【作者】 蔡清萍；

【导师】 徐冠南；

【作者基本信息】 第二军医大学 ， 普通外科学， 2000， 博士

【摘要】 近年来胰腺癌的发病率呈不断上升趋势，其恶性程度高、较早转移特性，缺乏有效的早期诊断以及术后易复发性、预后极差。根本原因在于对胰腺癌的发生。发展的机理了解甚少，缺乏有效的临床治疗手段。目前，关于癌基因及抑癌基因在肿瘤发生发展中的作用及其机制的研究是肿瘤基础研究中的热点。本研究以人胰腺癌标本及胰腺癌株为研究对象，研究了新近由Ohta发现的三联脆组（FHIT）基因在胰腺癌中的mRNA表达的变化规律、利用脂质体转染技术研究了FHIT过表达对胰腺癌发生发展的影响并对其作用机制进行了初步探讨。主要结论如下： 一、运用RT－PCR及Nested-PCR技术研究发现：胰腺癌及癌旁组织中FHIT cDNA均存在较高的异常率分别为 66．7％和 60％。与正常腺组织均有显著差异（P＜0．01）其cDNA异常主要在非编码区（外显子2-4）及编码区外显子5，以一个或数个外显子的丢失为主，FHIT基因的异常和FRA3B区密切相关。故推测FRA3B区可能存在对致癌因素敏感的特定基因序列。癌旁组织中 FHIT cDNA的高异常率，提示在组织学正常的胰腺癌切缘的组织细胞在基因水平上却可能存在异常而比正常细胞更易癌变，这可能与胰腺癌术后高复发率有关。FHIT基因与胰腺癌的发生发展密切相关。FHIT基因异常在胰腺的发生发展中起重要作用。有意义的是发现胰腺癌株1990有FHIT全基因丢失。 二、利用较高转染效率的脂质体介导方法对有FHIT全基因丢失的 一 二 一1990胰腺癌细胞株进行 FHIT转染。转染后，经 RT－PCR及 Western blot－ting证实获得阳性克隆，其生长明显受到抑制。转染后的细胞对裸鼠致瘤性明显受到抑制或消失。结果表明FHIT抑制了胰腺癌的增殖及致瘤性。TIlT为一抑癌基因。本研究为胰腺癌发生的机理及治疗提供了新的途径及线索。 三、通过对转染前后细胞形态学的观察，发现转染后细胞凋亡指数明显高于对照组，凋亡定性分析——琼脂糖凝胶电泳检查到特征性的ndder带，提示凋亡是FHIT抑制胰腺癌生长的途径。对转染前后细胞内AeA及 ApA的测定结果为 AoA明显下降，而 ApA有上升，ApA／AfoA之比变化更为明显，FHIT蛋白为一AoA水解酶，但其抑癌作用并不依赖其水解活性，故推测 FHIT与 AeA结合，FHIT可能为 AoA的特异性搬运载体，通过细胞信号传导途径诱导了调亡，同时 AoA及 APA可能是一未被发现的新的细胞信号传导分子。更多还原

【Abstract】 Recent the morbidity of pancreatic adenocarcinoma has a tendency to be increasing. It’s well know that the malignancy of pancreatic adenocarcinoma is much higher than any other alimentary tract carcinomas, it’s easy for pancre- atic adenocarcinoma to metaslasize, no validity diagnosis is found for this dis- ease, with a high relapse after operation, all of which contribate to the poor prognosis of pancreatic adenocarcinoma. Now, the study of the genes which are associated with the ctevelopment of carcinoma is becoming the hot spot in oncology field. The aim of this study is (i) to explore the change regularity of the FHIT gene in pancreatic adenocarcinoma and their adjacent tissues, (ii) determine the effect on cell growth and tumorigenicity of FHIT gene in 1990 pancreatic adeno careimoma cell line (iii) to probe the mechanize of FHIT in suppressing the development of panereatic adenocarcinoma. The results and conclusions are as following. 1. Using the method of RT-PCR and Nested-PCR the study showed that the altered frequence of human pancreatic adneocarcinoma and their adjacent tissues were both high (66.7% and 60%) and were much higher than that of normal pancreas tissues (P<0. 01). The most common altered region of FHIT eDNA in the tissues analysed was no coding area exon 2 exon 4 and the first coding exon 5, the alter about FHIT is usually deleted one or several exon. The high altered frequence of FHIT is connected closely to the fragile region, the FRA3B locus. The FRA3B locus may have a special gene sequence suscep- tible to other agents that interfere with DNA replication, such as nicotine, caf- feine, alcohol, or other known carcinogens. The high frequence of altered FHIT in pancreatic adenocarcinoma adjacent tissues implicates that this tissues are easily oncology leading to the high relapse frequence after operation. So, FHIT gene plays an important role in the development of pancreatic adenocar- cinoma. FHIT gene was found lost totally in 1990 cell line. 2. The method of liposome transfection can get a high validity of transfec- tion. FHIT was tranfected to 1990 cell line in which the FHIT gene was lost totally. Integration and expression of exogenous FHIT gene were confirmed by RT-PCR and Western blotting. The growth of 1990 pFHIT was suppressed clearly and its tumorigericity in nude mice was reliminated or supressed also, all of which indicated that FHIT suppressed the growth and tumorigenicity of the panereatic adenocarcinoma cell line and supported FHIT is a tumor sup- pressor gene. So, a new approach is available to probe the mechanize and ther- apy protocols about pancreatic adenocarcinoma. 3. When the morphology of 1990 and 1990 pFHIT were evaluated, the apoptosis in 1990 pFHIT increased clearly. By the analysis of its DNA on a- garose gel for electrophoresis, the characteristic DNA ladder of 200 bp oc- curred. It could be spectulated that FHIT suppress the development of paner- catic adehocarcinoma by the path of apoptosis. The level of Ap3A in 1990 pFHIT decreased clearly than that of 1990. However, the level of Ap4A in 1990 pFHIT increased, the ratio of Ap4A to Ap3A changed more clearly. FHIT protein is a Ap3A hydrolase l but its suppression on tunror didn, t dependon its hydrosis active. Taken together, a role for FHIT as a proapototic factor...is in agreement with the structura1 and biochedrical studies indicating thatFHIT. Ap3A complex is the active FHIT form involved in cel1ular signalinglinking the induction of apoptosis in 1990 pFHIT celIs更多还原