VEZT在胃癌组织中的表达研究及其载体的构建

Expression of VEZT in gastric cancer tissues and the construction of VEZT over-expression vector

目的 探讨胃癌相关候选抑癌基因VEZT在胃癌组织中的表达及其临床意义,构建其真核过表达载体,为寻求胃癌的靶基因治疗提供依据.方法 采用免疫组织化学SP法检测119例胃癌组织和癌旁正常胃组织中VEZT的表达,分析其与患者临床病理特征及预后的关系.用RT-PCR方法从293肾上皮细胞扩增出VEZT基因片段,利用DNA重组技术将目的基因连接于pEGFP-N1真核表达载体上,构建重组质粒载体,分别用1％酶切电泳及DNA测序、对比分析对重组质粒载体进行鉴定.结果 胃癌组织及癌旁正常组织中VEZT的阳性表达率分别为30.3％和60.5％,高分化腺癌VEZT阳性率高于中、低分化腺癌（χ^2=5.002,P＜0.05）,早期胃癌高于晚期胃癌（χ^2 =5.551,P＜0.05）,无淋巴结转移者高于有淋巴结转移者（χ^2=5.878,P＜0.05）,VEZT的表达与胃癌患者年龄、肿瘤大小无关.VEZT阳性胃癌患者的生存时间明显长于阴性患者（χ^2=6.908,P＜0.01）.酶切电泳分析得到的目的片段大小与理论值一致,DNA测序、对比证实目的基因序列无突变,成功构建真核表达质粒载体pEGFP-N1-hVEZT.结论 VEZT蛋白与胃癌的组织分化类型、TNM分期、浸润深度和淋巴结转移相关,VEZT高表达胃癌患者的5年生存率也较高.

Objective To investigate the expression of VEZT and its clinical pathological significance in gastric cancer tissues,and to construct the VEZT over-expression vector.Methods The expression of VEZT was examined in 119 cases of gastric carcinoma and their corresponding normal mucus tissues by SP immunohistochemical staining.The relationships between the VEZT expression levels and its clinicopathological characteristics,prognosis were also investigated.VEZT cDNA was extracted and amplificated from 293 cells by polymerase chain reaction （PCR）.Using DNA recombinant technique,the target gene was connected to the pEGFP-N1 vector to construct recombinant plasmid vector after the target gene and pEGFP-N1 were purified.The recombinant plasmid was identificated by 1% enzyme electrophoresis and DNA sequencing.Results The VEZT-positive expression in gastric carcinoma was 30.3% and 60.5% in normal gastric tissues.VEZT expression in high differentiated cancer tissues was higher than that in lower differentiated tissues（χ^2 =5.002,P 〈 0.05）,expression of VEZT in early gastric cancer was significantly higher than that in patients with advanced gastric cancer （χ^2 =5.551,P 〈 0.05）,expression of VEZT in patients without lymph node metastasis was significantly higher than that of lymph node metastasis （χ^2 =5.878,P 〈 0.05）.Patients with positive VEZT expression have better prognosis than the negative patients（χ^2 =6.908,P 〈 0.01）.The target fragment,consistent with the theoretical value,was verified by gel electrophoresis.DNA sequencing confirmed that the gene sequence had no mutation and the eukaryotic expression plasmid vector pEGFP-N1-hVEZT was successfully constructed.Conclusions VEZT protein was correlated with TNM staging,tumor stage,invasion depth and lymph node metastasis,positive VEZT expression predicting better five year survival.The VEZT over-expression plasmid vector was successfully constructed.