【作者】 洪士开；

【导师】 陆云飞；

【作者基本信息】 广西医科大学 ， 外科学， 2009， 博士

【摘要】 背景与目的最早在研究果蝇发育过程中发现,Twist参与调节多种发育过程,尤其在早期中胚层的形成方面起重要作用。1996年人类Twist基因被成功克隆,定位于7p21.2,编码203个氨基酸,分子量约28Kd。Twist蛋白是具有碱螺旋-环-螺旋(basic helix-loop-helix ,bHLH)结构的转录因子家族成员,其可以与另外一个同源或异源的bHLH因子结合,形成二联DNA结合域,特异性地结合E-box盒,从而激活或抑制特异性的靶基因转录。近年来的研究表明Twist是个新的癌基因,它可以通过p53依赖与非依赖的途径参与细胞的凋亡抵抗。目前研究表明,Twist过表达在肿瘤的发生、侵袭转移中发挥重要作用,但是其中的具体机制尚不清楚。有关Twist在胃癌发生、发展中的作用尚无深入的研究。上皮细胞间质转型( epithelial-mesenchymal transitions, EMT)是具有极性的上皮细胞转换成为具有活动能力的间质细胞并获得侵袭和迁移能力的过程,它存在于人体多个生理和病理过程中。EMT的发生涉及到多个信号转导通路和复杂的分子机制,与钙连接素、生长因子、转录因子、微环境等有关。EMT与肿瘤细胞的侵袭和转移关系密切。上皮钙粘连素(E-cadherin,E-cad)和神经钙粘连素(N-cadherin,N-cad)都是钙粘连素家族的重要成员,参与细胞间粘附。E-cadherin是影响肿瘤浸润转移较为重要的分子,其丢失可导致肿瘤细胞脱落与转移,与E-cadherin不同,N-cadherin可促进肿瘤浸润转移。Twist, E-cadherin, N-cadherin都是EMT的重要分子。基于上述考虑,本实验采用逆转录-聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR )、免疫组织化学技术对胃癌组织及其癌旁组织中Twist, E-cadherin,N-cadheri n的表达进行检测,为阐明胃癌的致病机制和防治提供实验基础和理论依据。方法1.采用RT-PCR方法检测58例人胃癌组织及58例相应癌旁组织中Twist, E-cadherin, N-cadherin mRNA的表达;2.采用免疫组织化学方法检测58例人胃癌组织及癌旁组织中Twist, E-cadherin, N-cadherin蛋白表达;3.采用SPSS 10.0统计软件进行统计学处理。采用t检验对计量资料进行统计学分析,采用x2检验对计数资料进行统计学分析,用相关进行相关分析,检验水准α=0.05.结果1.人胃癌组织RT-PCR方法检测结果显示: Twist, E-cadherin, N-cadherin分别于210bp、132bp、156bp处显带,为阳性表达。Twist, N-cadherin mRNA在胃癌组织中的阳性率分别为72.41%, 48.28%;明显高于癌旁组织的31.03%, 13.79%;E-cadherin mRNA在胃癌组织的阳性表达率为39.66%,明显低于癌旁组织的84.48%;差异有统计学意义(P<0.01)。2.人胃癌组织免疫组织化学染色结果显示: Twist, N-cadherin蛋白在胃癌组织中的阳性表达率分别为75.9%,44.8%;明显高于癌旁组织的32.8% ,12.1% ;E-cadherin蛋白在胃癌组织中的阳性表达率为36.2%,明显低于癌旁组织的91.3%;差异有统计学意义(P<0.01)。结论1. Twist、N-cadherin mRNA及蛋白在胃癌中过表达,E-cadherin mRNA及蛋白在胃癌中低表达;可能与胃癌发生发展有关;2. Twist, E-cadherin, N-cadherin与胃癌的浸润转移有关,三者共同参与了EMT过程。3.联合检测三项指标对于进一步预测胃癌的转移、判断其预后具有重要意义。更多还原

【Abstract】 Background and objective:Twist was originally identified in Drosophila as a protein involved in the regulation of diverse developmental processes, particularly in the formation of mesoderm.The human Twist gene is located at chromosome 7p21.2.The Twist protein consists of 201 amino acids and its molecular weight is about 28kd.Twist protein is a transcription factor that belongs to the family of basic helix-loop-helix(bHLH)protein.The mechanism of bHLH molecules require that they either homo-or heterodimerize with other bHLH molecules,form a bipartite DNA-binding domain and bind to a conserved E-box region on the promoter of genes which activate or inhibit transcription.Recent studies have shown that Twist is a potential oncogene and be involved in the protection of apoptosis via both p53-dependent and independent pathways.To date several studies revealed that Twist played crucial roles in tumorigenesis, invasion and metastasis,but its accurate molecular mechanisms remain unclear.However,there have been little available data on the role of Twist in the development and progession of gastric cancer;the molecule involved in the regulation of Twist and its mechanism was still unclear. We researched for the molecule involved in the regulation of Twist and its mechanisms.Epithelial-mesenchymal transitions (EMT) is a process that epithelial cell layers lose polarity and acquire expression of mesenchymal components and manifest a migratory phenotype. It exists in many physiological and pathological p rocesses in human body. The progress of EMT involves a number of signal transduction pathways and complex molecular mechanisms, and is related to calcium catenin,growth factors, transcription factors, microenvironment, and so on. EMT play an important role in tumor metastasis. E-cadherin and N-cadherin are members of the family of transmembrane glycoproteins expressed on epithelial cells and are responsible for calciumdependent cell -to-celladhesion. E-cad can lead to Loss of cell adhesion,cell adhesion may contribute to loss of contact inhibition of growth, which is an early step in the neoplastic process.Furthermore, loss of cadherin activity may result in cancer cell detachment and metastasis.It has been suggested that, unlike E-cadherin, N-cadherin may promote invasion and metastasis of carcinoma. Twist,E- and N-cadherin are important molecules that modulate cell migration and tumor,invasion.According to above metioned consider, in this study, Reverse transcription polymerase chain reaction (RT-PCR) technique and immunohistochemical method were used to detect the expression of Twist,E-cadherin and N-cadherin in gastric cancer. So that to provide experimental evidence for elucidating the etiopathogenesis, treatment and prevention in gastric cancer.Methods:1 .Reverse transcription polymerase chain reaction (RT- PCR) technique was used to examine the expressions of Twist,E-cadherin and N-cadherin mRNA in the 58 gastric cancer tissues and their 58 non-tumorous gastric tissues.2. Supervision TM immunohistochemical method was used to detect the expression of Twist,E-cadherin and N-cadherin proteins in 58 cases of gastric cancer tissues and their non-tumorous gastric tissues.3. SPSS 10.0 statistical software was applied to statistics the date. To categorical variable the sperman correlation analysis were used to analyze. To numerical variable the date was expressed with x士s and t test was used to compare these datas. The significant level was a =0.05.Results:1 .The positive expression of Twist,E-cadherin and N-cadherin locate in land 210bp,132bp,156bp. The positive expression of Twist,E-cadherin and N-cadherin mRNAs were 72.41%,39.66%, 48.28% in gastric cancer tissues, and were 31.03%, 84.48%, 13.79% in non-tumorous gastric tissue, respectively. (P<0.01).2. The positive expression rates of Twist,E-cadherin and N-cadherin proteins were 75.9%,36.2%,44.8% in gastric cancer tissues, and were 32.8%,91.3%,12.1% in non-tumorous gastric tissue,respectively. (P<0.01).Conclusions:1 .The higher expressions of Twist,N-cadherin and the lower expression of E-cadherin in gastric cancer had the close relation to carcinogenesis of gastric cancer.2. The expressions of Twist,E-cadherin and N-cadherin are related with the invasion,metastasis of gastric cancer,the genes of Twist,E-cadherin and N-cadherin participate with process of EMT together. 3.United measurement of the expression of Twist,E-cadherin and N-cadherin genes is helpful to predict metastasis of gastric cancer and to judge prognosis of gastric cancer.更多还原