β-catenin、nm23H_1、Tiam-1与胃癌生物学行为关系的研究
摘要
前言
肿瘤的发生、发展是一个多因素、多步骤、多阶段的过程,其中涉及多种癌基因、抑癌基因、错配修复基因、细胞黏附因子等的异常改变,而转移是肿瘤治疗失败的主要原因。肿瘤转移是一个多步骤的复杂过程,多种基因结构和功能的异常在这一过程中发挥重要作用。肿瘤细胞的侵袭、转移与其运动能力密不可分,细胞骨架在维持细胞形态和运动的同时,还可以整合内、外源信号,参与细胞分泌、接触抑制、增殖和凋亡等多种生物活动。β-catenin是细胞中的一种多功能蛋白质,很多肿瘤的预后与β-catenin有相关性。Tiam-1是一个细胞骨架调节因子,而β-catenin具有细胞黏附和信号传导功能。nm23是近年来研究较多的肿瘤转移抑制基因,但是,不同的研究发现,在不同类型的肿瘤细胞中,nm23的作用不同。即使同样是胃癌,其作用也不尽相同。本研究主要探讨三者在胃癌发展中的相互关系。
材料与方法
1.病例选择:收集2003-2005年中国医科大学附属第一医院肿瘤科术后大体标本,共63例,所有患者术前均未进行化疗或者放疗,年龄范围26-78岁,平均年龄56岁,其中高分化腺癌30例,低分化腺癌30例,未分化癌3例。同时收集中国医科大学附属第一医院内镜科活检标本,慢性浅表性胃炎30例,年龄范围25.68岁,平均年龄46岁。
2.取材:标本一部分于10%福尔马林中固定,石蜡包埋,另外留取新鲜标本,剪成小块立即放入液氮,并转入—80℃冰箱保存。
3.β-catenin鼠抗人单克隆抗体及nm23 H_1单克隆抗体工作液、PV9000试剂盒购自北京中杉金桥生物技术公司。DAB显色试剂盒购自福州迈新试剂公司。经Gene Bank检索,利用Primer 5.0软件设计β-catenin、
PREFACEThe pathogenesis of tumor is a process of multiple factors, multiple steps and many stages, which concerned with abnormalities of many oncogenes, tumor suppressor genes, mismatch repair genes, cellular adhesive factors etc. Tumor metastasis is a complex process of multiple steps, in which abnormalities of many gene structure and function play an important role. Cell skeleton can maintain cell shape and motion, also can participate in many biological activities such as cell secretion, contact inhibition, proliferation and apoptosis through integrating internal and external signals.p-catenin is a multifunctional protein in cell, and it is related to prognosis of many tumors. Tiam-1 is a regulater of cell skeleton, and P-catenin has cell adhesive and signal conducting function. nm23 is a tumor suppressor gene, however, different studies found that in different kind of tumor cells the effects of nm23 were different.Our purpose was to investigate the relationship of P-catenin, nm23 and Tiam-1 during the process of gastric carcinoma.MATERIALS and METHODS1. Cases: 63 samples after surgical operation were collected from Oncology Department of the first affiliated hospital of China Medical University from 2003 to 2005. All the patients didn't undergo chemotherapy or radiotherapy before surgical operation. The mean age is 56 years (range 26-78 years). Well-differentiated adenocarcinoma are 30 cases, poorly differentiated 30 cases, undifferentiated 3 cases. Meantime 30 biopsy samples of superficial gastritis were collected from Endoscopy Center of the first affiliated hospital of China Medical University. The mean age is 46 years (range 25-68 years).
2. Samples: a part of samples were kept in 10% formalin and then embedded in paraffin. Other samples were collected fresh and cut into pieces, snap frozen in liquid nitrogen, and kept frozen at -80 °C.3. Working fluid of p-catenin mouse-human monoclone antibody and nm23H, monoclone antibody, PV9000 Kits were bought from Zhongshan Jinqiao biotechnology corporation, Beijing. DAB coloration kits were supplied by Maixin technique corporation, Fuzhou. Primers of P-catenin, nm23H, and Tiam-1 were searched by Gene Bank and designed by Primer 5.0 software. The primers were synthesized by Sanbo Yuanzhi biotechnology limited corporation, Beijing. Taq enzyme of RT-PCR, dNTP and marker were all bought from TaKaRa biology coporation, Dalian.4. Methods: Immunohistochemistry stain with two-step method. nm23H, was located mainly in cytoplasm, and also on membrane. It was brown granular or mass. P-catenin was located mainly in cytoblast, and also in cytoplasm with brown granular or mass.It was positive when the stained cell number was over 10%. It was negative when there was no stained cell or the stained cell number was under 10%. RNA were extracted according to TRIZOL agent directions. RNA purity and concentration were measured with ultraviolet spectrophotometer. RNA purity: OD260/OD280^ 1.65-1.96.cDNA inverse transcription: RNA 3ul. Reaction system: 2xbuffer 15ul,MgSO4(25mM)6ul,AMV(22u/ul) 1.5ul,dNTPs(10mM) 1.5ul, oligodT(50uM) 1.5ul,RNase-inhibitor(40u/ul) 0.75ul,ddH2O 0.75ul, do the process as follows:Inverse transcription reaction: 65 °C for 1 minute30 °C for 5 minutesTemperature increased gradually within 15-30minutes. 65 °C for 30 minutes
98 °C for 5 minutes5 °C for 5 minutesPCR reaction system: cDNA 3ul, 2Xbuffer 2.5ul, MgSO4(25mM)6ul, dNTPs(2.5mM)2ul, Taq-E(5u/ul)0.2ul, ddH2O 17.1|il, primer of p-catenin 0.1^1(5pmol/^il)Dnm23H1 0.1 ul(5pmol/ul) and Tiam-1 0.1 ul (5pmol/ul).Run amplification as follows: pre-apomorphosis at 94 °C for 3 minutes, apomorphosis at 94°C for 40 seconds, anneal at 57°C for 1 minute, elongation at 72°C for 1 minute, repeat 35 times, finally elongation at 72°C for 7 minutes.p-actin was inner-control o Product of gene amplification went on electrophoresis by 2% agarose gel, and then took pictures. 5. Statistical treatment: x2 test was used with SPSS 12.0 software.RESULTSTable 1 Relationship between protein expression of P-catenin,nm23H, anddifferentiation degree_______________________________________________differentiation degree n |3 -catenin positive nm23H, positivewell-differentiation 30 io (33.3%) * 22 (73.3%) ** poor-differentiation 30 19 (63.3%) * 12 (40.0%) **undifferentiation________3 2 (73.3%)__________1 (33.3%)There is statistical difference of P -catenin between well-differentiated group and poor-differentiatedgroup (p<0.05).There is statistical difference of nm23Hl between well-differentiated group and poor-differentiated group
Table 2 Relationship between protein expression of |3-catenin, nm23H, and lymph node metastasislymph node metastasis n 0 -catenin positive nm23Hl positivePositive negative 38 25 23(60.5%) 8(32.0%) 16(42.1%) 19(76.0%)p value 0.0267 0.0081Table 3 Expression of p-catenin, nm23H, mRNA in gastric carcinoma and superficial gastritis n 0 -catenin positive nm23H , positivesuperficial gastritis gastric carcinoma 30 40 16 (53.32 24 (60.02 O 11 (36. 26 (65. 7%) 0%)p value 0.5770 0.0188 Table 4 Relationship between p-catenin,nm23H, mRNA and differentiation degreedifferentiation degree n P -catenin positive nm23H , positivewell-differentiation poor-differentiation 20 20 11 (55.09^ 13 (65.09< 0 ) / \ 0 ) 16 (80. 10 (50. 0%) 0%)p value 0.5186 0.0467
Table 5 Relationship between P-catenin,nm23Hi mRNA and lymph node metastasislymph node metastasis n &- catenin positive nm23H i positivepositive negative 23 17 14 10 (60.9%) (58.8%) 11 (47. 15 (88. 8%) 2%)p value 0.8961 0.0081 Table 6 Expression of |3-catenin, Tiam-1 mRNA in gastric carcinoma and superficial gastritis n 0- catenin positive Tiam-1 positivesuperficial gastritis gastric carcinoma 30 40 16 24 (53.3°/ (60.0°/ *) 8 (26.7%) 27 (67.5%)p value 0.5770 0.0007Table 7 Relationship between P-catenin,Tiam-l mRNA and differentiation degreedifferentiation degree n 0- ■catenin positive Tiam-1 positivewell-differentiation poor-differentiation 20 20 11 13 (55.0%) (65.0%) 10 (50 17 (85 .0%) .0%)p value 0.5186 0.0181 Table 8 Relationship between P-catenin, Tiam-1 mRNA and lymph node metastasislymph node metastasis n ￡ -catenin positive Tiam-1 positivepositive negative 23 17 14 (60.9%) 10 (58.8%) 19 (82.6%) 8 (47.1%)p value 0.8961 0.0176
DISCUSSIONp-catenin is a multifunctional protein, and has the function of cell adhesion and signal conducting. Majority |3-catenin in cell bind to E-cadherin, and bind to actin cell skeleton through a -catenin to participate in cell-cell adhesion and cell motion. Any change of the structure of E-cadherin-catenin complex will influence cell-cell junction. Increased intracellular P-catenin monomer binds to Tcf or Lef and enters into cytoblast. Tcf/Lef is DNA binding protein and acts as transcription factor to bind to corresponding DNA leading to continuous transcription of target gene.Diminishing or disappearing of P-catenin express in cytomembrane can cause that a -catenin can't bind to cadherin and thus affect normal cell-cell junction, therefore, adhesive function of tumor cells decrease and the tumor cells gain the function of metastasis and invasion. Nucleotide diphosphate kinase is coded by nm23 gene and exists extensively. It at least participates in two important functions in tumor growing, that is microtubular polymerization/disaggregation and signal conducting mediated by G-protein. In the process of signal conducting, GDP is dioxidated into GTP, and G-protein is activated, participate in tumor progress and metastasis. Tiam-1 gene can regulate cell adhesion mediated by E-cadherin, and participate in combination of adhesive complex together with Rho. The results showed that expression of p-catenin at cytomembrane disappeared in gastric carcinoma tissue, positive expression in cytoblasts was obviously higher than in superficial gastritis, and
expression was higher in poorly differentiated group and lymph node metastasis group than in well-differentiated group and no lymph node metastasis group. Positive expression of P-catenin mRNA exist both in gastric carcinoma tissue and superficial gastritis, and there was no statistical difference between them. There was no statistical difference in poorly differentiated group, well-differentiated group, lymph node metastasis and no lymph node metastasis group. Positive expression of nm23Hi in poorly differentiated group was obviously lower than in well-differentiated group,while positive expression of nm23Hi in lymph node metastasis group was obviously lower than in no lymph node metastasis group, suggested that expression of nm23 gene was related to the differentiation degree of gastric carcinoma and lymph node metastasis.nm23Hi may inhibit the expression of P-catenin.However,nm23Hi inhibiting Wnt conducting pathway may occur before or after P-catenin protein entering into cytoblast, rather than affect on mRNA. Positive expression of Tiam-1 mRNA in gastric carcinoma tissue was obviously higher than in normal tissue, and positive expression in poorly differentiated group and lymph node metastasis group was higher than in well-differentiated group and no lymph node metastasis group, suggested that Tiam-1 promote the process of malignancy.Although Tiam-1 can bind to E-cadherin at membrane-membrane junction, abnormal expression of P-catenin can cause abnormal linkage of cadherin-catenin between cells. While Tiam-1 promote tumor cells migration and participate in gene express control and cell proliferation, thus leading to tumor cell turning to malignancy* invasion and metastasis.
CONCLUSION1. Expression of P-catenin protein has positive correlation with differentiation degree of gastric carcinoma and metastasis. Expression of nm23Hi protein has negative correlation with differentiation degree of gastric carcinoma and metastasis.2. Expression of P-catenin mRNA has no correlation with differentiation degree of gastric carcinoma and metastasis. Expression of nm23Hi mRNA has negative correlation with differentiation degree of gastric carcinoma and metastasis.Expression of Tiam-1 mRNA is related with differentiation degree of gastric carcinoma and metastasis.3. In the progress of gastric carcinoma, the expression of nm23H| has inhibitory function to Wnt signal pathway, however, this inhibitory function may occur before or after P-catenin protein entering cytonucleus, rather than effect on mRNA.4. Tiam-1 in gastric carcinoma doesn't enhance cell adhesive function mediated by E-cadherin to inhibit metastasis.
肿瘤的发生、发展是一个多因素、多步骤、多阶段的过程,其中涉及多种癌基因、抑癌基因、错配修复基因、细胞黏附因子等的异常改变,而转移是肿瘤治疗失败的主要原因。肿瘤转移是一个多步骤的复杂过程,多种基因结构和功能的异常在这一过程中发挥重要作用。肿瘤细胞的侵袭、转移与其运动能力密不可分,细胞骨架在维持细胞形态和运动的同时,还可以整合内、外源信号,参与细胞分泌、接触抑制、增殖和凋亡等多种生物活动。β-catenin是细胞中的一种多功能蛋白质,很多肿瘤的预后与β-catenin有相关性。Tiam-1是一个细胞骨架调节因子,而β-catenin具有细胞黏附和信号传导功能。nm23是近年来研究较多的肿瘤转移抑制基因,但是,不同的研究发现,在不同类型的肿瘤细胞中,nm23的作用不同。即使同样是胃癌,其作用也不尽相同。本研究主要探讨三者在胃癌发展中的相互关系。
材料与方法
1.病例选择:收集2003-2005年中国医科大学附属第一医院肿瘤科术后大体标本,共63例,所有患者术前均未进行化疗或者放疗,年龄范围26-78岁,平均年龄56岁,其中高分化腺癌30例,低分化腺癌30例,未分化癌3例。同时收集中国医科大学附属第一医院内镜科活检标本,慢性浅表性胃炎30例,年龄范围25.68岁,平均年龄46岁。
2.取材:标本一部分于10%福尔马林中固定,石蜡包埋,另外留取新鲜标本,剪成小块立即放入液氮,并转入—80℃冰箱保存。
3.β-catenin鼠抗人单克隆抗体及nm23 H_1单克隆抗体工作液、PV9000试剂盒购自北京中杉金桥生物技术公司。DAB显色试剂盒购自福州迈新试剂公司。经Gene Bank检索,利用Primer 5.0软件设计β-catenin、
PREFACEThe pathogenesis of tumor is a process of multiple factors, multiple steps and many stages, which concerned with abnormalities of many oncogenes, tumor suppressor genes, mismatch repair genes, cellular adhesive factors etc. Tumor metastasis is a complex process of multiple steps, in which abnormalities of many gene structure and function play an important role. Cell skeleton can maintain cell shape and motion, also can participate in many biological activities such as cell secretion, contact inhibition, proliferation and apoptosis through integrating internal and external signals.p-catenin is a multifunctional protein in cell, and it is related to prognosis of many tumors. Tiam-1 is a regulater of cell skeleton, and P-catenin has cell adhesive and signal conducting function. nm23 is a tumor suppressor gene, however, different studies found that in different kind of tumor cells the effects of nm23 were different.Our purpose was to investigate the relationship of P-catenin, nm23 and Tiam-1 during the process of gastric carcinoma.MATERIALS and METHODS1. Cases: 63 samples after surgical operation were collected from Oncology Department of the first affiliated hospital of China Medical University from 2003 to 2005. All the patients didn't undergo chemotherapy or radiotherapy before surgical operation. The mean age is 56 years (range 26-78 years). Well-differentiated adenocarcinoma are 30 cases, poorly differentiated 30 cases, undifferentiated 3 cases. Meantime 30 biopsy samples of superficial gastritis were collected from Endoscopy Center of the first affiliated hospital of China Medical University. The mean age is 46 years (range 25-68 years).
2. Samples: a part of samples were kept in 10% formalin and then embedded in paraffin. Other samples were collected fresh and cut into pieces, snap frozen in liquid nitrogen, and kept frozen at -80 °C.3. Working fluid of p-catenin mouse-human monoclone antibody and nm23H, monoclone antibody, PV9000 Kits were bought from Zhongshan Jinqiao biotechnology corporation, Beijing. DAB coloration kits were supplied by Maixin technique corporation, Fuzhou. Primers of P-catenin, nm23H, and Tiam-1 were searched by Gene Bank and designed by Primer 5.0 software. The primers were synthesized by Sanbo Yuanzhi biotechnology limited corporation, Beijing. Taq enzyme of RT-PCR, dNTP and marker were all bought from TaKaRa biology coporation, Dalian.4. Methods: Immunohistochemistry stain with two-step method. nm23H, was located mainly in cytoplasm, and also on membrane. It was brown granular or mass. P-catenin was located mainly in cytoblast, and also in cytoplasm with brown granular or mass.It was positive when the stained cell number was over 10%. It was negative when there was no stained cell or the stained cell number was under 10%. RNA were extracted according to TRIZOL agent directions. RNA purity and concentration were measured with ultraviolet spectrophotometer. RNA purity: OD260/OD280^ 1.65-1.96.cDNA inverse transcription: RNA 3ul. Reaction system: 2xbuffer 15ul,MgSO4(25mM)6ul,AMV(22u/ul) 1.5ul,dNTPs(10mM) 1.5ul, oligodT(50uM) 1.5ul,RNase-inhibitor(40u/ul) 0.75ul,ddH2O 0.75ul, do the process as follows:Inverse transcription reaction: 65 °C for 1 minute30 °C for 5 minutesTemperature increased gradually within 15-30minutes. 65 °C for 30 minutes
98 °C for 5 minutes5 °C for 5 minutesPCR reaction system: cDNA 3ul, 2Xbuffer 2.5ul, MgSO4(25mM)6ul, dNTPs(2.5mM)2ul, Taq-E(5u/ul)0.2ul, ddH2O 17.1|il, primer of p-catenin 0.1^1(5pmol/^il)Dnm23H1 0.1 ul(5pmol/ul) and Tiam-1 0.1 ul (5pmol/ul).Run amplification as follows: pre-apomorphosis at 94 °C for 3 minutes, apomorphosis at 94°C for 40 seconds, anneal at 57°C for 1 minute, elongation at 72°C for 1 minute, repeat 35 times, finally elongation at 72°C for 7 minutes.p-actin was inner-control o Product of gene amplification went on electrophoresis by 2% agarose gel, and then took pictures. 5. Statistical treatment: x2 test was used with SPSS 12.0 software.RESULTSTable 1 Relationship between protein expression of P-catenin,nm23H, anddifferentiation degree_______________________________________________differentiation degree n |3 -catenin positive nm23H, positivewell-differentiation 30 io (33.3%) * 22 (73.3%) ** poor-differentiation 30 19 (63.3%) * 12 (40.0%) **undifferentiation________3 2 (73.3%)__________1 (33.3%)There is statistical difference of P -catenin between well-differentiated group and poor-differentiatedgroup (p<0.05).There is statistical difference of nm23Hl between well-differentiated group and poor-differentiated group
Table 2 Relationship between protein expression of |3-catenin, nm23H, and lymph node metastasislymph node metastasis n 0 -catenin positive nm23Hl positivePositive negative 38 25 23(60.5%) 8(32.0%) 16(42.1%) 19(76.0%)p value 0.0267 0.0081Table 3 Expression of p-catenin, nm23H, mRNA in gastric carcinoma and superficial gastritis n 0 -catenin positive nm23H , positivesuperficial gastritis gastric carcinoma 30 40 16 (53.32 24 (60.02 O 11 (36. 26 (65. 7%) 0%)p value 0.5770 0.0188 Table 4 Relationship between p-catenin,nm23H, mRNA and differentiation degreedifferentiation degree n P -catenin positive nm23H , positivewell-differentiation poor-differentiation 20 20 11 (55.09^ 13 (65.09< 0 ) / \ 0 ) 16 (80. 10 (50. 0%) 0%)p value 0.5186 0.0467
Table 5 Relationship between P-catenin,nm23Hi mRNA and lymph node metastasislymph node metastasis n &- catenin positive nm23H i positivepositive negative 23 17 14 10 (60.9%) (58.8%) 11 (47. 15 (88. 8%) 2%)p value 0.8961 0.0081 Table 6 Expression of |3-catenin, Tiam-1 mRNA in gastric carcinoma and superficial gastritis n 0- catenin positive Tiam-1 positivesuperficial gastritis gastric carcinoma 30 40 16 24 (53.3°/ (60.0°/ *) 8 (26.7%) 27 (67.5%)p value 0.5770 0.0007Table 7 Relationship between P-catenin,Tiam-l mRNA and differentiation degreedifferentiation degree n 0- ■catenin positive Tiam-1 positivewell-differentiation poor-differentiation 20 20 11 13 (55.0%) (65.0%) 10 (50 17 (85 .0%) .0%)p value 0.5186 0.0181 Table 8 Relationship between P-catenin, Tiam-1 mRNA and lymph node metastasislymph node metastasis n ￡ -catenin positive Tiam-1 positivepositive negative 23 17 14 (60.9%) 10 (58.8%) 19 (82.6%) 8 (47.1%)p value 0.8961 0.0176
DISCUSSIONp-catenin is a multifunctional protein, and has the function of cell adhesion and signal conducting. Majority |3-catenin in cell bind to E-cadherin, and bind to actin cell skeleton through a -catenin to participate in cell-cell adhesion and cell motion. Any change of the structure of E-cadherin-catenin complex will influence cell-cell junction. Increased intracellular P-catenin monomer binds to Tcf or Lef and enters into cytoblast. Tcf/Lef is DNA binding protein and acts as transcription factor to bind to corresponding DNA leading to continuous transcription of target gene.Diminishing or disappearing of P-catenin express in cytomembrane can cause that a -catenin can't bind to cadherin and thus affect normal cell-cell junction, therefore, adhesive function of tumor cells decrease and the tumor cells gain the function of metastasis and invasion. Nucleotide diphosphate kinase is coded by nm23 gene and exists extensively. It at least participates in two important functions in tumor growing, that is microtubular polymerization/disaggregation and signal conducting mediated by G-protein. In the process of signal conducting, GDP is dioxidated into GTP, and G-protein is activated, participate in tumor progress and metastasis. Tiam-1 gene can regulate cell adhesion mediated by E-cadherin, and participate in combination of adhesive complex together with Rho. The results showed that expression of p-catenin at cytomembrane disappeared in gastric carcinoma tissue, positive expression in cytoblasts was obviously higher than in superficial gastritis, and
expression was higher in poorly differentiated group and lymph node metastasis group than in well-differentiated group and no lymph node metastasis group. Positive expression of P-catenin mRNA exist both in gastric carcinoma tissue and superficial gastritis, and there was no statistical difference between them. There was no statistical difference in poorly differentiated group, well-differentiated group, lymph node metastasis and no lymph node metastasis group. Positive expression of nm23Hi in poorly differentiated group was obviously lower than in well-differentiated group,while positive expression of nm23Hi in lymph node metastasis group was obviously lower than in no lymph node metastasis group, suggested that expression of nm23 gene was related to the differentiation degree of gastric carcinoma and lymph node metastasis.nm23Hi may inhibit the expression of P-catenin.However,nm23Hi inhibiting Wnt conducting pathway may occur before or after P-catenin protein entering into cytoblast, rather than affect on mRNA. Positive expression of Tiam-1 mRNA in gastric carcinoma tissue was obviously higher than in normal tissue, and positive expression in poorly differentiated group and lymph node metastasis group was higher than in well-differentiated group and no lymph node metastasis group, suggested that Tiam-1 promote the process of malignancy.Although Tiam-1 can bind to E-cadherin at membrane-membrane junction, abnormal expression of P-catenin can cause abnormal linkage of cadherin-catenin between cells. While Tiam-1 promote tumor cells migration and participate in gene express control and cell proliferation, thus leading to tumor cell turning to malignancy* invasion and metastasis.
CONCLUSION1. Expression of P-catenin protein has positive correlation with differentiation degree of gastric carcinoma and metastasis. Expression of nm23Hi protein has negative correlation with differentiation degree of gastric carcinoma and metastasis.2. Expression of P-catenin mRNA has no correlation with differentiation degree of gastric carcinoma and metastasis. Expression of nm23Hi mRNA has negative correlation with differentiation degree of gastric carcinoma and metastasis.Expression of Tiam-1 mRNA is related with differentiation degree of gastric carcinoma and metastasis.3. In the progress of gastric carcinoma, the expression of nm23H| has inhibitory function to Wnt signal pathway, however, this inhibitory function may occur before or after P-catenin protein entering cytonucleus, rather than effect on mRNA.4. Tiam-1 in gastric carcinoma doesn't enhance cell adhesive function mediated by E-cadherin to inhibit metastasis.
引文
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2 Fang DC,Luo YH,Yang SM,Li XA,Ling XL,Fang L.Mutation analysis of APC gene in gastric cancer with microsatellite instabilly.World J Gastroenteol 2002;8:787-791
3 Song ZJ,Gong P,Wu YE.Relationship between the expression of iNOS,VEGF,tumor angiogenesis and gastric cancer. World J Gastroenteol 2002;8:591-595
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