【作者】 王朝阳；

【导师】 许丽艳； 李恩民；

【作者基本信息】 汕头大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 NGAL（neutrophil gelatinase-associated lipocalin）蛋白首先由Kjeldsen等人于1993年在中性粒细胞内发现，与炎症、胚胎发育、免疫应答、趋化作用、信号转导以及多种肿瘤的发生与发展等过程相关。我们在运用基因芯片技术筛选永生化食管上皮细胞系SHEE和由SHEE转化而来的食管癌细胞系SHEEC之间的差异表达基因时发现NGAL基因在SHEEC细胞中过表达，进一步研究发现NGAL基因可能与肿瘤的侵袭与转移相关，但确切的功能与机制不清楚。最近我们研究发现NGAL基因还可能参与细胞的分化。越来越多的研究结果提示NGAL可能是一种重要的炎症或肿瘤标志物，对判断与监测病情变化具有重要的意义。但是目前无论是NGAL蛋白本身还是抗NGAL的抗体均未商品化，所以本文通过原核表达的策略首先获得纯的NGAL蛋白、然后制各其抗体，最后通过对各种食管癌组织样品进行检测，探讨NGAL基因在食管癌中的表达模式，这对深入研究NGAL在生理和病理过程中所发挥的功能具有科学意义和应用价值。 主要研究内容和方法： 一、构建四种NGAL原核融合表达载体并比较分析蛋白表达产物。 二、Ni²⁺-金属鏊合层析技术纯化NGAL蛋白并制备多克隆抗体。 三、研究NGAL基因在食管癌组织及其血浆中的表达模式。 主要的研究结果： 一、构建了NGAL基因四种原核融合表达载体。 二、成功进行了四种NGAL融合蛋白的原核表达，并分析了它们的表达量、可溶性及其细胞定位，最后筛选出了适合大样表达NGAL蛋白的原核融合表达载体pDsbA2.0-NGAL。 三、成功进行了NGAL蛋白的纯化与鉴定，获得了较纯的NGAL蛋白。 四、成功制备出效价高、特异性较好的NGAL多克隆抗体，适用于IHC、Western blotting及ELISA等。 五、运用IHC、Western blotting及ELISA等实验技术，初步探讨了NGAL基因在食管癌组织中的表达模式。汕头大学医学院硕士研究生论文结论及意义:一、不同的融合伴侣对靶蛋白的表达量、细胞定位以及可溶性会产生不同的影 口向。二、NiZ七金属鳌合层析纯化技术是一种高效的纯化蛋白的技术。三、成功建立了检测NGAL表达模式的实验技术:免疫组织化学、western blotting与EL工SA技术，通过不同层面揭示了NGAI砂在食管癌中的表达模式:IHC实验显示染色阳性细胞定位于细胞胞浆且集中于高分化食管鳞状上皮细胞癌癌巢癌珠部分;Western blotting实验结果显示NGAL蛋白在部分食管癌组织中表达上调，但尚需增加样本量进一步确证;而ELISA实验结果初步显示在正常人群血浆/血清中能够检测到NGAL基因的蛋白表达产物，这为进一步探讨NGAL基因在食管癌中的生物学功能与意义奠定了实验基础。更多还原

【Abstract】 NGAL (neutrophil gelatinase-associated lipocalin) protein was found at first in neutrophil granulocyte in 1993 by Kjeldsen et al. And it involved in inflammation, embryonic development, immunity response, chemotaxis phenomena, signal transduction, occurence and progression procedure of many cancers. We had found NGAL gene was overexpressed in esophageal cancer cell line(SHEEC)by cDNA microarray screening different expression genes between immortal esophageal epithelial cell line(SHEE) and SHEEC transformed from SHEE, but not known its mechanisms. Furthermore research indicated that NGAL might be involved in tumor’s invasion and metastasis with unknown mechnisms, too. Recently some researches have shown that NGAL gene is associated with cell modulation and differentiation. So it can be predicted that NGAL maybe have some more important functions. Obviously, so many research results indicate NGAL may be an important disease marker, especially on cancers. So intensive research of NGAL function on physiology and pathology has important scientific significance and application merits. Now NGAL protein itself and its antibody both were not commercilized, which limited intensive application of NGAL on clinical research, at the same time restricted intensive understanding of NGAL function, too. So to construct the expression vectors of NGAL gene and to express NGAL protein, then to prepare anti-NGAL antibody, at last to detect NGAL expression level and discuss its expression pattern with the anti-NGAL antibody by IHC, Western blotting and ELISA experimental techniques have an important significance for furthermore research of NGAL function. Main Procedures and Methods:1. To construct four NGAL gene fusion expression vectors and to analyze comparatively their expressed proteins in E.coli.2. To purify NGAL protein with Ni2+- metal chelate affinity chromatography and to prepare anti-NGAL polyclonal antibody.3. To detect the expression level of NGAL in the esophageal cancer by IHC, Westernblotting and ELISA experimental techniques and to discuss the expression pattern of NGAL in the esophageal cancer, which serves for furthermore reseach work of NGAL gene function. Main Results:1. Four NGAL gene fusion expression vectors had been successfully constructed in prokaryote.2. Four NGAL fusion proteins were successfully expressed in prokaryote and their productivity,solubility as well as subcellular localization were all analysed, at last the pDsbA2.0 - NGAL expression vector which can be suitable for large-scale expression was screened.3. Purification and identification of NGAL protein had been done successfully, at last the NGAL protein with some extent purity and abundance had been gained.4. Anti-NGAL polyclonal antibody with satisfied titer ,sensitivity and specificity was prepared successfully and was suitable for IHC Western blotting as well as ELISA.5. Expression pattern of NGAL in the esophageal cancer was discussed primarily by IHC Western blotting and ELISA.Conclusion and significance:1. Different fusion partner has different effect on productivity, subcellular localizition and solubility of target protein.2. Ni2+- metal chelate affinity chromatography is a protein purification technique with very high efficacy.3. Detecting techniques for NGAL expression pattern were established successfully:IHC, Western blotting and ELISA.The expression pattern of NGAL in the esophageal cancer was shown through different aspect. IHC experiment manifested positive staining located in the cytoplasm or cellular membrane of the central esophageal carcinoma nest with high-grade differentiation. Western blotting experiment displayed that expression level of NGAL in some esophageal carcinomas were higher than those of nomal esophageal mucosas,but many more samples must be detected to verify this point. And ELISA experiment manifestedprimarily that NGAL expression can be detected in the normal plasm/serum. All of these had built up a good更多还原