【作者】 张洪兰；

【导师】 舒红；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 前言肺癌是最常见、最具有危险性的恶性肿瘤之一,五年生存率较低,而80%肺癌为非小细胞肺癌(non-small cell of lung cancer, NSCLC),其发生、发展是多因素多步骤参与的过程。由PTKs(蛋白质酪氨酸激酶)和PTPs(蛋白质酪氨酸磷酸酶)精确调控的蛋白质酪氨酸磷酸化水平在此过程中起重要作用。而很多PTKs都是由原癌基因或癌基因编码的,因此某些PTPs可能在体内起抑癌基因的作用,这个结论直到PTEN/MMAC1/TEPl(后简称PTEN)基因发现后才得到证实。作为第一个编码PTPs的抑癌基因即第10号染色体同源丢失性磷酸酶-张力蛋白基因(phoshatase and tension homology deleted on chromosome ten, PTEN),自1997年3月以来先后被国际上三个科研小组分别克隆后立即成为分子生物学、细胞信号转导和肿瘤病理等领域的研究热点,一些研究已经证明它与肿瘤的形成,尤其进展有关。Li和Steck等首先对为数不多的脑肿瘤、前列腺癌及乳腺癌肿瘤细胞株和一些原发肿瘤进行该基因的初步检测,均发现了不同形式的体细胞性突变(somatic mutation)。然而对于非小细胞肺癌发生、发展过程中是否有PTEN基因的突变报道较少,至于其蛋白水平的改变,结论尚不一致。方法收集有完整随访资料的61例非小细胞肺癌及20例癌旁正常肺组织蜡块,其中30例非小细胞肺癌组织抽提DNA, PCR扩增PTEN基因第3、5、7、8外显子,琼脂糖凝胶电泳鉴定扩增产物,有条带者DNA测序；所有蜡块均使用免疫组化SP法检测组织中PTEN、PI3K和P-Akt蛋白的表达情况；计数资料采用率表示,不同组间比较用x2检验,生存分析采用Kaplan-Meier法进行单因素分析及Log-rank检验,并用Cox比例风险模型进行多因素分析。结果测序结果显示,仅1例标本(临床Ⅳ期、低分化、T3N2M1期、术后生存12个月)发生了PTEN基因外显子5、7、8的纯和性丢失。余29例NSCLC标本中发现了2类2例突变位点的存在,这2类突变均位于内含子中：第7外显子3’-侧翼下游46bp处检测到的一个G→A(116033)点突变,在第8外显子5’-侧翼上游2bp和3bp之间(11859-11860)插入了一个T。免疫组化结果显示：NSCLC组织标本中PTEN蛋白的表达低于癌旁正常肺组织；肺腺癌标本中PTEN蛋白表达较高；PTEN蛋白表达随NSCLC患者最初发现时临床分期的增加及出现淋巴结转移而降低；PTEN蛋白在术后生存期≥4年的患者中表达较高(P<0.05)。PTEN蛋白表达在不同年龄组及不同分化程度组的NSCLC组织中差异无显著性(P>0.05)Spearson相关性检验显示：NSCLC组织中PTEN与PI3K/Akt蛋白表达成负相关,证实了PTEN和PI3K/Akt信号通路的负向调控关系。Cox比例风险模型显示：在众多的影响因子中仅吸烟和PTEN阴性表达为影响NSCLC患者预后的危险因子。结论本研究结果显示：PTEN基因的改变、蛋白的表达水平下调及PTEN-PI3K/Akt信号通路的失衡在NSCLC的发生发展中起到了一定的作用,维持了肿瘤的恶性生物学行为,促进了肿瘤的进展。表明PTEN基因有可能通过降低NSCLC的恶性表型成为NSCLC基因治疗的一个有效靶基因。更多还原

【Abstract】 IntrodutionLung Cancer is one of the most common and maligant tumors in the world and the patients have poor prognosis. About 80%of lung cancer is non small cell of lung cancer (NSCLC). Just like some tumors, the development of NSCLC is a complex process involving many genes and factors. The phosphorylation level of protein tyrosine, precisely regulated by PTKs and PTPs, plays an important role in the cellular signal transduction and cell cycle progression. As many PTKs are encoded by pro-oncogenes, it has been postulated that some of PTPs may act as tumor suppressor gene for a long time. But this assumption is not testified until the discovery ofPTEN tumor suppressor gene.Since characterized and cloned seperatedly by Jing Li, Peter A. steck and DaMing Li. in 1997, PTEN has been found to be inactivated by point mutations or homozygous deletions in many malignant tumors such as glioblastoma, protate carcinoma and breast carcinoma. Although much work has been done on the relationships between PTEN gene and some other tumors, there are few reports on the relationship between PTEN mutation and NSCLC. And so far, to our knowledge there is no unified view on PTEN protein expression during the occurrence and development ofNSCLC.MethodsIn the study, we collected 61 NSCLC samples with complete follow-up data and 20 cases near cancer tissue as control. At first, we extracted 30 cases of NSCLC tissue s DNA, then designed primers of PTEN 3.5.7,8 exons.consequently got the amplification production by PCR and identified by agarose gel electrophoresis.The next step, we sended the ones which have amplification productions to the detecting company, then we could obtain the sequence result. We also used immunohistochemistry to detect PTEN,PI3K and P-Akt protein expression in all samples. At last, we usedχ2 test to analyze the mutation and protein expression outcomes and analyzed survival aspects by single factor Kaplan-Meier assay and Cox-Model.ResultsIt was shown that two kinds of novel point mutations and one case of PTEN EXON5,7 and 8 loss of homozygosity in NSCLC samples used were identified by PCR and DNA sequencing methods. Among them, one case of base substitutions was found in 7-intron region(46bp of downstream of exon73’-flanking region,116033 G→A), while other 1 case of point mutation localized in 7-intron region(between 2 bp and 3 bp of upstream of exon85-flanking region insert a T,11859-11860).By immunohistochemistry, the expression level of PTEN protein in NSCLC were significantly lower than in tumor-sorrounding lung tissues(P<0.05). Besides the low expression level of PTEN protein was significantly associated with squamous cancer, late clinical stage,lymphangitic metastases and short survival time after operation(P< 0.05). However no significant relationship was found with age and histological grading(P>0.05).Spearman correlation test showed that the expression level of PTEN protein was signicantly positive correlation with PI3K and Akt protein expression. Now, it demonstrated the negative regulation between PTEN and PI3K/Akt signal pathway.Cox multiple factor proportion risk model showed that among many factors, only smoking and loss expression of PTEN were risk factors of patients survival time after operation.Conclusions Our results suggest that mutation,loss expression of PTEN gene and the disbalance between PTEN and PI3K/Akt signal pathway may be involved in the occurrence and development of NSCLC. which sustained the malignant phenotype of tumor and facilitated tumor development. Then we believe that PTEN may be implicated in the genetherapy of NSCLC.更多还原