【作者】 聂胜峰；

【导师】 兰斌；

【作者基本信息】 福建医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的:1.研究非甾体类抗炎药活化基因- 1(nonsteroidal anti- inflammatory drug - activated gene-1 ,NAG-1)在胃癌组织中的表达及与胃癌临床病理特征之间的关系,探讨NAG-1在胃癌发生、发展和侵袭中的作用。2.构建NAG-1的真核表达载体pEGFP-N1/NAG-1,为体外研究NAG-1基因功能建立实验基础。方法:1.采用半定量逆转录聚合酶链反应(RT-PCR)法检测76例原发性胃癌患者癌组织及相应胃正常黏膜组织中NAG-1mRNA的表达;2.采用免疫组织化学S-P方法检测胃癌及正常胃粘膜组织中NAG-1蛋白的表达情况;3.以正常胃粘膜组织cDNA为模板,用RT-PCR技术扩增NAG-1基因片段,将该片段插入表达载体pEGFP-N1中,并通过蓝白斑实验、酶切、PCR和DNA测序刷选和鉴定阳性克隆。结果:1.正常组织和胃癌组织中均表达NAG-1mRNA,胃癌组织中的相对含量为0.5563±0.2181,显著低于胃正常组织的0.9059±0.0751(P<0.05)。高、中分化胃癌组织中的NAG-1mRNA相对含量为0.6701±0.1637,显著高于低、未分化胃癌组织的0.4005±0.1859(p<0.05)。不同浸润深度、有无淋巴结转移、不同临床分期的胃癌组织间NAG-1mRNA相对含量的差异均无统计学意义(p>0.05)。2.胃癌组织的NAG-1蛋白的表达程度显著于低于胃正常粘膜组织(X2=47.186 ,p=0.000<0.05)。细胞分化程度低的癌组织NAG-1蛋白表达程度显著低于分化程度高的癌组织(X2= 7.506,p=0.006<0.05)。不同性别、有无淋巴结转移、不同临床分期的胃癌组织间NAG-1蛋白表达程度的差异均无统计学意义(p值>0.05)。3.对阳性克隆进行PCR、酶切及DNA测序鉴定,证实NAG-1目的片段已正确插入表达载体。结论:1. NAG-1mRNA在胃癌组织中的表达与浸润深度、淋巴结转移、TNM分期无关,而与肿瘤的分化程度相关,提示NAG-1mRNA的表达程度影响胃癌的分化。2. NAG-1蛋白的表达与肿瘤分化程度相关,NAG-1蛋白质在胃癌组织中的表达与浸润深度、淋巴结转移、TNM分期无关,提示NAG-1蛋白质的表达可能影响胃癌的分化。3. NAG-1mRNA在胃癌组织低于正常胃组织中的表达,NAG-1蛋白在胃癌组织中的表达明显低于它们在正常胃组织中的表达,提示NAG-1的表达改变可能与胃癌发生与发展有关。4.成功构建pEGFP-N1/NAG-1重组质粒,为进一步研究NAG-1基因功能建立实验基础。更多还原

【Abstract】 【Objective】:1.To study the expression of nonsteroidal anti - inflammatory drug - activated gene-1 in human gastric cancer and its relationship with the clinical staging ,differntiation degree ,and lymphatic metastasis of gastric cancer.2.To clone a fragment of NAG-1 gene into an expression vector pEGFP-N1 for expressing NAG-1 protein in vitro.【Methods】:1.The express of NAG-1 mRNA was detected by semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in the cancer tissues and corresponding adjacent normal tissues in 76 gastric cancer patients.2.The expression of NAG-1 protein expression in human gastric carcinoma tissues and normal gastric tissues were detected by S-P immunohistochemistry.3.Using nomal gastic tissue cDNA as template,amplified the NAG-1 gene with RT-PCR.Insert this fragment into the expression vector pEGFP-N1,and identified the positive clone with white and blue assay,endonuclease,PCR and DNA sequenceing identification.【Results】:1.NAG-1 mRNA was positive in both nomal tissues and gastric cancer tissues; expression in gastric cancer tissue significantly was lower than that in nomal tissues(0.5563±0.2181 vs 0.9059±0.0751,P<0.05).NAG-1mRNA expression in well and moderately differentiated cancer cells was significantly higher than those of poorly and non-differerntiated cancer cells(0.6701±0.1637 vs 0.4005±0.1859,p<0.05) , but the expression was not associated with the depth of tumor invasion,lymph node metastasis , and clinical stage (p>0.05).2Immuno-histochemistry showed the expression of NAG-1 protein in the cancer tissues was lower than that in the normal tissues (p<0.05);and the expression was not relationship with the gender, lymph node metastasis and clinical stage (all p >0.05).3. The expression of NAG-1 mRNA in gastric cancer tissues was lower than that in nomal tissues ;the expression of NAG-1 protein in the cancer tissues was lower than that in the normal tissues.they suggest that the expression of NAG-1 may relationship with the development and progression of gastic cancer.4. The positive clons were identified with PCR,endonuclease and DNA sequencing identification,and they proved that the target fragment of NAG-1 gene had been inserted into expression vector pEGFP-N1 rightly.【Conclusion】:1. The expression of NAG-1mRNA in gastric carcinoma tissues and normal gastric tissues had statistical difference;The expression of NAG-1mRNA had no correlation with the depth of tumor invasion、lymph node metastasis and TNM stage. It is associated with the differentiation of tumor. That suggest the carcinogenesis、progression and invasion of gastric carcinoma had correlation with the NAG-1mRNA expression.2. The expression of NAG-1 protein was significant lower in gastric carcinoma than normal gastric tissues. The expression of NAG-1 protein was associated with differentiation grades.That show NAG-1 protein suppressed the processes of carcinogenesis、progression and invasion of gastric carcinoma. Downregulation of NAG-1 protein promoted the processes of carcinogenesis、progression、invasion and metastasis of gastric carcinoma,Which made the gastric carcinoma cells more malignant and more invasive3. Cloning NAG-1 recombinant plasmid lays the foundation for expressing NAG-1 protein in vitro.更多还原