【作者】 施英瑛；

【导师】 张曼利；

【作者基本信息】 河北医科大学 ， 内科学， 2003， 硕士

【摘要】 目的：观察胃癌前病变（肠上皮化生IM、不典型增生Dys）和胃癌（GC）组织中TGF-β1、TGF-βRⅡ和Smad4蛋白表达水平变化及其与临床病理指标之间的关系，探讨TGF-β信号转导通路中TGF-β1、TGF-βRⅡ和Smad4在胃癌发生发展中的作用以及三者间的相互关系。方法：选择经胃镜活检病理组织学证实的肠上皮化生（IM）者20例、不典型增生（Dys）者20例及胃癌患者46例为试验组，其中胃癌患者全部手术。据病理和手术探查结果将46例胃癌按肿瘤组织学分化程度分为高中分化组（乳头状腺癌、管状腺癌，22例）和低分化组（低分化腺癌、印戒细胞癌，24例）；按有无区域淋巴结转移分为淋巴结转移组（25例）和无淋巴结转移组（21例）；按有无远处转移分为远处转移组（10例）和无远处转移组（36例）；按浸润深度分为累及浆膜组（24例）和未及浆膜组（22例）；按TNM分期（1988年国际抗癌联盟统一方案）分为TNMⅠ、Ⅱ期组（19例）和TNM Ⅲ、Ⅳ期组（27例）。另选20例胃镜观察及活检病理组织学基本正常者为对照组。所有入选者均排除心、脑、肝、肾、肠、胰腺等疾患。测试者均于行<WP=4>胃镜检查时取胃黏膜标本5块（胃窦2块、胃体2块、胃角1块），标本足够大，深达黏膜肌。10％中性福尔马林固定4小时以上，石蜡包埋，连续切片，片厚5μm。常规HE染色，光镜下确定病理组织学类型。每例标本行免疫组化SABC法检测TGF-β1、TGF-βRⅡ、Smad4蛋白表达水平，病理图象分析系统处理切片染色结果。结果：1.免疫组化染色显示：TGF-β1的阳性反应物位于胞浆内，在正常胃黏膜，TGF-β1阳性细胞较少，着色均匀，主要分布于黏膜表层；癌组织中，TGF-β1在胞浆内着色不均，呈棕褐色，颗粒状。TGF-βRⅡ的阳性反应物位于胞浆和（或）胞膜，在正常胃黏膜，TGF-βRⅡ呈弥漫或颗粒状染色；癌组织中TGF-βRⅡ表达较正常组织明显减弱甚至无表达。Smad4蛋白阳性反应物位于胞浆和（或）胞核，在正常胃黏膜，Smad4蛋白在胞浆和胞核中均有染色；胃癌细胞内，Smad4蛋白阳性染色较正常组织明显减弱甚至缺失，且阳性细胞胞核染色罕见。2. TGF-β1、TGF-βRⅡ和Smad4蛋白在各组中的表达水平变化：GC组TGF-β1蛋白表达水平（阳性面积％ 下同）为64.62±14.71，显著高于NC组（18.97±10.86，P<0.01）、IM组（24.42±13.45， P<0.01）和Dys组（33.60±14.61，P<0.01）；Dys组与NC间也有明显差异（P<0.05）；IM组与NC组间无明显差异（P＞0.05）。GC组TGF-βRⅡ表达水平为17.91<WP=5>±10.66，显著低于NC组（61.94±12.64，P<0.01）、IM组（58.07±11.14，P<0.01）和Dys组（53.35±14.71，P<0.01）；IM组与NC组、Dys组与NC组间差异无显著性（P＞0.05，P＞0.05）。GC组Smad4蛋白表达水平为23.57±14.31，显著低于NC组（70.33±12.13，P<0.01）、IM组（67.93±13.93，P<0.01）和Dys组（68.82±13.22，P<0.01）；IM组与NC组、Dys组与NC组间差异无显著性（P＞0.05，P＞0.05）。3. GC组TGF-β1、TGF-βRⅡ和Smad4蛋白表达与临床病理的关系：胃癌组织学分化程度：低分化组中TGF-β1表达水平（71.29±10.05）显著高于高中分化组（57.33±15.70,P<0.01）；TGF-βRⅡ和Smad4蛋白表达水平(14.09±9.63,16.64±12.81)显著低于高中分化组（22.08±10.35, P<0.01；30.22±12.20, P<0.01）。区域淋巴结转移：有区域淋巴结转移组TGF-β1表达水平（72.30±7.57）显著高于无区域淋巴结转移组（55.47±16.02,P<0.01）； TGF-βRⅡ表达水平(14.82±10.78)显著低于无区域淋巴结转移组（21.59±9.50,P<0.01）；Smad4蛋白表达水平在两组间差异无显著性（P>0.05）。 远处转移：远处转移组Smad4蛋白表达水平（14.95±7.22）显著低于无远处转移组（27.08±12.32,P<0.01）；TGF-β1和TGF-βRⅡ表达水平在两组间差异无显著性（P>0.05）。浸润深度：累及浆膜组TGF-β1表达水平（68.73±12.55）明显高于未及浆膜组（60.13±15.84,P<0.05）；Smad4蛋<WP=6>白表达水平（14.81±9.33）明显低于未及浆膜组（25.67±13.85,P<0.05）；TGF-βRⅡ表达水平在两组间差异无显著性（P＞0.05）。TNM分期：Ⅲ、Ⅳ期组TGF-β1表达水平（71.13±9.75）显著高于Ⅰ、Ⅱ期组（55.36±15.80,P<0.01）；TGF-βRⅡ表达水平(15.30±10.09)明显低于Ⅰ、Ⅱ期组（21.61±10.60, P<0.05）；Smad4蛋白表达水平在两组间差异无显著性（P＞0.05）。4. 相关性分析：GC组TGF-β1与TGF-βRⅡ、TGF-β1与Smad4间存在显著直线负相关（r=-0.625，P＜0.01，r=-0.297，P＜0.05）；TGF-βRⅡ与Smad4间无明显直线相关性（P＞0.05）。NC组、IM组和Dys组各组内TGF-β1与TGF-βRⅡ、TGF-βRⅡ与Smad4、TGF-β1与Smad4间皆无明显直线相关性（P＞0.05）。各组间TGF-β1与TGF-βRⅡ间存在显著直线负相关（r=-0.820，P＜0.01），TGF-β1与Smad4以及TGF-βRⅡ与Smad4间无明显直线相关性（P＞0.05）。结论：本研究结果表明：1. TGF-β1表达水平Dys组明显高于NC组，GC组显著高于NC组、IM组和Dys组，提示TGF-β1在胃癌前病变时已经异常升高，可能是胃癌形成过程中较早的一个生物学征象，检测TGF-β1可能有助于胃癌的早期更多还原

【Abstract】 Objective：To investigate both the function of TGF-β1，TGF-βRⅡand Smad4 which belong to TGF-βsignal passway and the relationship among the three protein in the carcinogenesis and development of gastric cancer. We evaluated the expression of TGF-β1,TGF-βRⅡand Smad4 protein in gastric mucous of the patients with gastric cancer or precancerous leisions.Methods：20 patients with intestinal metaplasia(IM),20 patients with dysplasia(Dys),46 patients with gastric cancer(GC) and 20 sex, age-matched health subjects were included in the present study. The diagnosis had been based on the gastric biopsies. According to the pathology report and surgery result, tumor were divided into such subgroups: well differentiation group(22 cases) and poorly differentiation group (24 cases);without regional lymph node invasion group(21 cases) and with regional lymph node invasion group(25 cases);without serosal invasion group (22 cases) and with serous invasion group(24 cases),without distant metastasis group(36 cases) and with distant metastasis<WP=9>group(10 cases), stage (TNM)Ⅰ,Ⅱgroup(19 cases) and stage Ⅲ,Ⅳgroup(27 cases).All of the subjects had no other diseases. No GC patients had received chemotherapy or radiation therapy or other adjuvant therapy. 5 specimens(2 from antrum,2 from fundal and 1 from cornu gland region, big enough and fetched muscle)were obtained when they were performed by gastroscopy. All of the specimens were fixed with 10% Formalin and embedded with paraffin. They were cut into 5-μm thick sections. A serial section from each specimen was stained with hematoxylin and eosin (H & E) for histological evaluation. Immunohistochemical staining of the sections for TGF-β1,TGF-βRⅡ and Smad4 protein was performed by using image analysis system to get the result.Results：1. TGF-β1 positive staining was observed in cytoplasm. Nomal epithelium positive cells was fewer and homogeneously stained,whereas in gastric cancerous tissue, more positive tumor cells were heterogeneously distributed and the color was deeper. TGF-βRⅡpositive staining was observed both in cytoplasm and cytomembrane. The positive cells were much more and the stain was also deeper in normal epithelium, while in gastric cancer tissue the positive cells were fewer and even no one is positive. In normal epithelium of the stomach, immunostaining of Smad4 was observed in cytoplasm and simultaneously in nuclei, while there was positive immunostaining for Smad4 only in the cytoplasm<WP=10>in cancer cells and the stain was much weaker even lack of stain. 2.The expression level(positive areas%) of TGF-β1 in GC group was 64.62±14.71 which has a significant difference from NC group(18.97±10.86, P<0.01), IM group(24.42±13.45, P<0.01) and Dys group(33.60±14.61, P<0.01).There was no statistical significance between NC and IM group(p>0.05).The expression of TGF-β1 were much higher in Dys group than that in GC group(p>0.05).There was a significant difference among TGF-βRⅡexpression in GC group(17.91±10.66) and NC group(61.94±12.64, P<0.01),IM group(58.07±11.14, P<0.01) and Dys group(53.35±14.72, P<0.01). We didn’t find statistical difference between NC and IM groups(p>0.05),IM and Dys groups(p>0.05). The expression of Smad4 protein in GC group was 23.57±14.31 which has a significant difference from NC group(70.33±12.13, p<0.01),IM group(67.93±13.93, p<0.01) and Dys group(68.82±13.22, p<0.01). We didn’t find statistical difference between NC and IM gro ups(p>0.05), IM and Dys groups(p>0.05). 3.In GC group, the expression of TGF-β1 was significantly higher in poorly differentiation(71.29±10.05) than that in well differentiation(57.33±15.70,P<0.01). TGF-βRⅡand Smad4 protein staining in poorly differentiation group(14.09±9.63,16.64±12.81) was lower than that in well differentiation(22.08±10.35,P<0.01；30.22±12.20, P<0.01). With lymph node<WP=11>invasion group, the expression of TGF-β1 was significantly higher(72.30±7.57) than that in without lymph node invasion group(55.47±16.02,P<0.01). TGF-βRⅡ was lower(14.82更多还原