【作者】 袁华敏；

【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， 2007， 硕士

【摘要】 近年来，许多研究表明，中性粒细胞明胶酶相关脂质运载蛋白编码基因NGAL(neutrophil gelatinase-associated lipocalin)是人类的一个重要的肿瘤相关基因，在结肠癌、乳腺癌、胰腺癌、肺癌、皮肤癌和食管癌等肿瘤中高表达，然而，在胃癌中NGAL基因的表达情况如何目前尚不清楚。在本文中，我们首先检测了NGAL基因在胃癌组织细胞中的表达情况，其次还研究了胃癌细胞中NGAL基因的表达调控机制。期望为全面认识NGAL基因的肿瘤生物学功能提供更多资料。研究内容和方法：1．采取免疫组织化学技术对55例胃癌及其相对应的正常胃腺上皮石蜡包埋组织样品中NGAL基因的表达情况进行检测。2．联合运用蛋白印迹和RT-PCR技术对两种胃癌细胞系BGC-823和SGC-7901中NGAL基因的表达情况进行检测。3．将NGAL基因5′侧翼调控区不同长度片段分别插入到质粒pGL3-Basic中，构建NGAL基因5′侧翼调控区系列荧光素酶报告基因表达载体。4．利用双荧光素酶报告基因检测系统研究确定，在胃癌细胞系BGC-823和SGC-7901中，NGAL基因启动子核心元件所在区域。5．结合突变与缺失技术，研究胃癌细胞中NGAL基因启动子区元件的TPA反应性。6．通过蛋白印迹技术，结合生物信息学预测分析，确定胃癌细胞中NGAL基因的转录激活因子，初步探讨TPA诱导NGAL基因表达的细胞信号转导通路。研究结果：1．免疫组织化学检测结果表明，NGAL基因在正常胃腺上皮中不表达或表达很弱，而在胃癌组织中表达很强，阳性信号弥散于整个胞浆。2．两个胃癌细胞系中NGAL基因表达有较大差别，BGC-823中NGAL基因表达水平明显高于SGC-7901。3．双荧光素酶报告基因检测结果表明，在两个不同的胃癌细胞系中，NGAL基因启动子核心元件所在区域不一样；在BGC-823细胞中，NGAL基因启动子核心元件位于基因5′侧翼区-110～-79；在SGC-7901细胞中，NGAL基因启动子核心元件位于基因5′侧翼区-152～-141。4．研究发现，在胃癌细胞系BGC-823和SGC-7901中，NGAL基因的启动子均有TPA反应性。5．在胃癌细胞系BGC-823和SGC-7901中，NGAL基因启动子的TPA反应元件位于基因5′侧翼区-85～-79。6．在生物信息学预测基础上，通过蛋白印迹实验确定，在胃癌细胞系BGC-823中，可能由ERK细胞转导信号途径介导了NGAL基因的TPA反应性，AP1可能是其中的转录激活因子。结论：1．胃癌组织细胞中，NGAL基因高表达。2．在胃癌细胞系BGC-823和SGC-7901中，NGAL基因的表达存在着较大的差别。3．在胃癌细胞系BGC-823和SGC-7901中，NGAL基因启动子的核心元件不同。4．在胃癌细胞中，NGAL基因的启动子有TPA反应性，其TPA反应元件位于基因5′侧翼区-85～-79。5．AP1可能是胃癌细胞中NGAL基因的转录激活因子，介导着TPA反应性。6．ERK的磷酸化激活可能介导了NGAL基因TPA反应性信号的转导。更多还原

【Abstract】 Recently, many researches suggest that neutrophil neutrophil gelatinase-associated lipocalin (NGAL) was an important gene related with tumor, which was up-expressed in colon cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer and esophageal cancer. However, the expression level of NGAL in gastric cancer remains unclear. In this study, we have detected the NGAL expression in gastric cancer tissues and cells, and investigated the regulational mechanism of NGAL gene expression in gastric cancer cells. This research will provide more clues for fully cognizing the oncobiology functions of NGAL.Materials and methods:1. Expression of NGAL protein was analyzed by immunohistochemistry in 55 paraffin sections of gastric cancer tissue and the corresponding sections of normal gastric glandular tissue.2. Expression of NGAL in BGC-823 and SGC-7901 cell lines was analyzed by western blotting and RT-PCR.3. The luciferase repoter gene expression vectors containing NGAL gene 5’flanking regulation region were constructed by subcloning the different length DNA fragments of NGAL gene 5’flanking regulation region into pGL3-Basic vector.4. In gastric cancer cell lines BGC-823 and SGC-7901, the key region of NGAL promoter was identified by Dual-Luciferase Reporter Assay System.5. By the use of mutation and deletion techniques, the TPA responsiveness of NGAL promoter was analyzed in gastric cell lines.6. By using western blotting and bioinformatics, we identified the transcriptional activator and approached the signal transduction pathways that mediate NGAL expression response to TPA in gastric cancer.Results:1. In immunohistochemical study, only weak positive or negative signal was presented in normal gastric gland epithelium cells. While in gastric cancer cells, strong positive staining could be observed, a whole-cytoplasm expression of NGAL was presented in the cancer cells.2. There was visible distinction of NGAL expression level in two kinds of gastric cancer cell lines and the expression level in BGC-823 cells was obviously stronger than that in SGC-7901 cells.3. By employing the Dual-Luciferase Reporter Assay System, we have found that the key element of NGAL promoter was located on the -110 to -79 segments from transcriptional start site in BGC-823 cells, but in SGC-7901 cells it was located on the -152 to -141.4. It was found that NGAL promoter has TPA responsiveness both in BGC-823 and SGC-7901 cells.5. In BGC-823 and SGC-7901 cells, the TPA response element of NGAL promoter remains in the region from -85 to -79.6. Results of bioinformational prediction and western blotting suggested that the phosphorylation of ERK might be involved in the TPA signal transduction and AP1 was the transcriptional activator mediating the TPA responsiveness of NGAL promoter in BGC-823 cells.Conclusions:1. NGAL gene was up-expressed in gastric cancer tissues and cells.2. There was significant difference of NGAL expression level between BGC-823 and SGC-7901 cells.3. In gastric cancer cells BGC-823 and SGC-7901, the core element of NGAL promoter was different.4. In gastric cancer cells, it was found that NGAL promoter has TPA responsiveness and the TPA response element located on the region from -85 to -79.5. AP1 might be the transcriptional activator mediating TPA responsiveness of NGAL promoter in gastric cancer cells.6. The phosphorylation of ERK might be involved in the signal pathway that mediates NGAL expression response to TPA.更多还原