转化生长因子-β_1、Smad_4和磷酸化细胞外信号调节激酶在胃癌组织中的表达及其临床意义
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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-β_1、β_2、β_3三种形态存在，三者的生物学作用相似，其中TGF-β_1含量最高且具有代表性。TGF-β_1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-β_1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。Smad_4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。Smad_4对ERK具有调节作用，能抑制TGF-β_1对Ras／ERK通路的激活；相反，ERK过度活化能灭活Smad_4基因，下调Smad_4，使TGF-β_1抑制作用减弱或消失。因此，TGF-β_1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-β_1受体缺陷、Smad_4蛋白缺失或／和ERK通路的过度激活时，可使TGF-β_1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 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)
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-activ
ated 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 cell
proliferation, 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 some
cancer 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
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
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)
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-activ
ated 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 cell
proliferation, 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 some
cancer 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
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32． Markus, Toshiguki J, Helmut F, et al. Differential localization of TGF-β isoforms in human gastric mucosa and overexpression in GC. Int J Cancer, 1997, 71(2) : 131-137
33． Ebert MP, Yu J, Miehlke S, et al. Expression of transforming growth factor beta-1 in gastric cancer and in the gastric mucoma of first-degree relatives of patients with gastric cancer. Br J Cancer, 2000, 82: 1795-1800
34． Maehara Y, Kakeji Y, Kabashima A, et al. Role of transforming growth factor-β1 in invasion and metastasis in gastric carcinoma. J. Clin. Oncol, 1999, 17(2) : 607-614
35. Saito H, Tsujitani S, Oka S, et al. The expression of transforming growth factor-β_1 is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma. Cancer, 1999, 86(8): 1455-1462
36. Kinugasa S, Abe S, Tachibana M, et al. Overexpression of transforming growth factor-β_1 in scirrhous carcinoma of the stomach correlates with decreased survival. Oncology, 1998, 55(6): 582-587
37．许洪元，陈莉，王元和等．胃癌组织TGF β_1和TGF βⅡ型受体表达的临床意义．中华消化杂志，1998，18(6)：346-348
38. McEarchern JA, Kobie JJ, mACK V, et al. Invasion and metastasis of amammary tumor involves TGF-beta signaling. Int J Cancer, 2001, 91(1): 76-82
39. Nakashio T, Narita T, Akiyama S, et al. Adhesion molecules and TGF-beta1 are involved in the peritoneal dissemination of NUGC-4 human gastric cancer cells. Int J Cancer, 1997, 70:612-618
40. Yashiro M, Chung YS, Nishimura S, et al. Establishment of two new scirrhous gastric cell lines analysis of factors associated with disseminated metastasis. Br J Cancer, 1995, 72(5): 1200-1210
41. Niki M, Okajima K, Isozaki H, et al. Measurement of the plasma transforming growth factor-β_1 (TGF-β_1) level in patients of gastric carcinoma-compared with the serum IAP level and the lymphocyte subsets (CD3, CD4, CD8). Nippon Shokakibyo Gakkai Zasshi, 1996, 93:303-311
42. Saito H, Tsujitani S, Oka S, et al. An elevated serum level of transforming growth factor-β_1 significantly correlated with lymph node metastasis and poor prognosis in patients with gastric carcinoma. Anticancer Res, 2000, 20:4489-4494
43．卢震亚，黄怀德，厉有名等．转化生长因子β_1基因在胃癌中的表达及其临床意义．实用癌症杂志，2001，16(4)：397-399
44．周新民，张学庸，胡家露等．人胃癌组织TGF-β_1蛋白的表达．第四军医学报，1998，19(1)：31-33
45． Flatten M, Wick W, Weller M. Malignant glioma biology: Role for TGF-β in growth motility, angiogenesis, and immune escapes. Microsc. Res. Tech, 2001, 52 (4) : 401-410
46． Santibanez JF, Guerrero J, Quintanilla M, et al. Transforming growth factor-betal modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in tansformedkeratinocytes. Biochem Biophys Res Commun, 2002, 296 (27) : 267-273
47． Saha D, Datta PK, Sheng H, et al. Synergistic induction of cyclooxygenase-2 by transforming growth factor-betal and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia, 1999, 1(6) : 508-517
48． Derynck R, Zhang Y. Intracellular signaling: the Mad way to do it. Curr Biol, 1996, 6(10) : 1226-1229
49． Heldin CH, M iyazono K, Dijke P. The TGF-β signaling from cell membrane to nucleus through SMAD proteins. Nature, 1997, 390 (6659) : 465-469
50． Massague J, Hata A, Liu F. TGF-β signaling through the Smad pathway. Trends cell Biol, 1997, 7 (4) : 187-192
51 . Nakao A, Imamura T, Kawabata M, et al. TGF-β recepter-mediated signaling through Smad2, Smad3 and Smad4． EMBO J, 1997, 16 (17) : 5353-5359
52． Zhang Y, Musci T, Derynck R. The tumor suppressor Smad4/DPC4 as a central mediator of Smad function. Curr Biol, 1997, 7(4) : 270-274
53． Hahn SA, Schutte M, Shamsul Hoque ATM, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21． 1． Science, 1996, 271 (5247) : 350-354
54． Wilentz RE, Su GH, Dai JL, et al. Immunohistochemical labeling for DPC4 mirrors genetic status in pancreatic adenocarcinomas a new marker of DPC4 inactivation. Am J Pathol, 2000, 156: 37-43
55． Wilentz RE, Iacobuzio-Donahue CA, Argani P, et al. Loss of expression of DPC4 in pancreatic intraepithelial neoplasia: evidence that DPC4 inactivation occurs late in neoplastic progression. Cancer Res, 2000, 60: 2002-2006
56． 魏红，杨竹林，李永国．原发和转移性肝癌中Smad4、 TGF-β1及TGF-βRI
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57． Hunt KK, Fleming JB, Abramian A, et al. Overexpression of tumor suppressor gene smad4/DPC4 induces p21wafl expression and growth inhibition in human carcinoma cells. Cancer Res, 1998, 58(24) : 5656-5661
58． Feng XH, Lin X, Derynck R. Smadi, Smada and Smad4 cooperate with spl to induce p15 (Ink4B) transcription in response to TGF-beta. EMBO J, 2000, 19(19) : 5178-5193
59． Schwarte-Waldhoff I, Volpert OV, Bouck NP, et al. Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis. Proc Natl Acad Sci USA, 2000, 97(17) : 9624-9629
60． Salovaara R, Roth S, Loukola A, et al. Frequent loss of SMAD4/DPC4 protein in colorectal cancers. Gut, 2002, 51 (1) : 56-9
61． Fukuchi M, Masuda N, Miyazaki T, et al. Decreased Smad4 expression in the transforming growth factor-beta signaling pathway during progression of esophageal squamous cell carcinoma. Cancer, 2002, 95(4) : 737-743
62． Takaku K, Miyoshi H, Matsunaga A, et al. Gastric and duodenal polyps in Smad4 (DPC4) knockout mice. Cancer Res, 1999, 59(24) : 6113-6117
63． Xu X, Brodie SG, Yang X, et al. Haploid loss of the tumor suppressor smad4/DPC4 initiates gastric polyposis and cancer in mice. Oncogene, 2000, 19(15) : 1868-1874
64． Wrana JL, Attisano L, Wieser R, et al. Mechanism of activation of the TGF-β receptor. Nature, 1994, 370: 341-347
65． 杨竹林，李永国，黄福生等．胃癌组织Smad4基因和转化生长因子β1及其 受体的表达及意义．中华胃肠外科杂志，2001，4(3) ：179-181
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79:143-180
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71. Ito Y, Sasak Y, Horimoto M, et al. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase in human hepatocellular carcinoma. Hepatology, 1998, 27: 951-958
72. Mandell JW, Hussaini, Zecevie M, et al. In situ visualization of intratumor growth factor signaling: immunohistochemical locali-zation of activated ERK/MAP kinase in glial neoplasma. Am J pathol, 1998, 153(5): 1411-1423
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74．李景和，文继舫，冯德云等．P-MAPK、cyclinD_1和P53蛋白在大肠癌中的表达及其相关性．临床与实验病理学杂志，2000，16(5)：391-393
75．白铁男，李雨成，张淑敏等．细胞外信号调节蛋白激酶在前列腺上皮内瘤组织中的表达及意义．中华泌尿外科杂志，2001，22(4)：239-240
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77． Lengye E, Singh B, Gum R, et al. Regulation of urokinase-type plasminogen activator expression by the v-mos oncogene. Oncogene, 1995, 11: 2639-2648
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21．周新民，张学庸，胡家露等．人胃癌组织TGF-β_1蛋白的表达．第四军医学报，1998，19(1)：31-33
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28． Yang BC, Zander DS, Mehta JL, et al. Hypoxia-reoxygenatino-induced apoptosis in cultured adult rat myocytes and the protective effect of platelets and Transforming growth factor-beta (1) . J Pharmacol Exp Ther, 1999, 291:733-738
29． Roberts AB. Molecular and cell biology of TGF-beta. Miner Electrolyte Metab , 1998,24:111-119
30． Asakura S, Kato H, Fujino S, et al. Immunohistochemical study of Transforming growth factor-beta and central fibrosis in T1 adenocarcinoma of the lung. Nippon Kyobu Geka Gakkai Zasshi, 1995,43:1924-1928
31． Naef M, Ishi Wata T, Friess H, et al. Differential localization of transforming growth factor beta isoforms in human gastric mucosa and overexpression in gastric carcinoma. Int J Cancer, 1997, 71 (2) : 131-137
32． Markus, Toshiguki J, Helmut F, et al. Differential localization of TGF-β isoforms in human gastric mucosa and overexpression in GC. Int J Cancer, 1997, 71(2) : 131-137
33． Ebert MP, Yu J, Miehlke S, et al. Expression of transforming growth factor beta-1 in gastric cancer and in the gastric mucoma of first-degree relatives of patients with gastric cancer. Br J Cancer, 2000, 82: 1795-1800
34． Maehara Y, Kakeji Y, Kabashima A, et al. Role of transforming growth factor-β1 in invasion and metastasis in gastric carcinoma. J. Clin. Oncol, 1999, 17(2) : 607-614
35. Saito H, Tsujitani S, Oka S, et al. The expression of transforming growth factor-β_1 is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma. Cancer, 1999, 86(8): 1455-1462
36. Kinugasa S, Abe S, Tachibana M, et al. Overexpression of transforming growth factor-β_1 in scirrhous carcinoma of the stomach correlates with decreased survival. Oncology, 1998, 55(6): 582-587
37．许洪元，陈莉，王元和等．胃癌组织TGF β_1和TGF βⅡ型受体表达的临床意义．中华消化杂志，1998，18(6)：346-348
38. McEarchern JA, Kobie JJ, mACK V, et al. Invasion and metastasis of amammary tumor involves TGF-beta signaling. Int J Cancer, 2001, 91(1): 76-82
39. Nakashio T, Narita T, Akiyama S, et al. Adhesion molecules and TGF-beta1 are involved in the peritoneal dissemination of NUGC-4 human gastric cancer cells. Int J Cancer, 1997, 70:612-618
40. Yashiro M, Chung YS, Nishimura S, et al. Establishment of two new scirrhous gastric cell lines analysis of factors associated with disseminated metastasis. Br J Cancer, 1995, 72(5): 1200-1210
41. Niki M, Okajima K, Isozaki H, et al. Measurement of the plasma transforming growth factor-β_1 (TGF-β_1) level in patients of gastric carcinoma-compared with the serum IAP level and the lymphocyte subsets (CD3, CD4, CD8). Nippon Shokakibyo Gakkai Zasshi, 1996, 93:303-311
42. Saito H, Tsujitani S, Oka S, et al. An elevated serum level of transforming growth factor-β_1 significantly correlated with lymph node metastasis and poor prognosis in patients with gastric carcinoma. Anticancer Res, 2000, 20:4489-4494
43．卢震亚，黄怀德，厉有名等．转化生长因子β_1基因在胃癌中的表达及其临床意义．实用癌症杂志，2001，16(4)：397-399
44．周新民，张学庸，胡家露等．人胃癌组织TGF-β_1蛋白的表达．第四军医学报，1998，19(1)：31-33
45． Flatten M, Wick W, Weller M. Malignant glioma biology: Role for TGF-β in growth motility, angiogenesis, and immune escapes. Microsc. Res. Tech, 2001, 52 (4) : 401-410
46． Santibanez JF, Guerrero J, Quintanilla M, et al. Transforming growth factor-betal modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in tansformedkeratinocytes. Biochem Biophys Res Commun, 2002, 296 (27) : 267-273
47． Saha D, Datta PK, Sheng H, et al. Synergistic induction of cyclooxygenase-2 by transforming growth factor-betal and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia, 1999, 1(6) : 508-517
48． Derynck R, Zhang Y. Intracellular signaling: the Mad way to do it. Curr Biol, 1996, 6(10) : 1226-1229
49． Heldin CH, M iyazono K, Dijke P. The TGF-β signaling from cell membrane to nucleus through SMAD proteins. Nature, 1997, 390 (6659) : 465-469
50． Massague J, Hata A, Liu F. TGF-β signaling through the Smad pathway. Trends cell Biol, 1997, 7 (4) : 187-192
51 . Nakao A, Imamura T, Kawabata M, et al. TGF-β recepter-mediated signaling through Smad2, Smad3 and Smad4． EMBO J, 1997, 16 (17) : 5353-5359
52． Zhang Y, Musci T, Derynck R. The tumor suppressor Smad4/DPC4 as a central mediator of Smad function. Curr Biol, 1997, 7(4) : 270-274
53． Hahn SA, Schutte M, Shamsul Hoque ATM, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21． 1． Science, 1996, 271 (5247) : 350-354
54． Wilentz RE, Su GH, Dai JL, et al. Immunohistochemical labeling for DPC4 mirrors genetic status in pancreatic adenocarcinomas a new marker of DPC4 inactivation. Am J Pathol, 2000, 156: 37-43
55． Wilentz RE, Iacobuzio-Donahue CA, Argani P, et al. Loss of expression of DPC4 in pancreatic intraepithelial neoplasia: evidence that DPC4 inactivation occurs late in neoplastic progression. Cancer Res, 2000, 60: 2002-2006
56． 魏红，杨竹林，李永国．原发和转移性肝癌中Smad4、 TGF-β1及TGF-βRI
的表达及意义．肿瘤防治杂志，2001，8(2) ：133-136
57． Hunt KK, Fleming JB, Abramian A, et al. Overexpression of tumor suppressor gene smad4/DPC4 induces p21wafl expression and growth inhibition in human carcinoma cells. Cancer Res, 1998, 58(24) : 5656-5661
58． Feng XH, Lin X, Derynck R. Smadi, Smada and Smad4 cooperate with spl to induce p15 (Ink4B) transcription in response to TGF-beta. EMBO J, 2000, 19(19) : 5178-5193
59． Schwarte-Waldhoff I, Volpert OV, Bouck NP, et al. Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis. Proc Natl Acad Sci USA, 2000, 97(17) : 9624-9629
60． Salovaara R, Roth S, Loukola A, et al. Frequent loss of SMAD4/DPC4 protein in colorectal cancers. Gut, 2002, 51 (1) : 56-9
61． Fukuchi M, Masuda N, Miyazaki T, et al. Decreased Smad4 expression in the transforming growth factor-beta signaling pathway during progression of esophageal squamous cell carcinoma. Cancer, 2002, 95(4) : 737-743
62． Takaku K, Miyoshi H, Matsunaga A, et al. Gastric and duodenal polyps in Smad4 (DPC4) knockout mice. Cancer Res, 1999, 59(24) : 6113-6117
63． Xu X, Brodie SG, Yang X, et al. Haploid loss of the tumor suppressor smad4/DPC4 initiates gastric polyposis and cancer in mice. Oncogene, 2000, 19(15) : 1868-1874
64． Wrana JL, Attisano L, Wieser R, et al. Mechanism of activation of the TGF-β receptor. Nature, 1994, 370: 341-347
65． 杨竹林，李永国，黄福生等．胃癌组织Smad4基因和转化生长因子β1及其 受体的表达及意义．中华胃肠外科杂志，2001，4(3) ：179-181
66． Park HJ, Kim BC, Kim ST, et al. Role of MAP kinases and their cross-talk in TGF-betal-induced apoptosis in FaOrat hepatoma cell line. Hepatology, 2002, 35(6) : 1360-1371
67． Widmann C, Gibson S, Jarpe MB, et al. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev, 1999,
79:143-180
68. Lopez-Ilasaca M. Signaling from G-protein-coupled receptor to mitogen-activated protein (MAP)-kinase cascades. Biochem Pharmacol, 1998, 56:269-277
69. Kiyokawa E, Takai S, Tanaka M, et al. Overpression of ERK, an EPH famility receptor protein tyrosine kinase, in various human tumors. Cancer Res, 1994, 54: 3645-3650
70. Couly S, Paterson H, Kemp P, et al. Activation of MAP kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH3T3 cells. Cell, 1994, 77:841-852
71. Ito Y, Sasak Y, Horimoto M, et al. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase in human hepatocellular carcinoma. Hepatology, 1998, 27: 951-958
72. Mandell JW, Hussaini, Zecevie M, et al. In situ visualization of intratumor growth factor signaling: immunohistochemical locali-zation of activated ERK/MAP kinase in glial neoplasma. Am J pathol, 1998, 153(5): 1411-1423
73. Albanell J, Codony-Servat J, Rojo F, et al. Activated extracellular signal-regulated kinases: association with epidermal growth factor receptor/transforming growth factor alpha expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments. Cancer Res, 2001, 61 (17): 6500-6510
74．李景和，文继舫，冯德云等．P-MAPK、cyclinD_1和P53蛋白在大肠癌中的表达及其相关性．临床与实验病理学杂志，2000，16(5)：391-393
75．白铁男，李雨成，张淑敏等．细胞外信号调节蛋白激酶在前列腺上皮内瘤组织中的表达及意义．中华泌尿外科杂志，2001，22(4)：239-240
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