【作者】 王强；

【导师】 戴显伟；

【作者基本信息】 中国医科大学 ， 外科学， 2009， 博士

【摘要】 胃肠道间质瘤PI3K/Akt信号转导通路的检测及其意义前言胃肠道间质瘤（Gastrointestinal stromal tumor,GIST）是胃肠道最常见的间叶源性肿瘤,占胃肠道肿瘤的1%-4%,每年发病率约2/10万。本病无明显性别、种族差异,可发生于消化道的任何部位,最常见的原发部位是胃和小肠,其次是结直肠和食管,还可以发生于网膜、系膜和后腹膜等胃肠道外部位。过去对GIST的组织发生、生物学行为、肿瘤的恶性程度都不十分明确,这就给临床诊治带来了很多混淆。近几年随着免疫组织化学、分子生物学、基因诊断和治疗的发展,以及甲磺酸伊马替尼（Glivec）的出现,GIST诊断与治疗取得了很大的进展。尽管如此,与其它肿瘤相比,GIST肿瘤性质不可预测性仍较为突出,其生物学特征仍较难以评估,如何控制肿瘤的侵袭与转移仍是目前治疗所面临的主要问题。肿瘤的发生涉及到多种细胞事件及生理过程,其中细胞信号传导通路的异常是细胞癌变的主要原因之一。磷脂酰肌醇3-激酶（PI3K）信号参与增值、分化、凋亡和葡萄糖转运等多种细胞功能的调节。近年来发现,IA型PI3K和其下游分子蛋白激酶B（PKB或Akt）所组成的信号通路在肿瘤的发生、发展、治疗及转归中发挥重要作用,并且已经成为肿瘤治疗的新靶点。进一步探讨该通路的调节以及与其他通路的交联,提高对该通路在人类各种常见肿瘤中的作用的认识,有助于更好地理解肿瘤细胞的恶性行为。该通路调节肿瘤细胞的增殖和存活,其活性异常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。PTEN是一种肿瘤抑制基因,PTEN基因的突变,失去了对细胞生长的负调控,可以激活PI3K/Akt途径,导致肿瘤发生。PTEN是最易发生突变的靶点基因之一,其突变频率与p53相当。PI3K作为联系胞外信号与细胞应答效应的桥梁分子,并不是完全独立的,而是在一系列上游或旁路信号分子的影响下,作用于下游的信号分子对细胞的凋亡起非常重要的调节作用。在许多研究中已经证实PI3K和Akt的抗凋亡作用,Akt主要作用于抗凋亡通路,近年来研究表明,PI3K/Akt能通过多种途径抑制细胞凋亡,促进细胞存活。主要通过直接或间接影响转录因子家族（Forkhead、NF-κB、p53等）发挥细胞存活调控作用。肿瘤血管生成是肿瘤发生转移的必要条件,PI3K/Ak信号转导通路参与了多种因子所介导的肿瘤血管生成过程。HIF-1作为肿瘤微环境中的一个至关重要的调节因子,在促进肿瘤血管生成和肿瘤的侵袭性方面起中心作用。HIF-1发挥作用主要是由HIF-1α亚基的表达和活性决定的。PI3K/Akt信号途径可以通过抑癌基因PTEN的调控来调节HIF-1α蛋白的表达,进而促进缺氧反应基因的转录,引起细胞对缺氧的一系列适应性反应。参与肿瘤细胞的代谢、血管扩张、新血管形成等。肿瘤的侵袭、转移是一个复杂多步骤的肿瘤细胞与宿主细胞及基质间相互作用的过程,侵袭和转移的程度是影响预后的主要因素,但目前仍然不清楚GIST发生发展和浸润转移的具体分子机制。近年来在许多肿瘤中发现,PI3K/AKt信号通路不断被激活,其功能渗及肿瘤细胞生存、增殖、迁移、侵袭和转移等方面。转化生长因子β（TGF-β）能以剂量和时间依赖的方式上调uPAmRNA和蛋白的表达,促进转移,而其上调uPA的作用也主要是通过PI3K/Akt/NF-κB通路所介导的。目前已经证实,uPA是NF-κB的靶基因,因此推测PI3K-Akt途径的活化导致NF-κB的上调,后者作用于其靶基因uPA导致肿瘤的转移。NF-KB的持续活化可能是促进uPA的合成主要因素之一,即uPA主要受NF-KB的调控,进而参与肿瘤的侵袭、转移。近年来,关于PI3K/Akt信号转导通路的报道日益增多,但是对于研究有关PI3K/Akt信号转导通路在GIST中系统的表达变化的报道仍然很少。本实验中我们通过对GIST组织PI3K/Akt信号通路中PI3K、Akt和PTEN的mRNA水平以及蛋白表达变化的检测,同时进一步讨论探讨PI3K/Akt信号转导通路激活后,其下游的细胞凋亡相关因子、促血管生成因子以及侵袭转移相关因子的表达变化,探讨其表达与GIST临床病理特征及预后的相关性,并探讨它们间的相互关系,以此揭示其在GIST发生发展过程中的作用,为进一步的肿瘤靶点治疗提供理论依据。材料与方法1、研究对象选择临床病理资料完整、随访结果确切的中国医科大学附属盛京医院1990年～2007年2月手术切除、病理证实的GIST新鲜组织标本124例。男性64例、女性60例,年龄9～79岁、平均54.6岁。肿瘤发生于胃62例（50.0%）、小肠28例（22.6%）、结直肠14例（11.3%）、食管9例（7.3%）、胃肠道外11例（8.9%）（其中网膜4例、肠系膜4例、后腹膜3例）。GIST蜡块组织标本124例。男性64例、女性60例,年龄9～79岁、平均54.6岁。肿瘤发生于胃62例（50.0%）、小肠28例（22.6%）、结直肠14例（11.3%）、食管9例（7.3%）、胃肠道外11例（8.9%）（其中网膜4例、肠系膜4例、后腹膜3例）。2、相关分子生物学试剂PTEN（1 0P01）:sc-73420;P-Akt1/2/3（Thr 308）-R:sc-1 6646-R;P-PI3-kinasep85α（Tyr 508）:sc-12929;HIF-1α（3C144）:sc-71247;NOS₂（C-1 1）:sc-7271;VEGF（C-1）:sc-7269;uPA（H-140）:sc-14019;MMP-2（8B4）:sc-13595;MMP-9（M-17）:sc-6841;TGF-β₁（500-M66）:sc-65378（Santa Cruz）;HRP标志的第二抗体（北京中杉公司）;硝酸纤维素膜（Takara公司）;RT-PCR试剂盒（Takara公司）;DAB显色底物（Takara公司）;DNA分子量标准（Takara公司）;PCR引物（Takara公司）;其他试剂为国产分析纯。3、RT-PCR检测相关因子在GIST及对照组的mRNA水平mRNA提取按QuickPrep micro mRNA Purification试剂盒说明书进行:主要包括:样品的准备,mRNA的分离,分别用高盐缓冲液和低盐缓冲液对已结合mRNA的oligo（dT）纤维颗粒的洗涤,mRNA的洗脱。用TaKaRa RNA PCR（AMV）V3.0试剂盒,按操作步骤进行反转录和PCR。反应产物经琼脂糖凝胶电泳后成像。4、Western印迹法检测相关因子在GIST及对照组的表达0.5g GIST加入1ml细胞裂解缓冲液（Cell signaling Technology,Beverly,MA）及1mmol的PMSF（苯甲基磺酰氟）匀浆。按常规步骤提取蛋白,测定蛋白浓度。电泳前,加入SDS样品缓冲液,100℃煮沸5 min,离心后上样,12%SDS-PAGE分离,然后将蛋白转至硝酸纤维素膜上,一抗孵育,碱性磷酸酶偶联的IgG作为二抗,O-dianidine及β-naphthyl acid phosphate作为碱性磷酸酶底物显色。5、免疫组织化学检测HIF-1α、NOS2、VEGF在GIST及对照组的表达取GIST组织经中性福尔马林固定、石蜡包埋后切成4μm薄片至玻片。玻片常规脱蜡,滴加1滴或50ul封闭血清,倾去血清,每张切片滴加1滴或50ul一抗,孵育后洗脱,然后每张切片滴加1滴或50ul Envision^TM二抗孵育,用新鲜配制的Envision^TM显色底物DAB液显色,1～2分钟。实验结果1、PI3K、Akt、PTEN在GIST及对照组mRNA和蛋白水平变化及相关性分析我们发现PI3K mRNA水平随NIH分级表达上调（P＜0.05）,PI3 K的阳性表达率与性别、年龄、组织学类型无关,但与黏膜受侵和肿瘤侵袭转移均显著相关（P＜0.05）,表明PI3K的表达与GIST细胞的转化过程以及肿瘤细胞的侵袭转移有关,因此PI3K的产物能调节细胞生长和凋亡。同时我们也发现AKt mRNA水平随NIH分级表达上调,其中在高、中危肿瘤组之间不明显,无统计学意义,其余各组有意义;Akt的阳性表达率与性别、年龄、组织学类型无关,与肿瘤侵袭转移和黏膜受侵显著相关,提示Akt的过表达促进了GIST的肿瘤侵袭转移。我们同时发现PTEN mRNA随NIH分级表达下调（P＜0.05）,随着肿瘤恶性程度的增加,PTEN的缺失率增高;PTEN的阳性表达率与性别、年龄、组织学类型无关,与肿瘤侵袭转移和黏膜受侵关系密切。进一步提示PTEN蛋白的缺失与GIST发生发展的关系密切。Western Blot实验的结果显示,在各指标蛋白水平的表达变化与mRNA水平基本一致。然而,Akt蛋白在GIST组织中的表达水平明显增高。这是由于在GIST组织中Akt蛋白表达增高是蛋白翻译和（或）翻译后水平而非基因转录水平上调的结果。本研究的124例GIST组织中,PI3K、Akt蛋白阳性表达之间呈显著正相关（r=0.292,P=0.03 1）;PI3K与PTEN蛋白阳性表达之间呈显著负相关（r=-0.412,P=0.036）;PTEN与Akt蛋白阳性表达之间呈显著负相关（r=-0.386,P=0.024）。可见PI3K下游的主要作用底物是AKt,可通过其正性调节来进一步激活AKt,活化的AKt可以通过磷酸化作用激活或抑制其下游靶蛋白,进而调节细胞的增殖、分化、凋亡以及迁移等。而PTEN与二者的表达呈负相关,表明PI3K/Akt与PTEN在GIST的发生发展中可能互相拮抗。在预后分析中,我们发现:PI3K、AKt与无瘤生存期负相关,PTEN、AKt与无瘤生存期负相关,PI3K高表达或AKt高表达或PTEN低表达患者无瘤生存期短。2、NF-κB、XIAP、bcl-2、survivin在GIST及对照组的mRNA水平变化及相关性分析NF-κB mRNA的表达变化与NIH分级,以及肿瘤的肿瘤侵袭转移、黏膜受侵密切相关,而与年龄、性别、组织学类型无关。XIAP mRNA随NIH分级表达上调;XIAP表达与GIST组织分化程度呈负相关,即分化程度越低,XIAP阳性表达率愈高,伴有肿瘤侵袭转移的GIST病例XIAP表达水平较高,同时XIAP的表达与黏膜受侵密切相关,而与年龄、性别、组织学类型无关。bcl-2 mRNA水平表达上调,与NIH分级、黏膜受侵有关,而与年龄、性别、组织学类型、转移复发及周围组织浸润无关。Survivin mRNA在GISI中的表达与NIH分级、肿瘤侵袭转移、黏膜受侵等因素有关,而其表达与年龄、性别以及组织学类型无关。由于NF-κB的调控作用,各指标之间mRNA的水平变化呈正相关。3、HIF-1α、NOS2、VEGF在GIST及对照组的表达及相关性分析HIF-1α阳性表达的黄色颗粒或棕黄色颗粒见于细胞核及胞浆中,正常GIST组织中HIF-1α的表达较弱。本组124例患者中（-）46例,（+）7例,（++）31例,（+++）28例,（++++）13例;总阳性表达率为63.7%（79/124）。HIF-1α的表达与GIST的NIH分级、肿瘤黏膜受侵及侵袭转移关系密切。Ⅰ、Ⅱ级与Ⅲ、Ⅳ级患者的HIF-1α的阳性率分别为46.2%、68.4%（P=0.006）;肿瘤黏膜受侵和无受侵组的患者HIF-1α阳性率分别为88.2%、54.4%（P=0.017）;有侵袭转移组的阳性率明显高于无侵袭转移组,阳性率分别为90.0%和51.2%,（P=0.026）;HIF-1α的表达未发现与其他临床病理特征有明显的相关性。NOS₂主要表达于细胞胞浆中,呈颗粒状分布。124例GIST中,NOS₂呈高表达状态（64.5%）,其阳性表达率明显高于正常GIST组织（17.7%）,差异有显著性（P＜0.05）（图2）。由表1可见:NOS₂的表达与GIST的NIH分级、肿瘤黏膜受侵及侵袭转移关系密切。Ⅰ、Ⅱ级与Ⅲ、Ⅳ级患者的NOS₂的阳性率分别为42.3%、70.4%（P=0.039）;肿瘤黏膜受侵和无受侵组的患者NOS₂阳性率分别为88.2%、55.7%（P=0.003）;有侵袭转移组的阳性率明显高于无侵袭转移组,阳性率分别为95.0%和50.0%（P=0.042）;NOS₂的表达未发现与其他临床病理特征有明显的相关性。结果提示,NOS₂可能参与了GIST发生发展形成的整个过程,为GIST发生发展中的危险因素之一,同时亦可能是判断GIST预后的一个可靠指标。VEGF阳性染色主要位于GIST细胞浆中,呈弥漫分布。VEGF蛋白的阳性表达率为58.1%（72/124）,阳性信号定位于GIST细胞胞浆和胞膜。VEGF的表达与GIST的NIH分级、肿瘤黏膜受侵及肿瘤侵袭转移关系密切。Ⅰ、Ⅱ级与Ⅲ、Ⅳ级患者的VEGF的阳性率分别为46.2%、61.2%（P=0.008）;肿瘤黏膜受侵和无受侵组的患者VEGF阳性率分别为79.4%、50.6%（P=0.006）;有侵袭转移组的阳性率明显高于无侵袭转移组,阳性率分别为80.0%和47.6%,（P=0.015）;VEGF的表达未发现与其他临床病理特征有明显的相关性。在GIST组织、肿瘤旁组织内可见血管内皮细胞胞质呈棕黄色染色,124例GIST组织中MVD值（23.15±8.92）高于肿瘤旁组织（8.3±2.76）,两组之间差别具有显著性（P＜0.05）,这说明GIST组织中血管生成较肿瘤旁组织活跃。CD117阳性定位于细胞膜,CD34阳性定位于细胞质,均呈棕黄色。本组CD117阳性表达率为91.1%（113/124）,CD34表达率为87.9%（109/124）,CD117和CD34共同表达率为80.6%（100/124）,二者均阴性的5例。CD117、CD34阳性表达率与性别、年龄、NIH分级、侵袭转移、黏膜受侵及组织学类型无关,其差异无显著意义（P＞0.05,见表1）。由表1可见,HIF-1α、NOS₂、和VEGF表达阳性者的MVD高于表达阴性者（P＜0.05）。且MVD与GIST的NIH分级、肿瘤黏膜受侵及侵袭转移关系密切,未发现与其他临床病理特征有明显的相关性。HIF-1α与NOS₂蛋白阳性表达之间呈显著正相关（r=0.392,P=0.026）;HIF-1α与VEGF蛋白阳性表达之间呈显著正相关（r=0.368,P=0.034）;NOS₂和VEGF蛋白阳性表达之间呈显著正相关（r=0.452,P=0.021）。4、uPA、MMP-2、MMP-9、TGF-β1在GIST及对照组的mRNA和蛋白水平变化及相关性分析在124例GIST中,uPA、MMP-2、MMP-9、TGF-β₁mRNA的表达与年龄、性别及组织学类型无显著关系。uPA、MMP-2、MMP-9、TGF-β₁mRNA的表达与肿瘤肿瘤侵袭转移、NIH分级及黏膜受侵有显著相关（P＜0.05）。各指标蛋白表达水平与mRNA水平基本一致。TGF-β₁可以介导PI3K/Akt信号转导通路上调uPA,二者表达呈正相关（r=0.356,P=0.028）,uPA和MMP-2之间表达呈明显正相关（r=0.323,P=0.035）,MMP-2与MMP-9的表达呈正相关（r=0.495,P=0.042）,提示两者在GIST的发生发展过程中的作用密切相关。讨论PI3K/Akt信号通路在广泛的人类肿瘤谱中失调,该通路某些成分的突变所导致的功能获得或功能缺失能够引起细胞转化,这条通路调节肿瘤细胞的增殖和存活,AKT是信号转导途径中的重要蛋白激酶,它是PI3K下游作用的靶蛋白,是PI3K/AKT信号转导通路的核心,PTEN是迄今发现的第一个具有磷酸酶活性的抑癌基因,可以通过控制活化的磷脂酰3,4,5-三磷酸肌醇（PIP3）来调控AKt的活性。因此,PTEN的突变使其丧失了调节AKt的能力,使细胞增殖失去控制,从而引起癌变。PTEN可以去磷酸化PIP3,拮抗PI3K的活性,降低细胞内PIP3的浓度,从而抑制AKt的激活。PI3K/AKt信号通路对于细胞增殖、分化和凋亡的调节是必要的。在GIST中,随其NIH分级PI3K、AKt表达上调,而抑癌基因PTEN的表达下调。三者阳性表达率与性别、年龄、组织学类型无关,与肿瘤侵袭转移和黏膜受侵关系密切。PI3K/Akt与PTEN在GIST的发生发展中可能互相拮抗共同作用。通过观察在GIST病人的蜡块组织标本中NF-κB以及凋亡相关因子XIAP、bcl-2以及survivin mRNA水平的变化,来探讨GIST中PI3K-Akt信号转导通路下游细胞凋亡相关因子的表达情况及其与GIST临床病理特征及预后的相关性,在GIST中,随着肿瘤恶性度的增加,PI3K、AKt的活性增高,活化的AKt可以直接磷酸化IKKa,使IKK激活,调节NF-κB活性。AKt是NF-κB依赖的基因转录的重要调节因素,对刺激癌细胞的生长有重要作用。GIST中PI3K/Akt信号转导通路的活化可以进一步激活NF-κB mRNA,从而进入细胞核内调控凋亡相关因子XIAP、bcl-2以及survivin mRNA水平的表达。NF-κB mRNA的表达变化与NIH分级,以及肿瘤的侵袭转移、黏膜受侵密切相关,而与年龄、性别、组织学类型无关。XIAP mRNA随NIH分级表达上调;XIAP表达与GIST组织分化程度呈负相关,即分化程度越低,XIAP阳性表达率愈高,伴有肿瘤侵袭转移的GIST病例XIAP表达水平较高,同时XIAP的表达与黏膜受侵密切相关,而与年龄、性别、组织学类型无关。bcl-2 mRNA水平表达上调,与NIH分级、黏膜受侵有关,而与年龄、性别、组织学类型、转移复发及周围组织浸润无关。Survivin mRNA在GIST中的表达与NIH分级、肿瘤侵袭转移、黏膜受侵等因素有关,而其表达与年龄、性别以及组织学类型无关。由于NF-κB的调控作用,各指标之间mRNA的水平变化呈正相关。肿瘤血管生成是肿瘤发生转移的必要条件,PI3K/Ak信号转导通路参与了多种因子所介导的肿瘤血管生成过程。HIF-1作为肿瘤微环境中的一个至关重要的调节因子,在促进肿瘤血管生成和肿瘤的侵袭性方面起中心作用。PI3K/Akt信号途径可以通过抑癌基因PTEN的调控来调节HIF-1α蛋白的表达,而AKt的活化可以增强HIF-1α的稳定性,进而促进缺氧反应基因的转录,引起细胞对缺氧的一系列适应性反应。VEGF可能通过对血管生成的调节肿瘤的发生、发展和转移中起着重要的作用。在许多实体肿瘤中VEGF的表达与肿瘤新生血管形成呈高度相关性。VEGF与肿瘤的生长、转移和预后密切相关,因此,以VEGF为靶点,抑制其表达,从而抑制肿瘤血管形成,阻断肿瘤的营养来源和转移通道,达到治疗肿瘤的目的,已成为近年来肿瘤治疗的新策略。VEGF可能通过PI3K/AKt途径激活并诱导NO合成,HIF-1α、NOS₂和VEGF三者之间可以通过PI3K/Ak信号转导通路相互调节、共同参与GIST的发生发展。因此,我们认为HIF-1α、NOS₂和VEGF表达可能是恶性肿瘤生长、浸润、转移的重要指标。它们可以作为靶点,抑制其表达,从而抑制肿瘤血管形成,阻断肿瘤的营养来源和转移通道,达到治疗肿瘤的目的。PI3K/AKt信号转导通路的激活可以活化HIF-1α,HIF-1α、NOS₂和VEGF以及MVD的表达与GIST的NIH分级、肿瘤黏膜受侵及肿瘤侵袭转移关系密切,HIF-1α、NOS₂和VEGF表达可能是恶性肿瘤生长、侵袭、转移的重要指标。肿瘤的侵袭、转移是一个复杂多步骤的肿瘤细胞与宿主细胞及基质间相互作用的过程,侵袭和转移的程度是影响预后的主要因素,TGF-β₁存在于肿瘤和某些正常细胞内,能调节正常细胞和转化后细胞的生长和分化。TGF-β₁ mRNA表达水平高的病灶具有较强的纵深浸润和广泛转移的倾向和能力,TGF-β₁本身还可作为一种运动因子,促进肿瘤细胞的运动和迁移。FGF-β₁可以介导PI3K/Akt信号转导通路上调uPA,uPA受PI3K/Akt信号转导通路介导的TGF-β₁调节,进一步激活MMP-2,uPA和MMP-2之间表达呈明显正相关。MMP-2同时也具有激活MMP-9的功能。提示两者在GIST的发生发展过程中的作用密切相关,可能通过协同作用促进肿瘤的发生、肿瘤细胞外基质的降解和肿瘤细胞的侵袭转移。本研究显示,TGF-β₁、uPA、MMP-2及MMP-9的表达与性别、年龄、组织类型不相关,而与肿瘤黏膜受侵、NIH分级、肿瘤侵袭转移相关,提示TGF-β₁、uPA、MMP-2及MMP-9参与了肿瘤的浸润和转移。结论1、在GIST中,随其NIH分级PI3K、AKt表达上调,而抑癌基因PTEN的表达下调。三者阳性表达率与性别、年龄、组织学类型无关,与肿瘤侵袭转移和黏膜受侵关系密切。PI3K/Akt与PTEN在GIST的发生发展中可能互相拮抗共同作用,PI3K、Akt蛋白阳性表达之间呈显著正相关（r=0.292,P=0.031）;PI3K与PTEN蛋白阳性表达之间呈显著负相关（r=-0.412,P=0.036）;PTEN与Akt蛋白阳性表达之间呈显著负相关（r=-0.386,P=0.024）。PI3K高表达或AKt高表达患者无瘤生存期显著缩短,两者均高表达,预后更差;PTEN低表达患者无瘤生存期短,PTEN低表达且AKt高表达,PTEN低表达且PI3K高表达无瘤生存期显著缩短。2、GIST中PI3K/Akt信号转导通路的活化可以进一步激活NF-κB mRNA,从而进入细胞核内调控凋亡相关因子XIAP、bcl-2以及survivin mRNA水平的表达。NF-κB、XIAP、bcl-2以及survivin mRNA的表达变化与NIH分级,以及肿瘤的肿瘤侵袭转移、黏膜受侵密切相关,而与年龄、性别、组织学类型无关。由于NF-κB的调控作用,各指标之间mRNA的水平变化呈正相关。3、在血运丰富的GIST中,PI3K/AKt信号转导通路的激活可以活化HIF-1α,HIF-1α、NOS₂和VEGF以及MVD的表达与GIST的NIH分级、肿瘤黏膜受侵及肿瘤侵袭转移关系密切,与其他临床病理特征没有有明显的相关性。我们的研究提示,HIF-1α、NOS₂和VEGF的表达呈显著正相关（r1=0.368,r2=0.452,r3=0.392,P1,P2,P3＜0.05）。由此认为,三者可能在GIST侵袭、转移及血管新生中有相互依赖的正调控作用,三者共同参与了GIST的发生发展。因此,我们认为HIF-1α、NOS₂和VEGF表达可能是恶性肿瘤生长、浸润、转移的重要指标。4、我们发现在GIST中TGF-β₁可以介导PI3K/Akt信号转导通路上调uPA,进而激活MMP-2及MMP-9,有效地降解细胞外基质。随着肿瘤浸润深度的加深,肿瘤侵袭转移程度的提高和NIH分级的进展,GIST肿瘤组织中TGF-β₁的表达逐渐升高。本研究显示,IGF-β₁、uPA、MMP-2及MMP-9的表达与性别、年龄、组织类型不相关,而与肿瘤黏膜受侵、NIH分级、淋巴转移远处转相关,提示TGF-β₁、uPA、MMP-2及MMP-9参与了肿瘤的浸润和转移。更多还原

【Abstract】 IntroductionGastrointestinal stromal tumors（GIST）are common mesenchymal tumors of the gastrointestinal tract with the incidence rate 2/100,000,which account gastrointestinal tract cancer of 1%-4%.There is no significant difference in race and gender.GIST appears in any part of the digestive tract,which commonly arise in the stomach and intestine.Colon/rectumi are the second predilection site,It also happens in the omentum,retroperitoneal mesangial and parenteral tract.On the past,the organization, biological behavior and the malignancy of GIST are not clear,which brings a lot of confusion to the clinical diagnosis and treatment.With the development of immunohistochemistry,molecular biology,gene engineering and the emergence of Imatinib mesylate（Glivec）,the diagnosis and treatment of GIST have made great progress in recent years.Although,Compared with other tumor,GIST is still unpredictabily,the biological characteristics of GIST are difficult to assess.how to control the invasion and metastasis is the main problems to treat GIST.Carcinogenesis are controlled by many cell incidences and Physiological processes,cell’s signal transduction pathway is one of important reasons.A lot of cell function such as Proliferation,differentiation,apoptosis and glucose transportation are regulated by Phosphatidylinositol 3-kinase（PI3K）.during passed few years,study showed IA type PI3K and cellueoprotein kinase B（PKB or Akt）were very important in generation,development,treatment and recurrence of cancer,it had became a new target for the treatment of cancer.The further study of this pathway regulation mechanism and the relationship with other pathway is conduced to increase the recognition of the effect in human normal tumor.It regulates cell proliferation and survive,the unusual activiting of PI3K/Akt pathway not only can induce cell carcinogenesis but also have relation with tumor cell immigration adhesion tumor blood vessels generation and extracellular matrix degradation.PTEN is a tumor suppressing gene,the mutation of PTEN can cause the lose of cell generation negative control and spring PI3K/Akt pathway witch induce carcinogenesis process.PTEN is one of the easiest mutation target gene,the rate is common to P53.PI3K as a bridge molecular combined the signal in vitro with cell response,witch is not completely independent,but have important regulating role in cell apoptosis through the effect that a series of upstream or bypassing signal factors act on the downstream signals.Many studies had showed the anti-apoptosis function of PI3K and Akt.Akt mainly acts in the anti-apoptosis pathway,PI3K/Akt can inhibit cell apoptosis and introduce cell survive through many way.It controlled cell survive through direct or indirect involved in the transcription factors such as Forkhead,NF-KB,P53 and so on.The angiogenesis of tumor is a requirement of the transference of tumor.PI3K/Akt signal path takes part in the tumor angiogenesis mediated by several factors.HIF-1, being a crucial regulatory factor,plays an Important role in the angiogenesis and invasion of tumors.The function of HIF-Ⅰis mainly due to the expression and activity of HIF-Ⅰαsubunit.The expression of HIF-Ⅰαprotein is regulated by cancer suppressor genes PTEN in PI3K/Akt signal path,promoting the transcription of hypoxia-reacted genes and arousing a series of adaptation for hypoxia.It is also concerned with the metabolism of tumors,dilation of vessels and angiogenesis et al.Invasion and transference of tumors is a complicated process containing the interaction between the tumor cells and host cells.The degree of invasion and transference of tumors is the major factor to the prognosis.But the molecular mechanism of GIST invasion and transference is still unknown.In recent studies, PI3K/Akt signal path was found activated consistantly.TGF-βcan up regulate the expression of uPAmRNA and its protein depending on the dosage and time,mainly mediated by the PI3K/Akt/NF-KB signal path.It has been identified that,Upa is the target gene of NF-KB.So it was confered that the activation of PI3K/Akt signal path leads to the up regulation of NF-KB,which induced the transference of tumor by targeting uPA.That is to say,uPA is mainly regulated by NF-KB,coming to take part in the invasion and transference of tumors. Recently,reports on PI3K/Akt signal path increased more,but few was found on their changes in GIST.We detected the mRNA changes of PI3K,Akt and PTEN,further discussing the changes of factors at expression level after PI3K/Akt signal path was activated.We discussed the relationship between theis expressions and the clinical pathologic characteristics and prognosis of GIST,in order to explore the functions in the occurrence and development of GIST,providing a basis for tumor targeting therapy in further.Materials and Methods1、SubjectsThe 124 fresh samples of GIST patients（64 males and 60 females）were acquired from the Shengjing Hospital affiliated to China Medical University from 1990 to 2007. All the patients were diagnosed by the examination of pathology after surgery removal. The clinical pathological materials were intact and the follow-up results were definite in all the 124 patients.The range of age of these patients was 9 to 79 years old and the mean age was 54.6 years old.The tumors of 62 patients（50.0%）were located in stomach,28（22.6%）in small intestine,14（11.3%）in colorectus,9（7.3%）in esophagus,and 11（8.9%）outside the gastrointestinal tract（4 in omentum,4 in mensentery,3 in retroperitoneum）.The paraffin samples of 57 GIST patients（29 males and 28 females）were acquired.The range of age of these patients was 16 to 62 years old and the mean age was 55.8 years old.The tumors of 29 patients（50.9%）were located in stomach,13（22.8%）in small intestine,7（12.3%）in colorectus,3（5.3%）in esophagus,and 5（8.8%）outside the gastrointestinal tract（2 in omentum,2 in mensentery,1 in retroperitoneum）.2、Reagents of molecular biologyPTEN（10P01）:sc-73420;p-Akt1/2/3（Thr 308）-R:sc-16646-R;p-PI3-kinase p85α（Tyr 508）:sc-12929;HIF-1α（3C144）:sc-71247;NOS₂（C-11）:sc-7271; VEGF（C-1）:sc-7269;uPA（H-140）:sc-14019;MMP-2（8B4）:sc-13595;MMP-9 （M-17）:sc-6841;TGF-β₁（500-M66）:sc-65378（Santa Cruz）;Second antibody marked by HRP（Beijing Zhongshan Company）;Cellulose Nitrate Membrane（Takara Company）;RT-PCR kit（Takara Company）;DAB Substrate（Takara Company）;DNA molecular weight marker（Takara Company）;PCR primer（Takara Company）;other were analytical reagents of China.3、mRNA levels in GIST group and control group detected by RT-PCRThe extraction of mRNA was followed the instruction of QuickPro micro mRNA Purification kit.The procedures included the preparation of samples,isolation of mRNA,washing of oligo（dT）fibrous particles binding mRNA with high salt buffer and low salt buffer respectively and the elution of mRNA.TaKaRa RNA PCR（AMV） V3.0 kit was used in reverse transcription and PCR reaction.The imaging of the reactive products was implemented after agarose gel electrophoresis.4、Expression of related factors in GIST group and control group detected by Western blottingThe homogenate was prepared after 1 ml cell lysis solution（Cell signaling Technology,Beverly,MA）and 1 mmol PMSF added to 0.5g GIST.The protein was extracted followed the regular steps and the concentration was detected.Before electrophoresis,the SDS buffer was added into the protein extraction,heated to boiling 5 min at 100℃,loaded after centrifugation,separated with 12%SDS-PAGE, transferred the protein to the Cellulose Nitrate Membrane and then incubated with the primary antibody.IgG coupled alkaline phosphatase used as the second antibody and O-dianidine andβ-naphthyl acid phosphate were used as the substrate for alkaline phosphatase for coloration.5、Expression ofHIF-1α,NOS₂ and VEGF in GIST group and control group detected by immunohistochemistry.The GIST tissues were fixed by formalin and then 4μm sections were prepared after embedded by paraffin.After dewaxed,one drop or 50μl blocking serum was added on the slide.Wiping off the serum,one drop（or 50μl）primary antibody was added on each slide,and then incubated.Then one drop（or 50μl）Envision^TMsecond antibody was added on each slide,and then incubated.Finally DAB coloration was implemented for 1 to 2 minutes. Results1、Expression of PI3K,Akt and PTEN on the levels of mRNA and protein in the GIST group and control group,and the correlation analysis among the three indexesThe up-regulated expression of PI3K mRNA presented with the increasing of NIH grades（p＜0.05）.The expression rate of PI3K was not associated with gender,age and histological type,while was significantly associated with the invasion of mucosa and lymph node metastasis（p＜0.05）.These indicate that the expression of PI3K is associated with the transformation and invasive metastasis of GIST cell and the product of PI3K may regulate cell growth and apoptosis.We also found that the up-regulated expression of Akt mRNA presented with the increasing of NIH grades except that between high risk group and moderate risk group.The expression rate of Akt was not associated with sex,age and histological classification,while was significantly associated with mucosa invasion and lymph metastasis.These indicate that the over-expression of Akt contribute the lymph metastasis of gastric cancer.In our study, the down-regulated expression of PTEN mRNA presented with the increasing of NIH grades（p＜0.05）,and the deficient rate of PTEN was higher with the severer malignancy of the tumor.The expression rate of PTEN was not associated with sex, age and histological classification,while was closely associated with mucosa invasion and lymph metastasis.These indicate that the deficiency of PTEN protein is associated with the development of GIST.The results of Western blotting showed that the changes of all the three indexes on the level of protein coincided with the changes on the level of mRNA.However,the expression of Akt protein increased significantly in the tissue of GIST caused by the changes of translation and（or）post-translation.In the 124 GIST samples,the expression of PI3K protein was positively correlated with the expression of Akt protein（r=0.292,P=0.031）.The expression of PI3K protein was negatively correlated with the expression of PTEN protein（r=-0.412,P=0.036）.The expression of PTEN protein was negatively correlated with the expression of Akt protein（r=-0.386, P=0.024）.These suggested that the main action substrate in the downstream of PI3K was Akt.The positive regulation of PI3K can activate Akt and activated Akt can activate or inhibit the expression of target protein by phosphorylation.Furthermore the proliferation,differentiation,apoptosis and migration were regulated.The expression of PTEN negatively correlated with PI3K and Akt suggest that the antagonist action may present between PI3K/Akt and PTEN in the development of GIST.2、Expression of NF-κB、XIAP、bcl-2 and survivin on the levels of mRNA and protein in the GIST group and control group,and the correlation analysis among the four indexesThe changes of the expression of NF-κB mRNA were closely associated with NIH grades,lymph node metastasis of tumors and the invasion of mucosa,but not associated with age,gender and histological type.The up-regulated expression of XIAP mRNA presented with the increasing of NIH grades and the expression of XIAP was negatively correlated with differentiation of GIST which showed that the lower the differentiation and the higher the expression rate of XIAP.The expression of XIAP was higher in the GIST patients with lymph node metastasis,and it was closely associated with the invasion of mucosa,but not with age,gender,histological type,metastasis, recurrence and invasion of peripheral tissues.The expression of Survivin mRNA were closely associated with NIH grades,lymph node metastasis of tumors and the invasion ofmucosa,but not associated with age,gender and histological type in the GIST group. Due to the regulation of NF-κB,the positive correlation occurred in the mRNA expression of the four indexes.3、The expression and correlation analysis of HIF-1α,NOS₂ and VEGF between GIST and control groupThe positive expression of HIF-1αin nucleus and cytoplasm is yellow or brown particles.The expression of HIF-1αis weaker in normal GIST tissue.In our 124 cases, （-）46 cases,（+）7 cases,（++）31 cases,（+++）28 cases,（++++）13 cases.The total positve expression rate is 63.7%（79/124）.The expression of HIF-1αis closely related to NH classification,mucosal invasion and lymphatic metastasis of GIST.The positive rate of HIF-1αinⅠandⅡis 46.2%and it is 68.4%inⅢandⅣ.The positive rate of HIF-1αin mucosal invasion and non-mucosal invasion are separately 88.2%and 54.4%. The positive rate in metastasis cases is much higher than non-metastaisis cases.They are 90.0%and 51.2%.The expression of HIF-1αhas nothing to do with other clinical pathological features. NOS₂ is mainly expressed in cytoplasm and distributed like particles.In our 124 cases of GIST,NOS₂ is high expression（64.5%）,the positive expression rate is much higher than normal GIST tissue（17.7%）and the difference is significant.The expression of NOS₂ is closely related to NH classification,mucosal invasion and lymphatic metastasis of GIST.The positive rate of NOS₂ inⅠandⅡis 42.3%and 70.4%inⅢandⅣ.The positive rate of NOS₂ in mucosal invasion and non-mucosal invasion are separately 88.2%and 55.7%.The positive rate in metastasis cases is much higher than non-metastaisis cases.They are 95.0%and 50.0%.The expression of NOS₂ has nothing to do with other clinical pathological features.So we concluded that NOS₂ maybe involved in the whole process of GIST mucosal cancer.NOS₂ is one of the dangerous factors and at the same time NOS₂ is a reliable indicator of prognosis.VEGF is mainly expressed in cytoplasm of GIST and it is diffuse distribution.The positive expression rate of VEGF is 58.1%（72/124）.The posive signal is located in cytoplasm and membrane of GIST cell.The expression of VEGF is closely related to NH classification,mucosal invasion and lymphatic metastasis of GIST.The positive rate of VEGF inⅠandⅡis 46.2%and 61.2%inⅢandⅣ.The positive rate of VEGF in mucosal invasion and non-mucosal invasion are separately 79.4%and 50.0%.The positive rate in metastasis cases is much higher than non-metastaisis cases.They are 80.0%and 47.6%.The expression of VEGF has nothing to do with other clinical pathological features.The cytoplasm of vascular endothelial cells is stained into brown particles in GIST and malignant tissue.In our 124 cases of gastric cancer,the value of MVD（23.15±8.92） is higher than adjacent tissue（8.3±2.76）.The difference is significant.So we can concluded that the angiogenensis of GIST tissue is more active than adjacent tissue. Positive CD117 is located in cell membrane,CD34 is located in cytoplasm,they are all brown paricles.The MVD value of positive expression is much higher than negative expression in HIF-1α,NOS₂ and VEGF.And MVD is closely related to NH classification,mucosal invasion and lymphatic metastasis,MVD has nothing to do with other clinical pathological features.The expression of HIF-1αand NOS₂ are of significant positive correlation （r=0.392,P=0.026）.The expression of HIF-1αand VEGF are of significant positive correlation（r=0.368,P=0.034）.The expression of NOS₂ and VEGF are of significant positive correlation（r=0.452,P=0.021）.4、The changing in mRNA and protein level and correlation analysis ofuPA,MMP-2,MMP-9 and TGF-β₁ in GIST and control group.In the 124 cases of GIST,the expression of uPA,MMP-2,MMP-9 and TGF-β₁ have no relationship with age,sex and histological type of patients.The expression of uPA,MMP-2,MMP-9 and TGF-β₁ is significantly related to lymphatic metastasis,NH classification and mucosal invasion.The protein level is consistent to mRNA.TGF-β₁ can mediate PI3K/Akt signal transduction pathway and increase the expression of uPA, they are of positive correlation（r=0.356,P=0.028）.The expression of uPA and MMP-2 are of significant positive correlation（r=0.323,P=0.035）.The expression of MMP-2 and MMP-9 are of significant positive correlation（r=0.495,P=0.042）.So we can conclude that the role of them are of significant correlation in GIST occurrence and development.DiscussionDisorder of PI3K/AKt signal transduction pathway expressed in a wide range of human tumors.the mutation of this pathway will cause the disfunction or acquire some functions witch can cause cell transformation.PI3K/AKt signal transduction pathways regulate tumor cells’ proliferation and survive.AKt which is the role of PI3K downstream target protein,and is the core of PI3K/AKT signal transduction pathway it is an important protein kinas of signal transduction pathway.PTEN is the first ever found a phosphates activity of tumor suppressor genes can be activated by controlling activity phosphatidyl 3,4,5-instill trisphosphate（PIP3）to control the activity of AKt. Thus,PTEN mutation can cause AKt loss the ability to control cell proliferation,then leads carcinogenesis.PTEN can be phosphorylated by PIP3,antagonistic activity of PI3K and reduce the concentration of intracellular PIP3,thereby inhibiting the activation AKt.In GIST,the NIH classification with its PI3K,AKt expression,and expression of tumor suppressor gene PTEN decrease.The positive expression rate of PI3K,AKt and PTEN have no relation with the sex,age,histology type,but have closely relation with tumor invasion,metastasis and mucosal involvement.PI3K/AKt and PTEN may have mutual antagonism effect during the development of GIST.We study the PI3K-AKt signal transduction pathway downstream apoptosis related factors expression in GIST and the relationship between the expression with GIST clinical pathological characteristics and the prognosis through the observation of NF-κB and apoptosis related factors such as XIAP,blc-2 and surviving mRNA changes in GIST paraffin block.In GIST the malignant level increase accompanied by the increase of PI3K and AKt activity,activated AKt can phosphorylated IKKa directly then activating IKK,through witch regulating NF-κB activity.AKt is the important regulation factor of NF-κB dependent transcription gene,it can stimulate tumor cell generation.PI3K-AKt signal transduction pathway activation can stimulate NF-κB mRNA,witch can go through into cell nucleus to regulate XIAP,bcl-2 and surviving mRNA expression in GIST.The expression of NF-κB mRNA and NH classification have closely relation with tumor invasion,metastasis and mucosal involvement,but have no relation with age,sex and histology type.XIAP mRNA expression up-regulate according to NH classification;XIAP expression has negative relation with GIST histological differentiation,that is XIAP expression level increase accompanied by the decrease of differentiation.The metastasis GIST patient express high level XIAP protein,and XIAP expression has relation with mucosal involvement,but has no relation with age,sex and histology type.Bcl-2 mRNA expression up-regulation has relation with NH classification and mucosal involvement,but has no relation with age, sex,histology type and invasion of around tissue.Survivin mRNA expression has relation with NH classification,tumor invasion and metastasis,mucosal involvement. But its expression has no relationship with age,sex and histological typeBecause of the role of regulation and control of NF-κB,the mRNA level between the target was a positive correlation Change.Tumor angiogenesis is a necessary condition for the transfer happen,PI3K/Ak signal transductional pathway is involved in the process of tumor angiogenesis that mediated by a variety of factors.As a tumor micro-environment of a critical regulatory factor,HIF-1 play a central role in promoting tumor angiogenesis and invasiveness of the tumor.PI3K/Akt signaling pathway can regulate HIF-1αprotein expression by regulation of PTEN tumor suppressor genet,and activation of AKt can enhance the stability of HIF-1α,thereby promote the transcription of hypoxia response genes,causing cells to hypoxia a series of adaptive responses.VEGF probably through the regulation of tumor angiogenesis,development and transfer plays an important role. In many solid tumors,the expression of VEGF and tumor angiogenesis was highly related to each other.VEGF are closely related with growth,metastasis and prognosis of tumor,therefore,take VEGF as target,inhibiting its expression,thereby inhibiting angiogenesis of tumor,blocking the transfer of nutrient sources and channels of tumor, to achieve the purpose of the treatment of tumor,has become the new strategy of treatment of cancer in recent years.VEGF may activate PI3K/AKt channels and induce NO synthesis,HIF-1α,NOS2,and VEGF can participate in the occurrence and development of GIST by the signal transduction pathway of PI3K/Akt.Therefore,we believe that expression of HIF-1α,NOS2,and VEGF may be the important target of malignant tumor’s growth,invasion and metastasis.They can be used as targets,inhibit their expression,thereby inhibit angiogenesis of tumor,blocki the transfer of nutrient sources and channels of tumor,to achieve the purpose of the treatment of tumor.The activation of PI3K/AKt signal transduction pathway can activate the expression of HIF-1α,HIF-1α,NOS2,VEGF,MVD and GIST of the NIH classification,submucosal invasion of tumor has closely relationship with invasion and metastasis of tumor,HIF-1α,NOS2 and VEGF expression may be the important target malignant tumor growth,invasion and metastasis.Tumor’s invasion and metastasis is a complex multi-step tumor cells and host stromal cells and the process of interaction,the degree of invasion and metastasis is a major factor affecting the prognosis,TGF-β1 exists in the tumor and some normal cells,can regulation of normal cells and transformed cell growth and differentiation.TGF-β1 mRNA expression in lesions with high levels of infiltration depth has a strong and extensive orientation and ability to transfer,TGF-β1 itself as a sport can also be factors,tumor cells to promote exercise and transfer. TGF-β1 can mediate PI3K/Akt signal transduction pathway to increases uPA,uPA is regulated by the PI3K/Akt signal transduction pathway-mediated of TGF-β1,further activation of MMP-2,uPA and MMP-2 expression was positively between related.At the same time,MMP-2 also has the activitical function of MMP-9.Prompting that both of them are closely related at the role of the development process of GIST,possible through the synergies promoting tumor incidence,tumor extracellular matrix degradation and tumor cell invasion and metastasis.This study shows that,the expression ofTGF-β1,uPA,MMP-2 and MMP-9 have no relationship with sex,age, and with the submucosal invasion of tumor,NIH grade,tumor invasion and metastasis-related,suggesting that TGF-β1,uPA,MMP-2 and MMP-9 involved in the tumor’s invasion and metastasis.Conclusion1、In gastrointestinal stromal tumors,the tumor suppressor gene-PTEN is reduced by PI3K and Akt increasing.The positive expression rate of PTEN,PI3K and Akt have no relationship with sex,age and histological type.On the other hand it has relationship with lymphatic metastasis and mucosal invasion.PI3K/Akt and PTEN maybe have the role of mutual antagonism in GIST occurrence and development.The expression of PI3K and Akt are of significantly positive correlation（r=0.292,P=0.031）.The expression of PI3K and PTEN are of significantly negetive correlation（r=-0.412, P=0.036）.The expression of PTEN and Akt are of significantly negetive correlation （r=-0.386,P=0.024）.2、In gastrointestinal stromal tumors,the activation of PI3K/Akt signal transduction pathway can further active NF-κB mRNA,and then it can enter nucleus and control the expression of apoptosis-related factor-XIAP,bcl-2 and survivin mRNA.The expression ofNF-κB,XIAP,bcl-2 and survivin mRNA is closely related to NH classification,mucosal invasion and lymphatic metastasis.They have no relationship with age,sex and histological type.Because of NF-κB regulation,the expression of them are of positive correlation.3、In rich blood supplying GIST,the activation of PI3K/Akt signal transduction pathway can further active HIF-1α,the expression of HIF-1α,NOS₂,VEGF and MVD is closely related to NH classification,mucosal invasion and lymphatic metastasis of GIST.They have no relationship with age,sex and histological type.Our study shows that the expression of HIF-1α,NOS₂ and VEGF are of significantly positive correlation （r1=0.368,r2=0.452,r3=0.392,P1,P2,P3＜0.05）.So we can conclude that they have interdependent positve regulation action in GIST invasion,metastasis and angiogenesis. They are all involved in GIST occurrence and development.So HIF-1α,NOS₂ and VEGF are important targets in malignant tumor growth,invasion and metastasis.4、We found that TGF-β₁ can mediate PI3K/Akt signal transduction pathway and increase the expression of uPA,then it can active MMP-2 and MMP-9,and effectively degrade extracellular matrix.The expression of TGF-β₁ is increasing with deepening the depth of invasion,enhancing the extent of lymphatic metastasis and progressing NH classification.Our study shows that the expression of TGF-β₁,uPA,MMP-2 and MMP-9 have no relationship with sex,age and histological type,they are closely related to NH classification,mucosal invasion and lymphatic metastasis.So TGF-β₁, uPA,MMP-2 and MMP-9 are involved in tumor invasion and metastasis.更多还原