【作者】 胡红艳；

【导师】 魏万里； 张丽娟； 杨承纲； 陈芸；

【作者基本信息】 昆明医学院 ， 病理学与病理生理学， 2010， 硕士

【摘要】 目的：为阐明ASPP基因家族与肺癌(Lung carcinoma)发生发展的关系,本文从基因水平检测肺癌组织中抑癌基因ASPP1、ASPP2启动子甲基化状态；从蛋白水平上检测ASPP1、ASPP2、p53在肺癌组织中的表达情况,并检测肺癌组织的凋亡指数(apoptosis index,AI),进而分析其间的相关性,探讨ASPP家族表达调控机制及其在p53凋亡通路中的作用,为肺癌的早期诊断,治疗及预后判断提供理论依据。方法：本研究实验组收集肺癌组织90例,对照组25例相应癌旁正常肺组织,构建肺癌组织芯片,免疫组织化学染色Envision法检测ASPP1、ASPP2及p53蛋白在肺癌组织芯片中的表达情况,并同时利用原位末端转移酶标记检测法(Terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL法)检测肺癌细胞的凋亡指数AI,应用甲基化特异性PCR(methylation-specific PCR,MSP)对抑癌基因ASPP1、ASPP2启动子CpG岛进行甲基化状态检测,分析ASPP1、ASPP2蛋白表达、启动子甲基化和p53细胞凋亡通路之间的关系。结果1.90例肺癌组织ASPP1和ASPP2阳性率分别为27.8％和11.1％；25例癌旁组织中ASPP1和ASPP2阳性率分别为52.0％和16.0％；ASPP1在肺癌组织和癌旁组织中表达阳性率两者比较,差异有统计学意义(χ2=5.188,P<0.05),而ASPP2在肺癌组织和癌旁组织中表达阳性率两者比较,差异无统计学意义(χ2=0.437,P>0.05)。2.90例肺癌组织中ASPP1蛋白的表达和分化程度、TNM分期及淋巴结转移呈正相关(P<0.05),与患者的性别、年龄、肿瘤大小、组织学类型无关(P>0.05)；而ASPP2蛋白的表达与肺癌的临床病理特征均无关(P>0.05)。3.39例p53表达阴性的肺癌组织中ASPP1表达阳性16例(41.02％),51例突变型p53肺癌组织中ASPP1表达阳性9例(17.64％),经Spearman’s相关分析,说明在肺癌组织中ASPP1蛋白表达与突变型p53蛋白表达呈负相关(r=-0.259,P<0.01),而39例p53表达阴性的肺癌患者中ASPP2蛋白表达阳性6例(15.38％),51例突变型p53中4例ASPP2表达阳性(9.75％),经Spearman’s相关分析,可认为在肺癌组织中ASPP2蛋白表达与p53蛋白的表达无相关性(r=-0.119,P>0.05)。4.比较90肺癌组织ASPP1蛋白阳性表达组与阴性组中AI值,发现ASPP1蛋白阳性组高于阴性组,提示ASPP1蛋白表达与细胞凋亡有关(t=2.337,P<0.05)；而90例肺癌组织中,ASPP2蛋白表达阳性组和阴性组之间AI值无显著性差异(t=1.083,P>0.05),可以为ASPP2蛋白的表达与肺癌细胞凋亡无关。5.NSCLC中ASPP1基因启动子甲基化率为42.22％(38／90),癌旁组织甲基化率为16.00％(4／25),有显著性差异(χ2=5.830,P>0.05)；ASPP1基因启动子甲基化与患者的年龄、性别、组织类型和分化程度无关(χ2=1.129、1.488、1.126、4.555,P>0.05),但与患者的TNM分期和淋巴结转移有关(χ2=6.919、5.213,P=<0.05)；90例肺癌组织中ASPP1甲基化者其蛋白表达率明显低于未甲基化者(χ2=5.188,P<0.05)；而ASPP2基因启动子在90例NSCLC和25例癌旁组织中均未检测到甲基化状态。结论1.ASPP1蛋白在肺癌组织中低表达,与肿瘤分化程度、临床分期和淋巴结转移有关,提示ASPP1表达缺失可能与肺癌发生、进展有关。2.ASPP2蛋白在肺癌组织和癌旁肺组织中的表达无明显差异,提示ASPP2在肺癌中可能不具有肿瘤抑制基因特性。3.在肺癌组织中,ASPP1促进肺癌细胞AI的增高,且其表达与突变型p53呈负相关,ASPP1可能是p53凋亡通路中的关键蛋白；而ASPP2可能与p53凋亡通路无关。4.肺癌组织中ASPP1启动子甲基化可能是ASPP1蛋白表达下调的原因,ASPP1高甲基化预示着NSCLC的高侵袭性,而ASPP2基因甲基化可能不是其蛋白的表达的调控机制。更多还原

【Abstract】 Objective:In order to research the relation between ASPP gene family and incidence of lung carcinoma, the article examined the promoter methylation statut of the ASPP1 and ASPP2 from lever of DNA in lung carcinoma, and the expression of ASPP1、ASPP2 and p53 from lever of protein, and the apoptosis index of the lung cancer tissues.Metholds:To construct tissue microarray of lung cancers,we collected 90 cases lung carcinoma tissues as experimental group and 25 normal adjacent lung tissue as control group. The expression state of ASPP1、ASPP2 and p53 protion were determined by immunohistochemical staining combined with tissue microarray of lung cancer, Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) was applied to detect the apoptosis in the tissue microarray, The apoptosis index(AI) were defined. The methylations of ASPPl and ASPP2 promoter region CpG island were analysised by methylation-specific PCR (MSP). Furthermore, We analysis the relationship among expression of ASPP1、ASPP2 protein, promoter region methylation,AI,and p53 apoptosis pathway.Results: 1.The position rate of ASPP1 in 90 cases lung cancer tissues was 27.8%, and the position rate of ASPP2 was 11.1%; the positive rate of ASPP1 in 25 normal adjacent lung tissues was 52.0%, and the ASPP2 positive rate was 16.0%; Significant difference was found between the expression of ASPP1 in cancer tissue and their normal adjacent tissues (x2=5.188,P<0.05); while the expression of ASPP2 has no significant difference (x2=0.437,P>0.05).2. The expression of ASPP1 in 90 cases lung cancer had positive relation with the differentiation, TNM stage and lymph node metastasis of the disease (x2:=4.680、6.330、6.045, P<0.05),but not related to sex, age,and histological type. Significant difference was not found between the expression of ASPP2 and clinical pathologic characteristic(P>0.05).3. The positive expression of ASPP1 was 16 cases in 39 cases that negative expression of p53,and 9 cases in 51cases that mutation p53 cancer tissues. The Spearman’s method was used, and the result was that there was negative correlation between the expression of ASPP1 and mutation p53 in lung cancer (r=-0.259,P<0.01),but the positive expression of ASPP2 was 6 cases in 39 cases that nagetive expression of p53 cancer tissues, and 4 cases in 51 cases mutation p53 tissues, the expression of ASPP2 was not associated with p53 state by used Spearman’s method(r=-0.119,P>0.05).4. The AI in the group of ASPP1 positive expression was significantly higher than in the group of ASPP 1 negative expression, this showed that there was close relationship between the expression of ASPP1 and cell apoptosis in lung carcinoma (t=2.337,P<0.05); but no significant different was found between the group of ASPP2 positive expression and the group of negative expression, so the expression of ASPP2 may has no relation with cell apoptosis in lung carcinoma(t=1.083,P>0.05)5. The positive rate of ASPP 1 methylation in lung tumor tissues was significantly higher than that in the adjacent lung tissues{42.22%(38/90) vs 16.00%(4/25), P =0.019}.The methylation state of ASPP 1 had close relation with TNM stage and lymphnode metastasis of the disease (x2=6.919、5.213,P=<0.05),but not related to age,sex, histological type and differentiation(x2=1.l29、1.488、1.126、4.555,P >0.05).The group with promoter methylation showed lower expression of ASPP 1 than that with unmethylation state (x2=5.188,P<0.05); Methylation in the promoter region of ASPP2 gene was not detected in lung cancer (n=90) or side-carcinoma tissues (n=25);Conclutions: 1. The lower expression of ASPP1 was correlation with TNM stage and lymph node metastasis in lung cancer, that shows the loss expression of ASPP1 may participate in lung carcinogenesis and development.2. There was no significant difference in expression of ASPP2 between lung cancer tissue and the paracarcinomatous tissues, it indicated that ASPP2 may not be as a tumor suppressor gene in lung carcinomas.3. ASPP1 can induce AI, and the expression of ASPP1 has a negative correlationship with mutation p53; ASPP1 may be as a key factor of p53 apoptosis pathway in lung cancer tissue;but ASPP2 may be independent of the p53 apoptosis pathway.4. The hypermethylation of ASPP1 gene might lead to the abnormal expression of ASPP1 protein; and it is respected of the aggressive clinical behavior and advanced tumor stage; while the ASPP2 gene promoter methylation may not be the cause of the abnormal expression of ASPP2 protein in tumorgenesis.更多还原