人大肠肿瘤TGF-β1和Smad4的表达及其与血管生成的关系
摘要
背景与目的：大肠癌是最常见的恶性肿瘤之一，它的发生率和死亡率均占全部恶性肿瘤的前列。与其他恶性肿瘤一样，大肠癌在其发生和发展过程中也具有浸润和转移的特性，其具体机制不甚明了。近来越来越多的研究发现，转化生长因子-β(Transforming growth factor β，TGF-β)分泌紊乱及其转导通路的异常与肿瘤的发生发展关系密切。
TGF-β是一类多功能的多肽类生长因子，对细胞的增殖与分化，细胞外基质的产生，细胞凋亡及机体的免疫系统均起着重要的调节作用。活化的TGF-β与其Ⅱ型受体(transforming growth factor β receptorⅡ，TβRⅡ)结合从而形成TGF-β／TβRⅡ复合物并迅速导致Ⅰ型受体(transforming growth factor β receptor Ⅰ，TβRⅠ)的磷酸化。磷酸化的TβRⅠ可磷酸化受体调节Smads(Smad2，Smad3)，并与共介导Smads(Smad4)结合形成异源复合物，转位到细胞核，调节靶基因的转录。TGF-β1是TGF-β家族的主要成员，多数肿瘤中可以观察到TGF-β_1的高表达，但其机制尚未完全阐明。Smad4是一种新近发现的抑癌基因，在TGF-β信号转导通路中具有重要作用，在人类多种肿瘤中表达水平降低。TGF-β信号转导通路的异常常见于一些来源于上皮细胞的肿瘤发展的晚期，并且与肿瘤的浸润及转移密切相关。
许多研究表明，肿瘤的生长是血管依赖性的，血管生成在肿瘤的浸润及转移中发挥了重要作用。肿瘤的血管生成受到许多癌基因，抑癌基因及生长因子的影响。文献报道，TGF-β1在多种肿瘤的血管生成中起重要作用，Smad4则可以抑制肿瘤的血管生成，但它们在大肠癌中的作用尚无定论。微血管密度(Microvesseldensity，MVD)是衡量血管生成的定量指标。CD34是特异性最强的血管内皮细胞
郑州大学2004年硕士论文
人大肠肿瘤TGF一阳和smad4的表达及其与血管生成的关系
标记物，可用来检测MVD。本文通过检测大肠腺癌中TGF一pl，Smad4的表达及
其相互关系，并探讨二者同MVD的关系，旨在阐明TGF一B信号转导通路障碍在
大肠癌发生及浸润和转移中的可能机制，从而为大肠癌的诊断、治疗及预后判断
提供一条新的途径。
材料与方法:(l)组织标本来源于郑州大学第一附属医院及郑州铁路局中心
医院1999一2003年间手术切除标本。共选取64例大肠腺癌标本、24例大肠腺瘤
标本、10例经病理证实为正常的癌旁组织标本，所有标本均经10%甲醛固定，常
规脱水后石蜡包埋。(2)免疫组化sp法(streptavidin-peroxidase，sP)检测ToF一日l，
Smad4及CD34在大肠正常粘膜、大肠腺瘤及大肠腺癌中的表达情况并计数MVD。
(3)统计工具为sPsslo.0版软件包，统计方法采用xZ检验、spe~an等级相关分
析、t检验以及方差分析，并以尸<0.05作为有统计学意义的判定标准。
结果:(l)TGF一m主要表达于细胞浆，偶见细胞核中也有表达，TGF一pl在正
常大肠粘膜及腺瘤组织中着色较均匀，染色浅淡;在大肠腺癌组织中成棕黄色或
棕褐色。Smad4的着色部位主要在细胞浆中，在细胞膜上也有着色，可见胞浆、
胞膜中的棕黄色和棕褐色的颗粒状物质。CD34阳性反应物质主要位于血管内皮细
胞的胞浆。TGF一州在大肠腺癌中的阳性表达率为70.31%，高于大肠腺瘤组的
33.33%和癌旁正常大肠粘膜组的30.00%，差异均具有统计学意义(P<0 .05)，而大
肠腺瘤组与癌旁正常大肠粘膜组比较，TGF一印的阳性表达率虽然升高，但不具有
统计学意义(P>0.05)。Smad4在大肠腺癌组中的阳性表达率为46.88%，与大肠腺
瘤组的83.33%和癌旁正常大肠粘膜组的90.00%相比明显降低护<0.05)，大肠腺瘤
与癌旁正常大肠粘膜组间比较无明显差异(外0.05)。大肠腺癌组MVD平均值为
26.85士8.27，在大肠腺瘤组和癌旁正常大肠粘膜组MVD平均值分别为14.36士7.43
和8.83士4.94，三组间两两相比均具有显著性差异(P<0.05)，大肠腺癌组MVD显著
高于大肠腺瘤组和癌旁正常大肠粘膜组，大肠腺瘤组的MVD也明显高于癌旁正常
大肠粘膜组。
(2)根据大肠腺癌组织分化程度不同将大肠腺癌分为高/中分化组和低分化
组，TGF一盯在高/中分化组和低分化组大肠腺癌的阳性表达率分别为69.05%、
72.73%，两组间比较无显著性差异(P>0.05)。Smad4在高/中分化组和低分化组
大肠腺癌的阳性表达率分别为50.00%、40.91%，呈一定的下降趋势，但两组间比
郑州大学2004年硕士论文
人大肠肿瘤TGF一娜和Smad4的表达及其与血管生成的关系
较亦无显著性差异(P>0.05)。MVD平均值在高/中分化组和低分化组大肠腺癌分
别为23.81士6.90和32.65士7.63，两组间比较有显著性差异(P<0.01)。
(3)根据癌细胞在肠壁内浸润深度的不同，可将大肠腺癌分为浸润未达外膜层
组和浸润达到或超过外膜层组。TGF一邵在此两组内的阳性表达率分别为48.00%和
84.62%，随浸润深度的增加呈上升趋势，两组间比较差异有显著性(P<0 .05)。Smad4
在此两组内的阳性表达率分别为64.00%和35.90%，随浸润深度的增加而呈一定的
下降趋势，两组间比较差异亦有显著性护<0.05)。两组的MVD平均值分别为19.93
士5.01和31.26士6.73，随浸润深度的增加呈一定的上升趋势，两组间比较差异有
显著性(尸<0.01)。
(4)根据大肠腺癌有?
Background and objectives: Colorectal carcinoma is one of the common malignant tumors that its incidence rate and death rate both rank the forefront of all the malignant tumors. As other tumors, colorectal carcinoma also has characteristics such as invasion and metastasis in process of its occurrence and development, but the mechanism is not fully understood. Recent studies have showed that secretive disorders and signaling pathway disturbance of transforming growth factor (TGF- ) are closely related to the progression and aggressiveness of malignant tumors.
TGF- signal transduction is involved in fundamental biological processes, such as cell growth regulation, differentiation, adhension, the formation of extracellullar matrix (ECM), immune regulation and cell apoptosis. Recent progress has led to the elucidation of a general TGF- B signal pathway: Firstly, TGF- ligand adheres to transforming growth factor B receptor I , II (T B R I ,T R II) and T R I is phosphorylated by T B RII. Then, Smad2 and Smad3 are activated by T B R I and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. TGF- B 1 is a major member of TGF- B family and it expresses a high level in many tumor tissues, but its mechanism is not clear. Smad4, a new anti-oncogene, plays a key role in TGF- B signal transduction and express a low level in many human tumor tissues. The disorders of TGF- B signal transduction are always closely to the invasion and metastasis of tumors.
Researches have indicated that the growth of tumor is dependent on angiogenesis.
Angiogenesis of tumors plays an important role in tumor cells' invasion and metastasis. Microvessel density (MVD) is quantitative indicator of angiogenesis. In addition, angiogenesis is also mediated by proto-oncogene, anti-oncogene and some growth factors. Studies have showed that TGF- P 1 plays an important role in angiogenesis of many tumors while Smad4 is an inhibitor of angiogenesis, but there exist lots of differences in colorectal carcinoma. In order to investigate the possible mechanism of colorectal carcinoma , an immunohistochemical streptavidin-peroxidase (SP) method was used to exmine the expression of TGF- P 1 , Smad4 and MVD in colorectal carcinoma and the correlation between these three proteins.
Materials and methods: (1) 64 surgically resected carcinoma of colorectal carcinoma samples, 24 adenoma samples and 1 0 normal mocosa samples of colorectal carcinoma which were adjacent to carcinoma mucosa, were confirmed pathologically. All the tissues were fixed in 10% neutral formalin and embedden in paraffin. (2) SP immunohistochemical staining technique was used to exmine the expression of TGF- P 1 , Smad4 and CD34 in normal mucosa, adenoma and colorectal carcinoma. (3) The data
were analyzed by software SPSS 10.0. x -test or t-test was used to analysize the difference between different groups. P value < 0.05 was considered as statistically significant.
Results: (1) TGF- P 1 protein was stained mainly in the cytoplasm of cells .Normal epithelial cells and adenoma were homogeneously stained by TGF- P 1 , whereas the positive tumor cells were heterogeneously distributed within colorectal carcinoma. Smad4 protein was stained mainly in the cytoplasm of cells, and occasionally evident in membrane of cells. CD34 protein was stained mainly in the cytoplasm of vascular endothelial cells. The positive rates of TGF- P 1 in colorectal carcinoma group, colorectal adenoma group and normal mocosa group were 70.31%, 33.33% and 30% respectively. There were significant differences (P<0.05) between normal mocosa group or adenoma group and carcinoma group, but no differences were observed between normal mocosa group and adenoma group. The positive rates of Smad4 in colorectal carcinoma group, colorectal adenoma group and normal mocosa group were 46.88%, 83.33% and 90% respectively. There were significant differences (P<0.05) between
normal mocosa group or adenoma group and carcinoma group, but no significance differ
TGF-β是一类多功能的多肽类生长因子，对细胞的增殖与分化，细胞外基质的产生，细胞凋亡及机体的免疫系统均起着重要的调节作用。活化的TGF-β与其Ⅱ型受体(transforming growth factor β receptorⅡ，TβRⅡ)结合从而形成TGF-β／TβRⅡ复合物并迅速导致Ⅰ型受体(transforming growth factor β receptor Ⅰ，TβRⅠ)的磷酸化。磷酸化的TβRⅠ可磷酸化受体调节Smads(Smad2，Smad3)，并与共介导Smads(Smad4)结合形成异源复合物，转位到细胞核，调节靶基因的转录。TGF-β1是TGF-β家族的主要成员，多数肿瘤中可以观察到TGF-β_1的高表达，但其机制尚未完全阐明。Smad4是一种新近发现的抑癌基因，在TGF-β信号转导通路中具有重要作用，在人类多种肿瘤中表达水平降低。TGF-β信号转导通路的异常常见于一些来源于上皮细胞的肿瘤发展的晚期，并且与肿瘤的浸润及转移密切相关。
许多研究表明，肿瘤的生长是血管依赖性的，血管生成在肿瘤的浸润及转移中发挥了重要作用。肿瘤的血管生成受到许多癌基因，抑癌基因及生长因子的影响。文献报道，TGF-β1在多种肿瘤的血管生成中起重要作用，Smad4则可以抑制肿瘤的血管生成，但它们在大肠癌中的作用尚无定论。微血管密度(Microvesseldensity，MVD)是衡量血管生成的定量指标。CD34是特异性最强的血管内皮细胞
郑州大学2004年硕士论文
人大肠肿瘤TGF一阳和smad4的表达及其与血管生成的关系
标记物，可用来检测MVD。本文通过检测大肠腺癌中TGF一pl，Smad4的表达及
其相互关系，并探讨二者同MVD的关系，旨在阐明TGF一B信号转导通路障碍在
大肠癌发生及浸润和转移中的可能机制，从而为大肠癌的诊断、治疗及预后判断
提供一条新的途径。
材料与方法:(l)组织标本来源于郑州大学第一附属医院及郑州铁路局中心
医院1999一2003年间手术切除标本。共选取64例大肠腺癌标本、24例大肠腺瘤
标本、10例经病理证实为正常的癌旁组织标本，所有标本均经10%甲醛固定，常
规脱水后石蜡包埋。(2)免疫组化sp法(streptavidin-peroxidase，sP)检测ToF一日l，
Smad4及CD34在大肠正常粘膜、大肠腺瘤及大肠腺癌中的表达情况并计数MVD。
(3)统计工具为sPsslo.0版软件包，统计方法采用xZ检验、spe~an等级相关分
析、t检验以及方差分析，并以尸<0.05作为有统计学意义的判定标准。
结果:(l)TGF一m主要表达于细胞浆，偶见细胞核中也有表达，TGF一pl在正
常大肠粘膜及腺瘤组织中着色较均匀，染色浅淡;在大肠腺癌组织中成棕黄色或
棕褐色。Smad4的着色部位主要在细胞浆中，在细胞膜上也有着色，可见胞浆、
胞膜中的棕黄色和棕褐色的颗粒状物质。CD34阳性反应物质主要位于血管内皮细
胞的胞浆。TGF一州在大肠腺癌中的阳性表达率为70.31%，高于大肠腺瘤组的
33.33%和癌旁正常大肠粘膜组的30.00%，差异均具有统计学意义(P<0 .05)，而大
肠腺瘤组与癌旁正常大肠粘膜组比较，TGF一印的阳性表达率虽然升高，但不具有
统计学意义(P>0.05)。Smad4在大肠腺癌组中的阳性表达率为46.88%，与大肠腺
瘤组的83.33%和癌旁正常大肠粘膜组的90.00%相比明显降低护<0.05)，大肠腺瘤
与癌旁正常大肠粘膜组间比较无明显差异(外0.05)。大肠腺癌组MVD平均值为
26.85士8.27，在大肠腺瘤组和癌旁正常大肠粘膜组MVD平均值分别为14.36士7.43
和8.83士4.94，三组间两两相比均具有显著性差异(P<0.05)，大肠腺癌组MVD显著
高于大肠腺瘤组和癌旁正常大肠粘膜组，大肠腺瘤组的MVD也明显高于癌旁正常
大肠粘膜组。
(2)根据大肠腺癌组织分化程度不同将大肠腺癌分为高/中分化组和低分化
组，TGF一盯在高/中分化组和低分化组大肠腺癌的阳性表达率分别为69.05%、
72.73%，两组间比较无显著性差异(P>0.05)。Smad4在高/中分化组和低分化组
大肠腺癌的阳性表达率分别为50.00%、40.91%，呈一定的下降趋势，但两组间比
郑州大学2004年硕士论文
人大肠肿瘤TGF一娜和Smad4的表达及其与血管生成的关系
较亦无显著性差异(P>0.05)。MVD平均值在高/中分化组和低分化组大肠腺癌分
别为23.81士6.90和32.65士7.63，两组间比较有显著性差异(P<0.01)。
(3)根据癌细胞在肠壁内浸润深度的不同，可将大肠腺癌分为浸润未达外膜层
组和浸润达到或超过外膜层组。TGF一邵在此两组内的阳性表达率分别为48.00%和
84.62%，随浸润深度的增加呈上升趋势，两组间比较差异有显著性(P<0 .05)。Smad4
在此两组内的阳性表达率分别为64.00%和35.90%，随浸润深度的增加而呈一定的
下降趋势，两组间比较差异亦有显著性护<0.05)。两组的MVD平均值分别为19.93
士5.01和31.26士6.73，随浸润深度的增加呈一定的上升趋势，两组间比较差异有
显著性(尸<0.01)。
(4)根据大肠腺癌有?
Background and objectives: Colorectal carcinoma is one of the common malignant tumors that its incidence rate and death rate both rank the forefront of all the malignant tumors. As other tumors, colorectal carcinoma also has characteristics such as invasion and metastasis in process of its occurrence and development, but the mechanism is not fully understood. Recent studies have showed that secretive disorders and signaling pathway disturbance of transforming growth factor (TGF- ) are closely related to the progression and aggressiveness of malignant tumors.
TGF- signal transduction is involved in fundamental biological processes, such as cell growth regulation, differentiation, adhension, the formation of extracellullar matrix (ECM), immune regulation and cell apoptosis. Recent progress has led to the elucidation of a general TGF- B signal pathway: Firstly, TGF- ligand adheres to transforming growth factor B receptor I , II (T B R I ,T R II) and T R I is phosphorylated by T B RII. Then, Smad2 and Smad3 are activated by T B R I and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. TGF- B 1 is a major member of TGF- B family and it expresses a high level in many tumor tissues, but its mechanism is not clear. Smad4, a new anti-oncogene, plays a key role in TGF- B signal transduction and express a low level in many human tumor tissues. The disorders of TGF- B signal transduction are always closely to the invasion and metastasis of tumors.
Researches have indicated that the growth of tumor is dependent on angiogenesis.
Angiogenesis of tumors plays an important role in tumor cells' invasion and metastasis. Microvessel density (MVD) is quantitative indicator of angiogenesis. In addition, angiogenesis is also mediated by proto-oncogene, anti-oncogene and some growth factors. Studies have showed that TGF- P 1 plays an important role in angiogenesis of many tumors while Smad4 is an inhibitor of angiogenesis, but there exist lots of differences in colorectal carcinoma. In order to investigate the possible mechanism of colorectal carcinoma , an immunohistochemical streptavidin-peroxidase (SP) method was used to exmine the expression of TGF- P 1 , Smad4 and MVD in colorectal carcinoma and the correlation between these three proteins.
Materials and methods: (1) 64 surgically resected carcinoma of colorectal carcinoma samples, 24 adenoma samples and 1 0 normal mocosa samples of colorectal carcinoma which were adjacent to carcinoma mucosa, were confirmed pathologically. All the tissues were fixed in 10% neutral formalin and embedden in paraffin. (2) SP immunohistochemical staining technique was used to exmine the expression of TGF- P 1 , Smad4 and CD34 in normal mucosa, adenoma and colorectal carcinoma. (3) The data
were analyzed by software SPSS 10.0. x -test or t-test was used to analysize the difference between different groups. P value < 0.05 was considered as statistically significant.
Results: (1) TGF- P 1 protein was stained mainly in the cytoplasm of cells .Normal epithelial cells and adenoma were homogeneously stained by TGF- P 1 , whereas the positive tumor cells were heterogeneously distributed within colorectal carcinoma. Smad4 protein was stained mainly in the cytoplasm of cells, and occasionally evident in membrane of cells. CD34 protein was stained mainly in the cytoplasm of vascular endothelial cells. The positive rates of TGF- P 1 in colorectal carcinoma group, colorectal adenoma group and normal mocosa group were 70.31%, 33.33% and 30% respectively. There were significant differences (P<0.05) between normal mocosa group or adenoma group and carcinoma group, but no differences were observed between normal mocosa group and adenoma group. The positive rates of Smad4 in colorectal carcinoma group, colorectal adenoma group and normal mocosa group were 46.88%, 83.33% and 90% respectively. There were significant differences (P<0.05) between
normal mocosa group or adenoma group and carcinoma group, but no significance differ
引文
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16 Weinder N, Semple J, Welch WR, et al. Tumor angiogenesis and metastasis relation in invasive breast carcinoma. N Eng J Med, 1991, 324: 1-8.
17 Heldin CH, Miyazono K, ten Dijke P. TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997,390(6659):465-471.
18 Imai Y, Tsurutani N, Oda H, et al. Genetic instability and mutation of the TGF-β receptor Ⅱ gene in ampullay carcinoma. Int J Cancer, 1998, 76:407-411.
19 Cardillo MR, Yap E. TGF-β 1 in colonic neoplasmia: a genetic molecular and immunohistochemical study. J Exp Clin Cancer Res, 1997, 16:281-288.
20 Massague J. TGF- β signal transduction. Annu Rev Biochem, 1998, 67: 753-791.
21 Sporm AB, Robert AB, Wakefield LM, et al. Transforming growth factor-β: biological function and chemical structure. Science, 1986, 233(4763): 532-534.
22 Miyazonok. TGF-beta/SMAD signaling and its involvement in tumor progression. Biol Pharm Bull, 2000, 23 (10): 1125-1130.
23 Nalf 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.
24 Markus, Toshiguki J, Hetmut F, et al. Differential lociation of TGF-β isoform in human gastric mucosa and overpression in GC. Int J Cancer, 1997, 71(2): 131-137.
25 王朝晖，张雪梅，战敏等．大肠癌转化生长因子β及其受体的表达．世界华人消化杂志，2001，9(4)：462-463．
26 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
27 Shim KS, Kim KH, Han WS, et al. Elevated serum levels of TGF-β1 in patients with colorectal carcinioma: its association with tumor progression and its significant decrease after curative surgical resection. Cancer, 1999, 85(3):554-561.
28 Tsushima H, Ito N, Tamura S, et al, Circulating transforming growth factor beta 1 as a predictor of liver metastasis after resection in colorectal cancer. Clin Cancer Res. 2001, 7(5):1258-1262.
29 Platten M, Wick W, Weller M. Malianant glioma biology: Role for TGF-β in growth motility, angiogenesis, and immune escapes. Microsc Res Tech, 2001, 52(4): 401-410.
30 Santibanez JF, Guerrero J, Quintanilla M, et al. Transforming growth factor betal modulates matrix metaloproteinase-9 production through the Ras/MAPK signaling pathway in transformedkeratinocytes. Biochem Biophys Res Commun, 2002, 296(27): 267—273.
31 Saha D, Datta PK, Sheng H, et al. Synergistic induction of cyclooxygenase-2 by transforming growth factor betal and epidermal growth factor inhibits apoptosiss in epithelial cells. Neoplasia, 1999, 1(6): 508-517.
32 Massague J. How cells read TGF-β signals. Nature Rev Molecular Cell Biol, 2000,1(3): 169-177.
33 Derynck R, Zhang Y, Feng XH. Smads: Transcriptional activators of TGF-β responses. Cell, 1998, 95(6): 737-740.
34 Steven A, Mark Z, Manju S, et al. Smad3 represses androgen receptor-mediated transcription. Cancer Res, 2001, 61 (5): 2112-2118.
35 Nakao A, Imamura T, Kawabata M, et al. TGF- β recepter-mediated signaling through Smad2, Smad3 and Smad4. EMBO J, 1997, 16 (17): 5353-60
36 Zhang Y, Musci T, Derynck R. The tumor suppressor Smad4/DPC4 as a central mediator of Smad function. Curr Biol, 1997, 7(4): 270-273
37 Hahn SA, Schutte M, Hoque AT, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science, 1996, 271 (5247): 350-353.
38 Hunt KK, Fleming JB, Abramian A, et al. Overexpression of tumor suppressor gene smad_4/DPC4 induces p21 wafl expression and growth inhibition in human
carcinoma cells. Cancer Res, 1998, 58(24): 5656-5661
39 Feng XH, Lin X, Derynck R. Smad_2, Smad_3 and Smad_4 cooperate with sp1 to induce p15 (Ink4B) transcription in response to TGF-beta. EMBO J, 2000, 19(19): 5178-5193
40 Salovaara R, Roth S, Loukola A, et al. Frequent loss of SMAD4/DPC4 protein in colorectal cancers. Gut, 2002, 51(1): 56-59.
41 Boulay JL, Mild G, Reuter J, et al. Combined copy status of 18q21 genes in colorectal cancer shows frequent retention of Smad7. Gene Chromosomes Cancer, 2001, 31(3); 240.
42 Miyaki M, Iijima T, Konishi M, et al. Higher frequency of Smad4 gene mutation in human colorectal cancerwith distant metastasis. Oncogene, 1999,18(20): 3098-3103.
43 Ohtaki N, Yamaguchi A, Goi T, et al. Somatic alterations of the DPC4 and Madr2 genes in colorectal cancers and relationship to metastasis. Int J Oncol. 2001, 18(2): 265-270.
44 Folkman J. What is the evidence that tumor are angiogenesis dependent? J Natl Cancer Inst, 1999,82; 4-6.
45 Basari S, Delellis Ra, Healt;ey GJ. Microvessel quantity and prognosis in invasive breast carcinoma. Hum Pathol, 1992, 23: 755-761.
46 Maedak, Change YS, gawa Y, et al. Prognosis value of vascular endothelial growth factor expression in gastric carcinoma. Cancer, 1996, 77:858-863.
47 Takahashi Y, Kitadai, bucana CD, et al. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res, 1995, 559(18): 3964-3968.
48 Nakasaki T, Wada H, Shigemori C, et al. Expression of tissue factor and vascular endothelial growth factor is associated with angiogenesis in colorectal cancer. Am J Hematol, 2002, 69(4): 247-254.
49 Nanashima A, Yamaguchi H, Sawai T, et al. Prognostic factors in hepatic metastasis of colorectal carcinoma: immunohistochemical analysis of tumor biological factors. Dig Dis Sci, 2001, 46: 1623-1628.
50 Kim KY, Teong SY, Won J, et al. Induction of angiogenesis by expression of soluble type Ⅱ transforming growth factor-beta receptor in mouse hepatoma. J Biol Chem, 2001, 276(42): 38781-38786.
51 Pepper MS. Transforming growth factor-beta: vasculogenesis , angiogenesis and vessel wall integrity. Cytokine Growth Factor Rev, 1997, 8: 21-43.
52 Relf M, LeJeune S, Scott PA, et al. Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth thctor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis. Cancer Res. 1997,57(5): 963-969.
53 Teraoka H, Sawada T, Nishihara T, et al. Enhanced VEGF production and decreased immuneogenicity induced by TGF-β1 promote liver metastasis of pancreastic cancer. Br J Cancer, 2002, 85(4): 612-617.
54 罗庚求，李景和，文继舫等．DPC4基因转染结肠癌细胞对VEGF表达的影响．临床与实验病理学杂志，2003，19(6)：626-629．
55 Xiong B, Gong LL, Zhang F. TGF betal expression and angiogenesis in colorectal cancer tissue. World J Gastroenterol. 2002, 8:(3): 496-498.
56 Zawel L, Dai J, Buckhaults S, et al. Human Smad3 and Smad4 are sequence specific transcription activators. Mol Cell, 1998, 1 (4): 611-617.
57 Dennier S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF beta inducible elements in the promotor of human plasminogen activator inhibitor type 1 gene. EMBOJ. 1998, 17(11): 3091-3100.
2 Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor in human disease.N Engl J Med, 2000,342(18): 1350-1358.
3 Robert AB. TGF-beta signaling from receptorstothe nucleus. Microbes Infect, 1999,1(15): 1265-1273.
4 Massague J. The transforming growth factor-beta family. Annu Rev Cell Biol, 1990, 6: 597-641.
5 Alexandrow MG, Moses HL. Transforming growth factor beta and cell c ycle regulation. Cancer Res, 1995, 55(7): 1452-1457.
6 Derynck R, Feng XH. TGF-β receptor signaling. Biochim Biophys Acta. 1997, 1333(1): 105-150.
7 Kretzschmar M, Doody J, Massague J. Opposing BMP and EGF signaling pathway converge on the TGF-β family mediator Smadl. Nature, 1997, 389: 618-622.
8 Kerbel RS. Tumor angiogenesis: past, present and the near future. Carcinogenesis, 2000, 21(3): 505-515.
9 Carmeliet P, Jain R. Angiogenesis in cancer and other disease. Nature, 2000, 407(6801): 249-257.
10 Saito H, Tsujitani S, Oka S, et al. The expression of transforming growth factor-betal 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.
11 Benckert C, Jonas S, Cramer T. Transforming growth factor beta 1 stimulates vascular endothelial growth factor gene transcription in human cholangiocellular carcinoma cells. Cancer Res. 2003,63(5): 1083-1092.
12 Breier G, Blum S, Peli J. Transforming growth factor-beta and Ras regulate the VEGF/VEGF-receptor system during tumor angiogenesis. Int J Cancer. 2002, 97(2):
142-8.
13 Schwarte-Waldhoff I, Volpert OV, Bouck NP, et al.SMAD/DPC4-mediated tumor suppression through suppression of angiogenesis. Proc Natl Sci USA, 2000, 97(17): 9624-9629
14 李景和，文继舫，曾庆富等．大肠癌转化生长因子β受体及其下游分子Mad2基因表达的研究．中华消化杂志，2000，20(2)：125-126．
15 李东印，马玉泉，李克．胃癌中E-钙粘蛋白的表达意义．世界华人消化杂志，2001，9(8)：974—975．
16 Weinder N, Semple J, Welch WR, et al. Tumor angiogenesis and metastasis relation in invasive breast carcinoma. N Eng J Med, 1991, 324: 1-8.
17 Heldin CH, Miyazono K, ten Dijke P. TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997,390(6659):465-471.
18 Imai Y, Tsurutani N, Oda H, et al. Genetic instability and mutation of the TGF-β receptor Ⅱ gene in ampullay carcinoma. Int J Cancer, 1998, 76:407-411.
19 Cardillo MR, Yap E. TGF-β 1 in colonic neoplasmia: a genetic molecular and immunohistochemical study. J Exp Clin Cancer Res, 1997, 16:281-288.
20 Massague J. TGF- β signal transduction. Annu Rev Biochem, 1998, 67: 753-791.
21 Sporm AB, Robert AB, Wakefield LM, et al. Transforming growth factor-β: biological function and chemical structure. Science, 1986, 233(4763): 532-534.
22 Miyazonok. TGF-beta/SMAD signaling and its involvement in tumor progression. Biol Pharm Bull, 2000, 23 (10): 1125-1130.
23 Nalf 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.
24 Markus, Toshiguki J, Hetmut F, et al. Differential lociation of TGF-β isoform in human gastric mucosa and overpression in GC. Int J Cancer, 1997, 71(2): 131-137.
25 王朝晖，张雪梅，战敏等．大肠癌转化生长因子β及其受体的表达．世界华人消化杂志，2001，9(4)：462-463．
26 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
27 Shim KS, Kim KH, Han WS, et al. Elevated serum levels of TGF-β1 in patients with colorectal carcinioma: its association with tumor progression and its significant decrease after curative surgical resection. Cancer, 1999, 85(3):554-561.
28 Tsushima H, Ito N, Tamura S, et al, Circulating transforming growth factor beta 1 as a predictor of liver metastasis after resection in colorectal cancer. Clin Cancer Res. 2001, 7(5):1258-1262.
29 Platten M, Wick W, Weller M. Malianant glioma biology: Role for TGF-β in growth motility, angiogenesis, and immune escapes. Microsc Res Tech, 2001, 52(4): 401-410.
30 Santibanez JF, Guerrero J, Quintanilla M, et al. Transforming growth factor betal modulates matrix metaloproteinase-9 production through the Ras/MAPK signaling pathway in transformedkeratinocytes. Biochem Biophys Res Commun, 2002, 296(27): 267—273.
31 Saha D, Datta PK, Sheng H, et al. Synergistic induction of cyclooxygenase-2 by transforming growth factor betal and epidermal growth factor inhibits apoptosiss in epithelial cells. Neoplasia, 1999, 1(6): 508-517.
32 Massague J. How cells read TGF-β signals. Nature Rev Molecular Cell Biol, 2000,1(3): 169-177.
33 Derynck R, Zhang Y, Feng XH. Smads: Transcriptional activators of TGF-β responses. Cell, 1998, 95(6): 737-740.
34 Steven A, Mark Z, Manju S, et al. Smad3 represses androgen receptor-mediated transcription. Cancer Res, 2001, 61 (5): 2112-2118.
35 Nakao A, Imamura T, Kawabata M, et al. TGF- β recepter-mediated signaling through Smad2, Smad3 and Smad4. EMBO J, 1997, 16 (17): 5353-60
36 Zhang Y, Musci T, Derynck R. The tumor suppressor Smad4/DPC4 as a central mediator of Smad function. Curr Biol, 1997, 7(4): 270-273
37 Hahn SA, Schutte M, Hoque AT, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science, 1996, 271 (5247): 350-353.
38 Hunt KK, Fleming JB, Abramian A, et al. Overexpression of tumor suppressor gene smad_4/DPC4 induces p21 wafl expression and growth inhibition in human
carcinoma cells. Cancer Res, 1998, 58(24): 5656-5661
39 Feng XH, Lin X, Derynck R. Smad_2, Smad_3 and Smad_4 cooperate with sp1 to induce p15 (Ink4B) transcription in response to TGF-beta. EMBO J, 2000, 19(19): 5178-5193
40 Salovaara R, Roth S, Loukola A, et al. Frequent loss of SMAD4/DPC4 protein in colorectal cancers. Gut, 2002, 51(1): 56-59.
41 Boulay JL, Mild G, Reuter J, et al. Combined copy status of 18q21 genes in colorectal cancer shows frequent retention of Smad7. Gene Chromosomes Cancer, 2001, 31(3); 240.
42 Miyaki M, Iijima T, Konishi M, et al. Higher frequency of Smad4 gene mutation in human colorectal cancerwith distant metastasis. Oncogene, 1999,18(20): 3098-3103.
43 Ohtaki N, Yamaguchi A, Goi T, et al. Somatic alterations of the DPC4 and Madr2 genes in colorectal cancers and relationship to metastasis. Int J Oncol. 2001, 18(2): 265-270.
44 Folkman J. What is the evidence that tumor are angiogenesis dependent? J Natl Cancer Inst, 1999,82; 4-6.
45 Basari S, Delellis Ra, Healt;ey GJ. Microvessel quantity and prognosis in invasive breast carcinoma. Hum Pathol, 1992, 23: 755-761.
46 Maedak, Change YS, gawa Y, et al. Prognosis value of vascular endothelial growth factor expression in gastric carcinoma. Cancer, 1996, 77:858-863.
47 Takahashi Y, Kitadai, bucana CD, et al. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res, 1995, 559(18): 3964-3968.
48 Nakasaki T, Wada H, Shigemori C, et al. Expression of tissue factor and vascular endothelial growth factor is associated with angiogenesis in colorectal cancer. Am J Hematol, 2002, 69(4): 247-254.
49 Nanashima A, Yamaguchi H, Sawai T, et al. Prognostic factors in hepatic metastasis of colorectal carcinoma: immunohistochemical analysis of tumor biological factors. Dig Dis Sci, 2001, 46: 1623-1628.
50 Kim KY, Teong SY, Won J, et al. Induction of angiogenesis by expression of soluble type Ⅱ transforming growth factor-beta receptor in mouse hepatoma. J Biol Chem, 2001, 276(42): 38781-38786.
51 Pepper MS. Transforming growth factor-beta: vasculogenesis , angiogenesis and vessel wall integrity. Cytokine Growth Factor Rev, 1997, 8: 21-43.
52 Relf M, LeJeune S, Scott PA, et al. Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth thctor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis. Cancer Res. 1997,57(5): 963-969.
53 Teraoka H, Sawada T, Nishihara T, et al. Enhanced VEGF production and decreased immuneogenicity induced by TGF-β1 promote liver metastasis of pancreastic cancer. Br J Cancer, 2002, 85(4): 612-617.
54 罗庚求，李景和，文继舫等．DPC4基因转染结肠癌细胞对VEGF表达的影响．临床与实验病理学杂志，2003，19(6)：626-629．
55 Xiong B, Gong LL, Zhang F. TGF betal expression and angiogenesis in colorectal cancer tissue. World J Gastroenterol. 2002, 8:(3): 496-498.
56 Zawel L, Dai J, Buckhaults S, et al. Human Smad3 and Smad4 are sequence specific transcription activators. Mol Cell, 1998, 1 (4): 611-617.
57 Dennier S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF beta inducible elements in the promotor of human plasminogen activator inhibitor type 1 gene. EMBOJ. 1998, 17(11): 3091-3100.