【作者】 宋小妹；

【导师】 张庆瑜；

【作者基本信息】 天津医科大学 ， 内科学， 2008， 硕士

【摘要】 胃癌是消化系统最常见的恶性肿瘤,死亡率居消化道各类癌症死亡率之首。研究其发病机理、探索有效治疗方法一直是医学研究最重要的课题之一。随着肿瘤生物学和分子遗传学理论与技术的发展,目前已认识到,胃癌是一种多基因异常疾病,和人体其他部位的肿瘤一样,胃癌发生发展主要是原癌基因的激活与抑癌基因的失活并由此产生一系列基因异常和动态演进的结果,因此寻找与胃癌发病相关的基因,深入了解胃癌的分子病理,在此基础上开拓胃癌治疗的新策略和靶点,成为肿瘤研究领域的一项重要内容。IA型PI3K/Akt通路与肿瘤的发生发展密切相关。该通路调节肿瘤细胞的增殖和存活,其活性增加不仅能导致细胞恶性转化,而且与肿瘤细胞的迁移、黏附、肿瘤血管生成以及细胞外基质的降解等相关。本课题重点研究了胃腺癌中IA型PI3K/Akt通路的异常表达,并应用RNA干涉技术干预该通路中PI3Kp85α的异常激活,观察干预PI3Kp85α后对胃腺癌恶性表型的影响及对裸鼠皮下SGC7901胃腺癌模型的治疗效果。课题分三部分进行:第一部分应用组织芯片技术结合免疫组化检测IA型PI3K/Akt通路主要成员PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中的表达情况。第二部分在体外应用RNA干涉技术以oligofectamine介导siRNA靶向PI3Kp85α转染人胃腺癌细胞系SGC7901后,应用realtime PCR检测目的基因PI3Kp85α的敲低情况,并用Western blot和免疫荧光技术检测了RNAi敲低PI3Kp85α的表达后,IA型PI3K/Akt通路主要成员PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2的表达变化:用MTT、annexin V标记、流式细胞术分析了转染前后细胞增殖、凋亡、细胞周期分布的变化:“划痕”法迁移实验和2-D Matrigel法研究细胞运动迁移能力:用Transwell和3-D Mattigel法分析转染前后细胞侵袭能力的变化。第三部分建立裸鼠胃腺癌皮下动物模型,每4天瘤内注射oligofectamine和siRNA的混合物一次并测量肿瘤体积,动态观察肿瘤生长情况,治疗32天后切除肿瘤。用免疫组化法检测RNA干涉技术敲低SGC7901裸鼠胃腺癌PI3Kp85α的表达后该通路主要成员的表达变化,并应用TUNEL法检测细胞凋亡,对体外实验的结果进一步验证。结果:第一部分组织芯片结合免疫组化技术显示IA型PI3K/Akt通路中PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中均呈高表达,其表达水平随肿瘤的演进程度及恶性级别增高而增强。第二部分Realtime PCR结果提示,转染PI3Kp85αsiRNA后可明显敲低染PI3Kp85αmRNA的表达。Western blot和免疫荧光检测发现除PI3Kp85α的蛋白明显敲低外,IA型PI3K/Akt通路中pAkt、Ki67、Bcl-2、MMP-2的表达水平均明显降低。MTT结果显示PI3Kp85αsiRNA组与转染nonsense siRNA组及对照组相比细胞增殖率明显受抑(P＜0.01);Annexin V检测显示PI3Kp85αsiRNA组细胞凋亡率明显升高,进一步应用流式细胞术检测细胞周期分布发现S期细胞比例降低,细胞阻滞于G0/G1期;“划痕”法迁移实验和2-D Matrigel法显示PI3Kp85αsiRNA组较nonsense siRNA组和对照组的细胞的运动迁移能力降低(P＜0.01);Transwell和3-D Matrigel法显示其侵袭能力减弱(P＜0.01)。第三部分成功建立SGC7901胃腺癌细胞系裸鼠皮下肿瘤后,每4天测量一次肿瘤体积,并在肿瘤局部多点注射siRNA和oligofectamine混合物,发现和对照组相比,PI3Kp85αsiRNA治疗组肿瘤生长缓慢,从治疗后的第20天起,肿瘤体积出现明显差别,这种差别在观察末期(32天)最显著。应用免疫组化法检测肿瘤标本,发现PI3Kp85α的蛋白明显敲低的同时,IA型PI3K/Akt通路中pAkt、Ki67、Bcl-2、MMP-2的表达水平均明显降低。应用TUNEL法检测原位细胞凋亡发现PI3Kp85α治疗组凋亡细胞明显增加。体内和体外实验的结果是一致的。结论:1.免疫组化显示PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中都有过表达,表明IA型PI3K/Akt信号通路在胃腺癌中是激活的,从而导致细胞增殖和侵袭。其中各种蛋白的表达相互关联也验证了这一点。2.应用RNA干涉技术以Oligofectamine介导siRNA靶向PI3Kp85α可有效敲低这个基因在人胃腺癌SGC7901细胞内的表达,同时抑制IA型PI3K/Akt通路的活性,从而抑制了细胞的增殖和侵袭,并诱发细胞凋亡。3.裸鼠皮下胃腺癌模型应用oligofectamine介导的PI3Kp85αsiRNA治疗,同样可有效敲低PI3Kp85α的表达,抑制IA型PI3K/Akt通路的活性,从而抑制肿瘤的生长,诱导细胞凋亡,与体外试验结果一致。4.通过RNA干涉技术敲低PI3Kp85α的表达对胃腺癌的治疗是有效的。PI3Kp85α可成为胃腺癌基因治疗的候选基因,具有潜在的临床应用前景。更多还原

【Abstract】 Gastric carcinoma is the most common and incurable digestive systerm tumor,which mortality is the highest.Studying its pathogenesis and trying to find more effective therapeutic modalities are the major research projects in medical field. Gastric carcinoma,just like tumors in other sites of the body,are involved in the processes of uncontrolled cell proliferation,cell dedifferentiation and dysregulation of cell apoptosis.With the development of molecular biology and molecular genetics,it has been found that some of the important oncogenes and tumor suppressor genes are related to the genesis of gastric carcinoma.However,up to date,it is still not clear which are the primary initiating molecular events and which are the secondary promoting molecular events.Therefore,seeking more genes associated with the genesis of gastric carcinoma,a comprehensive understanding of the molecular pathology of gastric carcinoma,and optimizing treatment strategies and developing novel therapeutic approaches for gastric carcinoma on this basis,have become an important research project for carcinoma.IA Type PI3K/Akt signaling pathway which regulates the proliferation and survival of tumor cells is closely related to the genesis and development of gastric adenocarcinoma.Its activity abnormality not only can induce cells malignant transfor -mation,but also influence the migration and adhesion of tumor cells,vascularization and degradation of extracellular matrix.The present study focused on the abnormal IA Type PI3K/Akt signaling pathway activity in gastric adenocarcinoma and siRNA targeting PI3Kp85αwas used to observe its inhibitory effect on the growth of human gastric adenocarcinoma SGC7901 cells and therapeutic efficacy of nude mice subcutaneous SGC7901 gastric adenocarcinoma model.The study was divided into 3 parts.In the first part,Using tissue microarray techniques and immunohistochemistry assay to survey the expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 in IA type PI3K/Akt signaling pathway during gastric adenocarcinoma malignant progression. In the second part,RNAi technology was used to observe its inhibitory effect on the growth of human gastric adenocarcinoma SGC7901 cells,siRNA targeting PI3Kp85αwas transfected into SGC7901 cells mediated by oligofectamine. PI3Kp85αmRNA expression were detected by realtime PCR after transfection.The expression of PI3Kp85αand other main downstream members including pAkt,Ki67, bcl-2,MMP-2 in IA type PI3K/Akt pathway was also studied by Western blotting and immunofluorescence staining after transfection.The phenotypic change of SGC7901 cells including proliferation,apoptosis and cell cycle after RNAi trasfection was studied by MTT assay,annexin V staining,flow cytometry;their motion and migra -tion ability was studied by scratch assay and 2-D Matrigel assay;the invasion ability was detected by Transwell analysis and 3-D Matrigel assay.In the third part,subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice.Every 4 days the mixture of oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured.On 32^ndday after the first injection,tumors were resected.The expression of PI3Kp85α,pAkt,Ki67,Bcl-2, MMP-2 in IA type PI3K/Akt signaling pathway were studied by immunohistochemis -try.Apoptosis in tumors were detected by TUNEL method.Results:The first part:The expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 in IA type PI3K/Akt signaling pathway were overexpressed during gastric adenocarcinoma malignant progression.The second part:SGC7901 cells were transfected with siRNA targeting PI3Kp85αmediated by oligofectamine in vitro.SGC7901 mRNA expression were obviously knocked down after transfection with siRNA.SGC7901 ceils transfected with siRNA targeting PI3Kp85αshowed lowering proliferation activity by MTT, while the expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 were downregulated by Western blot analysis and immunohistochemical staining.The transfected cells had higher apotosis rate by annexin V staining and most cells arresting in the Go/G1phase by flow cytometry.The migration and invasive ability was attenuated by scratch assay,2-D Matrigel assay,Transwell analysis and 3-D Matrigel assay.The third part:Subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice for in vivo study.As compared with the control group,the tumors in mice treated with siRNA targeting PI3Kp85αgrew slowly and the different ce of tumor volumes became significant since the 20th day after the first time of siRNA therapy until the 32^ndday tumors were resected.PI3Kp85α,pAkt,Ki67, Bcl-2,MMP-2’s expression were downregulated by immunohistochemistry. Meanwhile,cell apoptosis was increased by TUNEL method.Conclusion:1.PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2’s overexpression can lead to IA type PI3K/Akt signaling pathway activation in gastric adenocarcinoma and result in cell proliferation and invasion.Meanwhile,the several proteins in this signaling pathway links to each other and maybe they are modulated with each other.2.Using RNAi technology,siRNA targeting PI3Kp85αmediated by oligofectamine efficiently knocks down the expression of PI3Kp85αin human gastric adenocarcinoma SGC7901 cells,inhibits activation of IA type PI3K/Akt signaling activity,results in decrease of cell proliferation activity and attenuateion of invasive and migration ability,and induces apoptosis.3.The established subcutaneous SGC7901 gastric adenocarcinoma models in nude mice are treated with siRNA targeting PI3Kp85α.The tumor growth is inhibited and cell apoptosis is induced.The findings of in vivo study is in accordance with those in vitro study4.Using RNAi technology to knock down the expression of PI3Kp85αin gastric adenocarcinoma is efficient in gastric adenocarcinoma therapy.PI3Kp85αcan be a candidate gene for gene therapy of gastric adenocarcinoma,which will have potential application prospects in clinics.更多还原