【作者】 许春进；

【导师】 陈玉龙； 徐峰；

【作者基本信息】 郑州大学 ， 内科消化， 2003， 硕士

【摘要】 背景与目的：胃癌是最常见的恶性肿瘤之一。据统计中国胃癌患者的死亡率占全部恶性肿瘤的首位。与其它恶性肿瘤一样，胃癌的发生机制不明。最近研究发现，转化生长因子-β（transforming growth factor β，TGF-β）家族的分泌紊乱及其传导通路的中断与肿瘤的发生发展关系密切。在TGF-β家族中，人类有TGF-β₁、β₂、β₃三种形态存在，三者的生物学作用相似，其中TGF-β₁含量最高且具有代表性。TGF-β₁对正常上皮多表现为抑制，是诱导上皮细胞凋亡的一个重要因子。TGF-β₁抑制信号的传递除通过激活受体及下游的Smads蛋白外，尚能通过激活C-Jun N—末端激酶（C-Jun N-terminal kinase，JNK）通路、P38丝裂原活化蛋白激酶(P38 mitogen-activated protein kinase，P^38MAPK)通路及Ras／细胞外信号调节激酶（extracellular signal-regulated kinase，ERK）通路。Smad₄蛋白是协同Smad（Co-Smad），在TGF-β信号传导中起关键作用，是细胞内信号传导蛋白Smads的功能活动中心。Ras／ERK通路主要是促进细胞的增殖和分化；而JNK通路及P^38MAPK通路的活化则诱导细胞凋亡。Smad₄对ERK具有调节作用，能抑制TGF-β₁对Ras／ERK通路的激活；相反，ERK过度活化能灭活Smad₄基因，下调Smad₄，使TGF-β₁抑制作用减弱或消失。因此，TGF-β₁抑制作用的发挥是各种传导通路和传导蛋白分子间交叉对话（cross-talk）即相互调节的结果。当组织细胞中存在TGF-β₁受体缺陷、Smad₄蛋白缺失或／和ERK通路的过度激活时，可使TGF-β₁隐匿其生长抑制作用，甚至成为刺激肿瘤细胞生长的因子，使本应停止增殖或凋亡的细胞不停地进入细胞周期，从而引起肿瘤的发生。本文通过检郑州大学2003年硕士毕业论文TGF- pl、smad4和P-ERK在胃瘤组织中的表达和意义多苍试飞跳不二蕊袭男止岌奋气次娜筑万蛋争感么玲臼牙之雄芝奋^价撰芝公洲么盆吩醉介纪汾毛筋冻争邪了戈称男彭瑟韶研夯亩介被东扩晦测TGF一pl，Sm碱和ERK的活化形式磷酸化细胞外信号调节激酶 （phosPho万lated一extracellularsi，al一regulated klnase，p一ERK）蛋白在胃癌中的表达情况及三者之间的相互关系，旨在阐明TGF一p传导通路障碍在胃癌发生发展中的作用，为着手从TGF一p传导通路角度防治肿瘤提供理论依据。 材料与方法:（1）选取19%一1 998年郑州大学第一附属医院67例胃癌根治术标本为实验组。全部病例均经手术和病理证实。另选取距肿瘤边缘大于scm的正常粘膜组织24例为对照组。所有标本经10%甲醛固定，常规脱水后石蜡包埋。（2）免疫组化sABC（strept Avidin Biotin eomplex）法检测TGF一p;、sm别为和p一ERK在胃癌组织中的表达情况。（3）统计工具应用sPsslo.0版软件包，应用了检验和sPe^an等级相关分析，一1<r<1，并以尸<0.05作为有统计学意义的判断标准。 结果:1.TGF一pl和Sm碱主要在细胞浆中表达，细胞核中偶见散在阳性物质表达。TGF一pl在正常胃粘膜组织中着色较均匀，染色浅淡，阳性率为4.2%;胃癌组织中TGF一p;抗原性物质呈棕黄色或棕褐色。TGF一pl在胃癌组织中的阳性率为67.2%。胃癌组织与正常粘膜组织间差异具有显著性（尸<0.01）;Sm别为在正常粘膜中阳性表达率为95.8%。在胃癌组织中其阳性表达率为44.8%，两组间存在显著性差异（P<0.ol）;p一ERK表达于细胞核中，正常粘膜组织中阳性表达率为167%，而在胃癌组织中其阳性表达率为76.1%，两组间差异显著 （尸<0.01）。 2.在分化较好组和分化较差组胃癌中，TGF一旦1蛋白的阳性表达率分别为51.5%和82.4%;sm碱蛋白的阳性表达率分别为60.6%和29.4%;p一ERK蛋白的阳性表达率分别为63.6%和88.2%，各组间比较具有显著差异性（P<0.ol或尸<0.05）。 3.根据癌细胞浸润的深度，将胃癌组分为:未浸润至浆膜组与浸润至浆膜或以外两组。TGF一pl在此两组中的阳性表达率分别为46.7%、83.8%，随浸润深度增加呈一定的上升趋势，两组间比较具有显著性差异（P<0.ol）。Sm别为在此两组中的阳性表达率分别为63.3%、29.7%，随浸润深度增加呈一定的下降趋势，组间比较具有显著性差异（P<0.01）。p一ERK在两组中的阳性表达率分别为60.0%、892%，组间比较具有显著性差异（尸<0.01）。郑州大学2003年硕士毕业论文TGF-川、smad4和P-ERK在胃瘤组织中的表达和意义 4.按有无淋巴结转移分为:无淋巴结转移组和有淋巴结转移组。在两组中TGF一pl的阳性表达率分别为50.0%、79.5%，Smad4的阳性表达率分别为64.3%、30.8%，p一ERK的阳性表达率分别为60.7%、87.2%，组间比较均有显著性差异 （尸<0 .01或P<0.05）。 5.按TNM分期将胃癌分为:I/n期组和m/W期组。在两组中TGF一p;的阳性表达率分别为30.4%、86.4%，Smad4的阳性表达率分别为65.2%、34.1%，p一ERK的阳性表达率分别为43 .5%、93 .2%，三者组间比较均有显著性差异 （P<0 .01或尸<0.05）。 6.TGF一p;与p一ERK的蛋白表达呈正相关（二0.503;p<。.01）;p一ERK与Smad4的蛋白表达呈负相关（二一0.270;尸<0.05）;Smad礴与TGF一pl的蛋白表达呈负相关（r=一0.521:P<0.01）更多还原

【Abstract】 Background and objectives: Gastric carcinoma is one of the common digestive carcinomas. Studies showed that it was the leading cause of cancer death. As other tumors, the mechanism of gastric carcinogensis has not been cleared. Recent studies showed that secretive disorders and signaling pathway disturbance of transforming growth factor superfamily are stronglly related to the progression and aggressiveness of cancers. Transforming growth factor- β1(TGF-β1) superfamily include three isoforms that have similar biological function in humans: TGF-β 1, TGF-β2, TGF-β3. TGF-β1 is the most prevalent of them and regulates cell growth and differentiation hi both normal and tumor cells, as well as being a potent inhibitor of epithelial cell growth. Although the Smad family of proteins recently has been shown to be a key participant in TGF-β signaling transduction, other signaling transduction pathways, such as Ras/ extracellular signal-regulated kinase (ERK), C-Jun N-terminal kinase (INK) and P38 mitogen-activated protein kinase (P38MAPK) pathways have also been shown to be activated by TGF- 3 . Smad4 is distinct from the R-Smads and is defined as a common mediator Smads (Co-Smad) because it forms hetero-oligomeric complexes with activated R-Smads and appears essential for R-Smad fuction. Smad4 acts as a central mediator in TGF- superfamily signaling transduction. Activated Ras/ERK pathway mainly exerts its effects on promoting cellproliferation, differentiation and migration. JNK and p38MAPK are activated by TGF-β1 to induce cell apoptosis in turn. Smadt acts inhibiting of Ras/ERK signaling pathway. Hyperactivation of the Ras/ERK signaling pathway may decrease TGF-β1antiproliferative responses by inactivating SrnacU gene and down-regulating SmacU protein. So cross-talk among TGF-β1 signaling transduction pathways regulates commitment to TGF-β1-induced growth inhibition. TGF- β1 resistance that is associated with functional inactivation or expression absence of either the TGF-β receptors or of signal transducers of the Smad family and with hyperactivation of the Ras/ERK signaling pathway accelerate cell cycle progression resulting in carcinogensis. hi order to investigate the possible mechanism of gastric carcinoma, an immunohistochemical SABC (Strept Avidin Biotin Complex) method was used to examine the expression of three kinds of proteins.Materials and Methods: (1) 67 surgically resected gastric carcinoma samples and 24 normal mucosa samples of stomach were collected. Normal mucosa samples of stomach which were adjacent to carcinoma mucosa were confirmed pathologically as normal mucosa. All the tissues were fixed in 10% neutral formalin and embedden in paraffin. (2) SABC inmunostaining technique was used to examine the expression of TGF-β14 mSmadt and β-ERK in normal mucosa and gastric carcinoma. (3) The data was analysized by software SPSS 10.0. x2-test and spearman correlation were used to compare difference between groups. β value < 0.05 was considered as statistically significant.Results: (1)TGF-β1 protein was stained maily in the cytoplasm of cells and occasionally evident in fibroblasts and smooth muscle cells. Normal epithelial cells were homogeneously stained by TGF-β1, whereas the positive tumor cells were hetergeneously distributed within gastric carcinoma. In normal gastric mucosa and gastric carcinoma, the positive expression rates of TGF-β1 protein were 4.2% and 67.2%,respectively. There were significant differences (P<0.01) between normal mucosa group and gastric carcinoma group. Normal gastric mucosa cells were homogeneously stained by Smad 4. hi the majority of cancer cells, Smad4 positive staining was observed in the cytoplasm and simultaneously in the nuclei of somecancer cells. The positive expression rates of SmaoU in normal mucosa and gastric carcinoma were 95.8% and 44.8%, respectively. When gastric carcinoma group compared with normal mucosa group, there were significant differences (P<0.01). p-ERK protein was stained更多还原