【作者】 罗庚求；

【导师】 文继舫；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 前言:胃癌是一种严重危害人们生命健康的常见肿瘤,它的发病机制尚不清楚。Twist蛋白是属于碱性的螺旋-环-螺旋蛋白家族中的高度保守的转录因子,在胚胎的发生发展中发挥重要作用。Twist被认为是癌基因蛋白,影响肿瘤细胞的凋亡。Twist作为上皮-间质转型(epithelial-mesenchymal transition,EMT)过程中的关键调控因子,对肿瘤的侵袭和转移有重要影响。尽管目前已积累了一些有关Twist的研究,但是Twist对胃癌细胞侵袭和转移有何影响却知之较少。目的:研究Twist基因转染对胃癌细胞侵袭和转移的影响,探讨Twist在胃癌发生、发展中的作用及其机制。方法:通过细菌转化、质粒提取、酶切、凝胶电泳、基因测序鉴定Twist正义质粒;利用脂质体介导转染技术转染SGC7901胃癌细胞株,经G418筛选得到稳定高表达Twist的TwistS-SGC7901(PcDNA3-Twist转染的SGC7901细胞)细胞模型;采用RT-PCR方法检测三组细胞中Twist的mRNA水平;采用免疫荧光和和Western blot分析法检测三组细胞中Twist的表达;经RT-PCR分析Twist转染对EMT标记物(Ecadherin,α-SMA)表达的影响;采用MTT比色法测定转染细胞的黏附能力,Boyden小室测定法测定转染细胞的迁移能力和侵袭能力;腹腔移植法建立裸鼠胃癌细胞转移模型。采用免疫荧光检测三组细胞中β-catenin的表达及定位;用EMSA方法检测Tcf-4/Lef的DNA结合活性;用RT-PCR分析Tcf-4下游靶基因cyclinD1的表达;用Western blot分析Tcf-4下游靶基因vimentin、mmp-2的表达。用ELISA方法分析Tcf-4下游靶基因VEGF的表达。通过细菌转化、提取Twist反义质粒;利用脂质体介导转染技术转染MKN45胃癌细胞株,经G418筛选得到稳定低表达Twist的TwistAS-MKN45(PcDNA3-TwistAS转染的MKN45细胞)细胞模型;采用RT-PCR方法检测三组细胞中Twist的mRNA水平;采用Western blot分析法检测三组细胞中Twist的表达。用EMSA方法检测Tcf-4/Lef的DNA结合活性;用RT-PCR分析Tcf-4下游靶基因cyclinD1的表达;Western blot分析Tcf-4下游靶基因vimentin和mmp-2的表达。用ELISA方法分析Tcf-4下游靶基因VEGF的表达。用Western-blot分析了E-cadherin的表达。利用免疫组织化学方法分析了Twist和Ecadherin在76例胃癌中的表达水平及其和临床病理参数之间的关系。结果:成功建立了Twist稳定高表达的细胞模型(TwistS-SGC7901);与SGC7901细胞及PcDNA3-SGC7901细胞相比较,TwistS-SGC7901细胞的黏附能力、迁移能力、侵袭能力和转移能力明显提高(p＜0.05);Twist能够促进SGC7901细胞发生EMT;Twist正义质粒转染能够增强SGC7901细胞TCF-4转录因子的DNA结合活性;并且能够提高TCF-4转录因子下游靶基因cyclinD1、VEGF、vimentin和mmp-2的表达。成功建立了Twist稳定低表达的细胞模型(TwistAS-MKN45);Twist反义质粒转染能够降低MKN45细胞TCF-4转录因子的DNA结合活性;并且能够降低TCF-4转录因子下游靶基因cyclinD1、VEGF、vimentin和mmp-2的表达。Twist蛋白阳性信号定位于细胞质及细胞核;Twist蛋白在胃癌组织中的阳性表达率为60.52%,远癌断端胃粘膜的表达阴性,且与胃癌分化程度、浸润范围以及淋巴结转移有关(P＜0.05),但是和年龄、性别无关。Ecadherin蛋白在胃癌组织中的阳性表达率为47.37%,低于远癌断端胃粘膜的表达水平(100%),Ecadherin蛋白的表达与胃癌的分化程度、浸润深度和淋巴结转移密切相关(P＜0.05),与年龄、性别无关。Twist蛋白的表达与Ecadherin蛋白呈负相关。结论:1.Twist基因高表达能够增强SGC7901细胞的黏附能力、迁移能力、侵袭能力和转移能力。2.Twist基因能促进SGC7901细胞EMT。3.Twist基因促进SGC7901细胞的侵袭和转移可能通过正调控Wnt信号通路而发挥作用。4.Twist、E-cadherin在胃癌组织中的表达和胃癌的分化程度、浸润深度以及淋巴结转移有关。Twist可能通过抑制Ecadherin的表达促进胃癌的转移。更多还原

【Abstract】 Introduction Gastric carcinoma(GC)is a frequent neoplasm that severely threatens people’s health,and its mechanism is still unknown. The Twist protein is a highly conserved transcription factor that belongs to the family of basic helix-loop-helix proteins,and it plays an important role in the embryogenesis.Twist is regarded as an oncogene protein, regulating the apoptosis of tumor cells.It has been shown that Twist is a key regulator of epithelial-mesenchymal transition and responsible for the invasion and metastasis of tumor.Despite some research of Twist,we know little of whether Twist contributes to the invasion and metastasis of gastric carcinoma.Objective Our investigation attempts to study the effect of transfected Twist gene on invasion and metastasis of human gastric carcinoma cells and the role of Twist gene in gastric carcinogenesis.Methods After bacteria transformation,use restriction enzyme technique,gel electrophoresis technique and gene sequencing identification the Twist sense plasmid;The SGC7901 cells were transfected with PcDNA3-Twist sense plasmid by lipofectamine technique,and gained the stable TwistS-SGC7901 cell model with high expression of Twist protein;The mRNA of Twist were detected by RT-PCR;The expression of Twist protein were detected by immunofluorescence and Western blotting;The expression of EMT marker(Ecadherin,α-SMA)were detected by RT-PCR;Adherent ability of transfected cells were detected by MTT method;The ability of migration and invasion of transfected cells was examined by Bodern method;Nude mice metastasis models were established by abdominal cavity transfer method;The expression ofβ-catenin protein were detected by immunofluorescence;The Tcf-4 DNA binding ability was detected by EMSA;The expression of Tcf-4 downstream target gene cyclinD1 was detected by RT-PCR;The expression of Tcf-4 downstream target gene vimentin and mmp-2 was detected by Western blotting;the expression of VEGF was analysised with ELISA.After bacteria transformation,extracted the Twist antisense plasmid; The MKN45 cells were transfected with PcDNA3-Twist antisense plasmid by lipofectamine technique,and gained the stable TwistAS-MKN45 cell model with low expression of Twist protein;The mRNA of Twist were detected by RT-PCR;The expression of Twist protein were detected by Westem blotting;The Tcf-4 DNA binding ability was detected by EMSA;The expression of Tcf-4 downstream target gene cyclinD1 was detected by RT-PCR;The expression of Tcf-4 downstream target gene vimentin and mmp-2 was detected by Western blotting;the expression of VEGF was analysised with ELISA;The expression of E-cadherin protein were detected by Western blottingWe analyzed the expressions of Twist and Ecadherin proteins and their correlation with clinical features on formalin-fixed,paraffin-embed-ed tissue sections from 76 patients with Gastric cancer using immunohistochemistry method.Results The cell models(TwistS-SGC7901)steadily expressing high Twist protein were obtained;Compared with SGC7901 and PcDNA3-SGC7901 cells,the cell adherence,migration,invasion and metastasis ability of TwistS-SGC7901 cells were increased obviously (P＜0.05);Twist played an important role in promoting SGC7901 cells epithelial-mesenchymal transition;SGC7901 cells transfected with Twist sense plasmid increased Tcf-4 transcription factor’ DNA binding ability, and also promoted the expression of Tcf-4 downstream target genes cyclinD1,VEGF,Vimentin and mmp-2.The cell models(TwistAS-MKN45)steadily expressing low Twist protein were obtained;MKN45 cells transfected with Twist antisense plasmid inhibited Tcf-4 transcription factor’ DNA binding ability,and also inhibited the expression of Tcf-4 downstream target genes cyclinD1, VEGF,Vimentin and mmp-2.Twist protein positive signal was localized in cytoplasm and in nucleus.40 out of 76(60.52%)gastric cancer tissues expressed Twist protein.But no signals were detected in gastric mucosa.The expression level of Twist protein was related to tumor differention degree,invasion extention,lymph node metastasis;but no significant correlation was observed between Twist and age or gender.30 of 76(47.37%)gastric cancer tissues expressed Ecadherin protein,but all the gastric mucosa expressed Ecadherin protein.The expression level of Ecadherin protein was related to tumor differention degree,invasion extention and lymph node metastasis.But no significant correlation was observed between Ecadherin and age or gender.However,the Twist level was negative related to the Ecadherin level.Conclusion1 Overexpression of Twist could promote the cell adherence, migration,invasion and metastasis ability of SGC7901 cells.2 Twist could induce EMT of SGC7901 cells.3 Twist promoted the invasion and metastasis of SGC7901 cells probably through the regulation of Wnt signaling dependent mechanism.4 The expression of Twist and E-cdhefin in gastric cancer was related to tumor differention degree,invasion extention and lymph node metastasis.Twist could promoted the invasion and metastasis of gastric cancer by inhibiting the expression of Ecadherin protein.更多还原