterozygosity and microsatellete instability at 39 loci and DNA hepermethylation on CpG insland in microdissected speciments from patients with hepatocellular carcinoma. Hepatology . 2000, 32(5): 970-979.
59.Pulling LC,Klinge DM,Belinshy SA.P16 INK4a and beta–catenin alteration in rat liver tumors individual by NNK. Carcinogenesis. 2002,30(3):561-568.
60.Stio Y, Kanai Y,Sakamoto M, et al .Expression of mRNA for DNA methytransferases and methyl-CpG-bind proteins and DNA methylation status on CpG island and pericentromeric satellite regions during human hepatocarinogenesis. Heoatology, 2002, 33(3): 561-568.
61.Park TJ, Kim HS,Byun KH, et al .Sequential changes in hepatocarcinogenesis induced by diethyl nitrosamine plus thioacetamide in fisher 344 rats ,induction of gankyrinexpression in liver fibrosis:pRb degradation in cirrhosis and methylation of P16 INK4a in hepatocellular carcinoma.Carciogenesis.2001,30(3):138-150.
62.Leon SA,Shapiro B,Sharoff DM, et al .Free DNA in the serum of cancer patients and effect of therapy .Cancer Res. 1997, 37(3): 646-650.
63.Zou HZ,Yu BM,Wang ZW, et al .Detection of aberrant P16 methylation in the serum of colorected cancer patients .Clin Cancer Res.2002,8:188-191.
64.Nunes DN,Kowalski LP,Simpson AJ, et al .Circulations tumor –derived DNA may permit the early diagnisis of head and reck sguamous cell .Int J Cancer .2001,92:214-219.
65.Jahr S,Hentze H,Englisch S, et al .DNA fragments in the blood plasma of
cancer patients quantitations and evidence for their origin from apoptotic and necrotic cell.Cancer Res. 2000,61(4):1659-1665.
66.Giacona MB,Ruben GC,Lczkowski KA, et al .Cell-free DNA in human blood plasma :length measurements in patients with pancreatic cancer and health control .Pancreas.1998,17:89
67.Steoun M,Maurice P,vasioukhin V, et al .The origin and mechanism of circulating DNA.Ann NY A cad Sci.2000,906:165.
68.Anker P,Struon M. Tumor-related alterations in circulating DNA, Poriential for diagnosis and detection of minimal residual disease .Leukemia,2001,15(2):289.
69.Suh SI,Pyun HY,Cho JW, et al .5-Aza-2 deoxycytidine leads to down regulation of aberrant P16 INK4a RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines.Cancer Letter. 2000, 160(1):81.
70.Chao Hong Xia,Sun Jianreng, Lu Jixin , et al .Slineal and experimental study on endometrial carcinoma .Chinese Journal of Obstetrics and Gynecology,2000,35(4):229.