【作者】 曹智新；

【导师】 张庆慧； 耿明；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 目的:检测Wnt-5a mRNA在胃癌及正常胃组织中的表达以及Wnt-5a、β-catenin和E-cadherin蛋白在胃癌、癌旁组织和正常胃组织中的表达,研究Wnt-5a与胃癌发生发展及各临床病理因素间的关系,结合β-catenin、E-cadherin蛋白在胃癌中的表达并分析三者的相关关系,进一步探讨正常胃组织向胃癌转变的分子生物学机制,探讨胃癌中Wnt-5a的早期诊断和预后价值,为临床治疗和药物开发提供新的作用靶点。方法:采用实时定量RT-PCR技术,对10例手术切除的新鲜胃癌和正常切缘组织(距肿瘤边缘5cm以上)进行Wnt-5a mRNA的检测;采用免疫组化SP法对84例胃癌、相应癌旁组织及其中20例正常胃组织中Wnt-5a、β-catenin和E-cadherin蛋白的表达进行检测。采用配对t检验、卡方检验及Spearman等级相关等方法进行统计学分析。结果:1.Wnt-5a mRNA在胃癌及正常组织中表达的相对含量分别为:5.919±1.869、1.281±0.744,两者间差异具有统计学意义(t=-2.374,P＜0.05),其中80%(8/10例)胃癌组织Wnt-5a mRNA显示上调。2.Wnt-5a蛋白在正常胃组织中均为阴性表达,在胃癌和癌旁组织中的阳性表达率分别为40.54%(34/84例)、14.29%(12/84例);其中100%(5/5例)肠上皮化生和62.5%(5/8例)不典型增生的癌旁组织Wnt-5a阳性表达;与正常组比较,Wnt-5a在胃癌组中显著高表达,差异有统计学意义(P＜0.01),但在癌旁组中的表达差异无统计学意义(P＞0.05);Wnt-5a在胃粘液腺癌中下调表达,与管状腺癌、乳头状腺癌、印戒细胞癌比较,差异具有统计学意义(P＜0.05),但四种组织学分型之间的组间比较差异不显著(P＞0.05);Wnt-5a在TNMⅢ、Ⅳ期病例组的表达显著高于Ⅰ、Ⅱ期(P＜0.001),在淋巴结转移组的表达显著高于无淋巴管转移组(P＜0.01),在低分化组与高中分化组的表达差异无统计学意义(P＞0.05)。3.β-catenin在正常胃组织100%出现细胞膜的正常表达;在胃癌及癌旁组织中则出现胞质下降表达以及胞质和/或胞核的异常表达,其阳性率分别为70.23%(59/84例)、38.1%(32/84例),其中胃癌组β-catenin的核阳性率为19.0%(16/84例);与正常组比较,β-catenin在胃癌和癌旁组中的异常表达均有统计学意义(P＜0.01);统计分析显示β-catenin的异常表达与TNM分期和淋巴结转移相关(P＜0.05),与肿瘤分化程度无明显相关(P＞0.05)。4.E-cadherin在正常胃组织膜表达阳性率为100%,在胃癌及癌旁组织膜下降/缺失表达率分别为57.14%(48/84例)、15.48%(13/84例);与正常组比较,E-cadherin在胃癌组的下调表达差异显著(P＜0.05),在癌旁组的下调无统计学意义(P＞0.05);E-cadherin在胃癌中的下调表达相关于TNM分期、淋巴结转移及肿瘤的分化程度(P＜0.05)。5.Wnt-5a在胃癌组织中的阳性表达与β-catenin的异常表达呈负相关关系(r=-0.367,P=0.001),与E-cadherin的膜下降/缺失表达之间呈正相关关系(r=0.262,P=0.016)。结论:1.Wnt-5a mRNA及蛋白在胃癌中均上调表达,Wnt-5a在胃癌中可能发挥癌基因样作用,且Wnt-5a基因的异常活化可能部分参与了胃癌发生的始动过程;其高表达是胃癌发生及演进过程中的频发事件,并与胃癌的临床分期及淋巴结转移相关,该蛋白极可能成为判断胃癌预后不良的指标。2.β-catenin在胃癌胞浆和/或胞核中异常高表达,可能参与了胃癌的发生发展过程;β-catenin的异常表达与胃癌的临床分期及淋巴结转移相关。3.E-cadherin在胃癌中显示有意义的下降表达,参与了胃癌的侵袭转移;其异常表达相关于肿瘤的TNM分期、分化程度及淋巴结转移。4.Wnt-5a在胃癌中的高表达与β-catenin在胃癌中的异常表达呈负相关关系,提示Wnt-5a与β-catenin在胃癌发生的不同时相表达,二者可能经由不同的信号通路在胃癌发生中发挥作用。5.Wnt-5a在胃癌中的高表达与E-cadherin下调表达间存在正相关关系,前者除涉及胃癌的发生发展外还可能协同E-cadherin在胃癌的浸润转移中发挥作用。更多还原

【Abstract】 Objective To detect the expression of Wnt-5a mRNA in gastric carcinoma and the corresponding peri-tumor tissue,and the expression of Wnt-5a,β-catenin and E-cadherin protein were also examined in gastric carcinoma(GC),peri-tumor tissue and normal stomach tissue.Approach the relationship between expression of Wnt-5a with the occurrence of GC and clinicopathologic characters,and discuss the correlation with the expression of Wnt-5a,β-catenin and E-cadherin in order to explore the mechanism of molecular biology for the occurrence and development of GC.We therefore wanted to investigate the role of Wnt-5a expression in tumor,with focus on diagnosis and prognosis in GC,Wnt-5a is not only a good prognostic indicator of gastric cancer but also a candidate for a molecular target of therapy for gastric cancer.Methods Real time RT-PCR was performed in ten fresh gastric carcinoma sample and the corresponding normal tissue to detect mRNA expression of Wnt-5a gene. Eighty-four gastric carcinoma cases and the corresponding peri-tumor tissues and twenty normal stomach mucosa were examined Wnt-5a,β-catenin and E-cadherin by SP immunohistochemical method.The results was treated by matched-pairs T test, chi-square test and Spearman Correlation analysis.Results 1.Real time RT-PCR analysis showed that Wnt-5a mRNA was overexpressed in GC, and the level of Wnt-5a mRNA in tumor and normal region were5.919±1.869,1.281±0.744 respectively,with a significant difference(P<0.05).Wnt-5a mRNA was up-regulation in 80%(8/10 cases) GC.2.Wnt-5a protein was negative in all normal gastric tissue,the positive expression rate of Wnt-5a protein in peri-tumor tissue and GC was 14.29%(12/84cases),40.54% (34/84cases) respectively.Wnt-5a protein was positive in 5 cases which develop intestinal metaplasia and 5/8 cases which develop atypical hyperplasia of all the peri-tumor tissue.Compare with the normal tissue,expression of Wnt-5a has a significant highly positive rate in gastric carcinoma(P<0.05),but no significance in peri-tumor tissue(P>0.05);negative expression of Wnt-5a in mucinous adenocarcinoma has a significant difference compare with that in tubular adenocarcinoma,papillary adenocarcinoma and signet-ring cell carcinoma,but no significant difference with histology type in group comparison(P>0.05).Abnormal expression of Wnt-5a was significantly associated with advanced TNM stages (P<0.001),and lymph node metastasis(P<0.01),but no relation with tumor grades(P>0.05).3.The membranous expression rate ofβ-catenin in normal gastric mucosa was 100%. it was observed in cytoplasm and/or cellular nucleus in GC and peri-tumor tissue, and the positive rate were respectively:70.23%(59/84),38.10%(29/84),of the total carcinoma casesβ-catenin cellular nucleus expression was observed in 19.05%(16 cases).Abnormal expression ofβ-catenin has statistical significance (P<0.01) in tumor and peri- tumor tissue compare with normal tissue,and the abnormal expression ofβ-catenin was associated with advanced tumor stages (P<0.05) and lymph node metastasis(P<0.05),but no correlation with tumor grades(P>0.05).4.The membranous expression rate of E-cadherin in normal mucosa was 100%,The low/loss membranous expression rate of E-cadherin in peri-tumor tissue and gastric carcinoma were 57.14%(48/84) and 84.5%(13/84) respectively.Compare with normal tissue,down-regulation of E-cadherin has a significant highly positive rate in gastric carcinoma(P<0.05),but no significance in peri-tumor tissue(P>0.05); Reduced expression of E-cadherin was significantly associated with advanced tumor stages,lymph node metastasis and the tumor grades(P<0.05,respectively).5.The expression of Wnt-5a was correlated withβ-catenin(r=-0.367,P<0.01) and E-cadherin(r=0.262,P<0.05) in GC.Conclusions:1.Wnt-5a mRNA and protein were up-regulation in GC.Wnt-5a may play a tumor stimulator function,and Wnt-5a gene may have been activated in early period of tumorigenesis.The high expression of Wnt-5a may be a frequent event in the occurrence and development of GC,and was associated with advanced tumor stages and lymph node metastasis.Wnt-5a protein may become an useful prognosis indicator of GC.2.β-catenin was detected in cytoplasm and/or cellular nucleus in gastric carcinoma and peri-tumor tissue,which indicate thatβ-catenin may participate in the occurrence of gastric caicinoma.Expression ofβ-catenin was associated with advanced tumor stages and lymph node metastasis.3.Reduced membranous expression of E-cadherin was a significant event in GC and associated with advanced tumor stages,lymph node metastasis and the tumor grades.4.The expression of Wnt-5a was negative correlated withβ-catenin in GC, suggesting that the expression of Wnt-5a andβ-catenin is mutually exclusive.The two molecules may suppress the expression of each other,and may play important role in the occurrence of GC via different signal pathway.5.The expression of Wnt-5a was positively correlated with E-cadherin in GC.Wnt-5a may besides play direct function in the occurrence and progression of GC and also may be in coordination with the expression of E-cadherin to enhance the invasion and metastasis of GC.更多还原