【作者】 宋伟；

【导师】 于世英；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2009， 博士

【摘要】 目的(1)观察Snail以及E-cadherin蛋白在胃癌组织中的表达及其与胃癌临床病理特征的关系；(2)探讨E-cadherin基因启动子在胃癌中的异常甲基化状态及其与临床病理资料的联系；(3)RNA干扰技术抑制转录因子Snail表达，观察人胃腺癌SGC-7901细胞上皮-间充质转分化表型和体外侵袭能力的影响；(4)建立稳定转染高表达绿色荧光蛋白(GFP)基因的胃癌细胞株并成功建立动物模型，为胃癌的生长及血管生成研究提供直接观察的必备条件。方法(1)应用免疫组织化学S-P法检测152例手术切除胃癌组织以及30例癌旁无瘤组织中Snail蛋白、E-cadherin蛋白的表达；(2)采用甲基化特异性聚合酶链反应(Methylation Specific-PCR，MSP)技术检测54例胃癌以及癌旁正常组织中E-cadherin基因启动子异常甲基化状态；(3)构建针对Snail的小干扰RNA的RNA干扰载体(Snail siRNA vector)和表达不针对任何已知mRNA的siRNA的阴性对照RNA干扰载体(control siRNA vector)，分别转染SGC-7901细胞，筛选得到Snail表达受抑制的SGC-7901-siSnail细胞和Snail表达未受影响的SGC-7901-siControl细胞。，分别采用RT-PCR和Western blot技术检测SGC-7901-nontransfection、SGC-7901-siSnail、SGC-7901-siControl三组细胞中Snail、a-平滑肌肌动蛋白(a-SMA)和E-cadherin表达，用Boyden chamber模型检测细胞侵袭能力。(4)将携带GFPcDNA的质粒载体转染至胃癌细胞株SGC-7901内，经过G418筛选和克隆化培养，并种植于裸鼠皮下，用荧光显微镜检测癌细胞荧光表达情况。结果(1)Snail在胃癌组织的表达显著高于癌旁无瘤组织，其表达与分化程度、组织学类型、淋巴结转移以及临床分期有关(p＜0．05)，与性别、年龄、肿瘤大小无关(p＞0．05)；E-cadherin在胃癌组织表达显著低于癌旁无瘤组织，其表达与分化程度、组织学类型、淋巴结转移有关(p＜0．05)，与性别、年龄、肿瘤大小以及临床分期无关(p＞0．05。(2)E-cad基因启动子异常甲基化在胃癌组织中发生频率分别为48．1％(26／54)，与癌旁正常组织11．11％(6／54)具有显著性差异(p＜0．05)。E-cad基因启动子异常甲基化频率与性别以及年龄无关；与分化程度、病理类型、浸润深度以及淋巴结转移等相关。(3)SGC-7901-si Snail组和未转染组相比，Snail和a-SMA表达显著减弱(p＜0．01)，E-cadherin表达显著增强(p＜0．01)，Boyden chamber穿膜细胞数显著减少(p＜0．01)；SGC-7901-siControl组Snail、a-SMA和E-cadherin表达及Boyden chamber穿膜细胞数和未转染组无显著差异(p＞0．05)。(4)转染GFPcDNA的癌细胞3d后大部分死亡，荧光显微镜下有散在或小团状的点状荧光。继续用G418筛选及克隆化培养20d后，只剩一个高表达绿色荧光蛋白的细胞团，再经150d(50代)培养后仍高表达绿色荧光蛋白。将SGC-7901-GFP细胞株接种于裸鼠前肢皮下，荷瘤鼠生长良好。结论(1)E-cadherin蛋白低表达与Snail蛋白高表达可能是胃黏膜恶性转变以及胃癌发生浸润转移的重要生物学标志；联合检测E-cadherin蛋白与Snail蛋白对预测胃癌浸润转移有重要意义；(2)胃癌中存在E-cadherin基因启动子较高频率的异常甲基化，可能是E-cadherin表达缺失的原因之一；(3)通过RNA干扰阻滞Snail表达能有效地抑制SGC-7901细胞上皮-间充质转分化及体外侵袭能力。Snail可能在胃腺癌上皮-间充质转分化及侵袭过程中扮演重要角色，抑制Snail表达可能成胃腺癌治疗的可行策略；(4)SGC-7901-GFP细胞株的建立以及动物模型的成功建立有助于观察和了解肿瘤侵袭和转移发生及其规律，为下一步的实验做好准备。更多还原

【Abstract】 Objective (1)To investigate the expression of Snail and E-cadherin protein in gastriccarcinoma and their relationship with clinical pathological features;(2) to investigate thefrequency of aberrant methylation of E-cadherin in gastric carcinoma;(3) to investigate theeffect of RNA interference (RNAi) targeting transcription factor Snail on epithelial-mesenchymal transition (EMT) and invasion ability of human gastric carcinoma cellSGC-7901 in vitro;(4) to study the establishment of stable high level green fluorescentprotein (GFP)-expressing cell line of gastric carcinoma and pass on its culture continuously.Methods (1)Snail and E-cadherin protein was detected in 152 gastric carcinoma and 30normal gastric tissues by using immunohistochemical method.(2) Aberrant methylationof E-cadherin was examined by methylation specific-PCR (MSP) in gastric carcinoma andnormal gastric tissues (n=54).(3) RNA interference plasmid that can express smallinterfering RNA (siRNA) targeting Snail (Snail siRNA vector) or express siRNA that doesnotmatch any known human coding mRNA (control siRNA vector)wasdesigned,constructed,and lipotransfected into SGC-7901 cell line.SGC-7901-siSnail cellsexpressing Snail suppressed or SGC-7901-si-Control cells expressing Snail uninfluencedwas selected by neomycin resistance.In SGC-7901-nontransfection,SGC-7901-siSnail andSGC-7901-siControl group SGC-7901 cells,Snail,alpha-smooth muscle actin (a-SMA)and E-cadherin expression were examined by reverse transcription-polymerase chainreaction (RT-PCR) and Western blot,invasion ability was examined by Boyden chambermodel.(4) SGC-7901 cell line of gastric carcinoma was transfected by plasma vector with integrated GFP cDNA.GFP-expressing cancer cells were detected by fluorescentmicroscopy through G418 selecting and cloned culture.Results (1) The expression of Snail protein in gastric carcinoma was significantlyhigher than that in normal gastric tissues (p＜0.05);Expression level of Snail wassignificantly related to differential degree、histology type、clinical stage、lymph nodemetastasis,but not correlate with sex、age、tumour size.The expression of E-cadherinprotein in gastric carcinoma was significantly lower than that in normal gastric tissues(p＜0.05);Expression level of E-cadherin was significantly related to differential degree、histology type、lymph node metastasis and but not correlate with sex、age、tumour size、clinical stage.(2) Aberrant methylation of E-cadherin was present in 48.1%(26/54) and11.11%(6/54) in gastric carcinoma and normal gastric tissues respectively.There wassignificant difference between gastric carcinoma and normal gastric tissues group.Therewas significant correlation between E-cadherin methylation frequencies and pathology type,clinical staging,depth of tumour invasion and lymphatic metastasis.No significantassociation was found between E-cadherin methylation frequencies and sex,age.(3)SGC-7901-nontransfection group:Both Snail and a-SMA expression were strong positive,but E-cadherin expression was poor positive.SGC-7901-si Snail group Compared with thatin SGC-7901-nontransfection group,Snail and a - SMA expression,and the numbers ofSGC-7901 cells permeating septum of Boyden chamber were decreased,but E-cadherinexpression increased significantly (p＜0.01).SGC-7901-si Control group:Compared withthat in SGC-7901- nontransfection group,Snail,a-SMA and E-cadherin expression,andNCS were similar.(4) Most of the cancer cells were dead 5 days after being transfected byGFP,scattered or clustered green fluorescence can be seen by microscopy.In culture for 65days,almost all of the clone 2-8 cells expressed high-intensity GFP fluorescence andstability.Conclusions (1)E-cadherin and overexpression of Snail might be impomalignanttransformation and invasion and matastasiscombined detection of E-cadherinherin and Snail has positiinvasion and metastasis in gastric carcinoma.(2) Methylation of E-cadherinmay play a role in the pathogenesis of gastric carcinoma.(3) RNAi targeting Snail caninhibit EMT and invasion of SGC-7901 cells efficiently in vitro.Snail might play a crucialrole in EMT and invasion of lung carcinoma and suppression of Snail expression might bea promising strategy for the treatment of human lung carcinoma.(4)SGC-7901-GFP cellline can provide a basis for establishing an ideal animal model for research of tumorinvasion and metastasis.更多还原