【作者】 周立；

【导师】 于健春；

【作者基本信息】 中国协和医科大学 ， 外科学， 2008， 博士

【摘要】 背景:胆囊癌来源于胆囊上皮,其发病隐匿,发展迅速,预后不良,严重威胁人类健康和生命安全。近年来我国胆囊癌的发病率呈增高趋势,更加凸现出对于其发生和发展进行深入研究的必要性。迄今,国内外学者研究发现胆囊癌细胞中多种肿瘤相关基因存在表达异常或突变,如K-ras、P53、P27^kipl、cyclin D1和β-catenin等。但是,近年在肝细胞癌中成功克隆的新肿瘤相关基因LAPTM4B（lysosome-associatedprotein transmembrane 4β）在胆囊癌中的表达及其临床、病理和预后意义,以及其相关作用机制尚不清楚。目的:本研究在相关前期文献报道的基础上首次探讨LAPTM4B基因在胆囊癌中的表达及其临床、病理和预后意义,并进一步着眼于该基因在胆囊癌中的作用及其相关分了机制,从而达到深化对胆囊癌发病和进展的了解及探索胆囊癌新的治疗靶点的目的。方法:第一部分LAPTM4B编码蛋白在胆囊癌中的表达及其临床病理和预后意义免疫印迹:应用LAPTM4B-EC2多克隆抗体检测配对癌和癌旁组织LAPTM4B编码蛋白的表达。免疫组织化学染色:应用特异性识别LAPTM4B-35蛋白的LAPTM4B-N_1-99多克隆抗体检测其表达,并与患者临床病理指标相联系。生存分析:Kaplan-Meier法统计生存率,Log-rank检验进行单因素分析,Cox检验进行多因素分析。第二部分LAPTM4B编码蛋白在胆囊癌细胞系GBC-SD中的表达免疫细胞化学染色:应用特异性识别LAPTM4B-35蛋白的LAPTM4B-N_1-99多克降抗体检测其在胆囊癌细胞系GBC-SD中的表达。免疫印迹:应用LAPTM4B-EC2多克隆抗体验证LAPTM4B编码蛋白在该细胞系中的表达。第三部分LAPTM4B基因在胆囊癌细胞中的作用机制质粒扩增及鉴定:大肠杆菌DH5α转化法扩增包含LAPTM4B完整开放阅读框架（ORF）的质粒pcDNA3-AE和空载质粒Mock（pcDNA3）,核酸内切酶BamH1和EcoR1双酶切及测序鉴定。质粒转染:以脂质体Lipofectamine^TM 2000将上述质粒瞬时转染至胆囊癌细胞系GBC-SD。MTT检测:检测转染和亲本细胞增殖状况。流式细胞术:检测转染和亲本细胞细胞周期和凋亡。Transwell实验:检测转染和亲本细胞的迁移和侵袭。过河实验:检测转染和亲本细胞的迁移。免疫印迹:应用多种特异性抗体检测相关蛋白在转染和亲本细胞中的表达。结果:第一部分:免疫印迹法检测的3对癌和癌旁组织中,LAPTM4B-35蛋白在2例患者的癌组织中阳性表达,而癌旁组织中均未见阳性条带。免疫组织化学染色发现LAPTM4B-35蛋白在57例（76%）胆囊癌患者的癌组织中阳性表达,其表达与肿瘤组织学类型、淋巴结累及、分期和分化程度以及患者预后密切相关。癌旁组织未见阳性染色。第二部分:免疫细胞化学染色显示LAPTM4B-35蛋白在胆囊癌细胞系GBC-SD中呈阳性表达,免疫印迹检测证实了这一发现但同时提示其表达较弱。第三部分:扩增后双酶切及测序鉴定表明扩增结果正确。转染pcDNA3-AE后细胞呈现明显增殖加速和细胞周期演进、迁移和侵袭增强及表阿霉素所诱导细胞凋亡的显著减轻。这些表型出现的同时伴有c-myc、c-fos、c-jun、cyclin D1、cyclin E、MMP-2、MMP-9、uPA、Bcl-2和Bcl-xL表达上调及P16、P27、Bax、Bid、剪切型caspase-3和剪切型caspase-9表达下调。然而,转染Mock质粒后及亲本细胞则无类似效应。结论:LAPTM4B-35蛋白在大多数胆囊癌患者中表达阳性并具有重要的临床病理和预后意义。LAPTM4B-35蛋白在胆囊癌细胞系GBC-SD中呈阳性表达。LAPTM4B基因可促进胆囊癌细胞增殖、迁移和侵袭并可抑制其凋亡。这些作用可通过调节其下游多种基因的表达而实现。因此,LAPTM4B基因在胆囊癌中具有重要意义并有可能作为新的治疗靶点。更多还原

【Abstract】 Background:Gallbladder carcinoma,which originated in epithelium of the gallbladder,carried concealed onset,rapid progression and dismal prognosis,thus seriously threatening the human health and life safety.In recent years,the increasing tendency of gallbladder carcinoma incidence in China further presented the necessity for thorough investigations on its pathogenesis and development.So far,domestic and abroad researchers have found that abnormal expression and mutation of many tumor associated genes,such as K-ras, P53,P27^（kip1）,cyclin D1 andβ-catenin,existed in gallbladder carcinoma cells. Nevertheless,expression and its clinicopathological and prognostic significances,and relevant mechanisms of LAPTM4B（lysosome-associated protein transmembrane 4β）,a novel tumor associated gene successfully cloned in hepatocellular carcinoma,remains unclear.Objective:The present study first,under the basis of previous relative literatures,explores the expression and clinical,pathological and prognostic significances,and further focusing on the effects and related molecular mechanisms of the gene on gallbladder carcinoma,in order to deepen the understanding on the pathogenesis and progression of gallbladder carcinoma and to probe into its new therapeutic targets.Methods:Part 1 Expression and clinicopathological and prognostic significances of LAPTM4B encoded proteins in gallbladder carcinomaWestern Blot:The expression of LAPTM4B encoded proteins is detected in paired cancerous and para-cancerous tissues,using polyclonal antibody LAPTM4B-EC2.Immunohistochemical staining:The expression of LAPTM4B-35 protein is detected by polyclonal antibody LAPTM4B-N_1-99,that specifically recognizes the protein,and is correlated with clinicopathological parameters of patients.Survival analysis:Survival rates are calculated by Kaplan-Meier method.Log-rank and Cox regression tests are adopted for uni-and multi-variate analyses.Part 2 Expression of LAPTM4B encoded proteins in gallbladder carcinoma cell line GBC-SDImmunocytochemical staining:The expression of LAPTM4B-35 protein is detected by polyclonal antibody LAPTM4B-N_1-99,that specifically recognizes the protein,in gallbladder carcinoma cell line GBC-SD.Western Blot:The expression of LAPTM4B encoded proteins in gallbladder carcinoma cell line GBC-SD is confirmed using polyclonal antibody LAPTM4B-EC2.Part 3 Mechanisms of LAPTM4B gene in gallbladder carcinoma cellsPlasmid amplification and identification:The plasmids pcDNA3-AE containing the complete open reading frame（ORF） of LAPTM4B and Mock（pcDNA3） are transformed into E.Coli DH5αand further amplified.The products are identified by digestion of endonucleases,BamH1 and EcoR1,and DNA sequencing.Plasmid transfection:Aforementioned plasmids are trainsiently transfected into gallbladder carcinoma cell line GBC-SD by liposome Lipofectamine^TM 2000.MTT assay:To detect proliferation of transfected and parent cells.Flow cytometry:To detect cell cycle distribution and apoptosis of transfected and parent cells.Transwell test:To detect migration and invasion of transfected and parent cells.Crossing river test:To detect migration of transfected and parent cells.Western Blot:To detect expression of related proteins in transfected and parent cells, using many kinds of specific antibody.Results:Part 1:LAPTM4B-35 protein is positively expressed in cancerous tissues of 2 patients out of 3 pairs of cancerous and para-cancerous tissues detected by Western Blot,whereas no any band is observable in para-cancerous tissues.Immunohistochemical staining shows that LAPTM4B-35 protein is positively expressed in cancerous tissues of 57（76%） patients with gallbladder carcinoma.Besides,its expression is closely correlated with histological type,lymph node involvement,staging and differentiation of the tumor,as well as patient prognosis.No positive staining is found in para-cancerous tissues.Part 2:Immunocytochemical staining reveals that LAPTM4B-35 protein is positively expressed in gallbladder carcinoma cell line GBC-SD.Western blot detection confirms this finding but simultaneously shows that its expression is a bit weak.Part 3:Digestion of two endonucleases and DNA sequencing indicate correct amplification.Cells transfected with pcDNA3-AE shows significantly accelerated proliferation and cell cycle progression,enhanced migration and invasion,and attenuated apoptosis induced by epirubicin.These phenotypes are accompanied by upregulated expression of c-myc,c-fos,c-jun,cyclin D1,cyclin E,MMP-2,MMP-9,uPA,Bcl-2 and Bcl-xL,and downregulated expression of P16,P27,Bax,Bid,cleaved caspase-3 and cleaved caspase-9.However,no similar effects are presented in cells transfected with the Mock plasmid and parent cells.Conclusions:LAPTM4B-35 protein is positively expressed in a majority of patients with gallbladder carcinoma and is of important clinicopathological and prognostic significances.LAPTM4B-35 protein is positively expressed in gallbladder carcinoma cell line GBC-SD.LAPTM4B gene promotes proliferation,migration and invasion,and inhibits apoptosis of gallbladder carcinoma cells.These effects are achieved via regulation of expression of multiple downstream genes.Therefore,LAPTM4B gene is of important implications in gallbladder carcinoma and may be a novel therapeutic target.更多还原