TRAF6在急性颅脑损伤中作用的研究-医学、外科学专业论文.docxVIP

T TRAF6 在急性颅脑损伤中作用的研究 PAGE PAGE 13 PAGE PAGE 10 中文摘要 目的:通过检测急性脑损伤组织中TRAF6的表达水平，及观察TRAF6对NFκB报 告基因活化和对星形胶质瘤细胞趋化因子CCL18产生的影响，为寻找新的急性脑 损伤后炎症反应的重要调控靶点奠定实验基础及理论依据。 方法:选取2010年7月～2010年9月间滨州医学院附属医院神经外科重型颅脑损 伤（GCS9’）手术病人30例，分别取无功能周围正常脑组织和损伤的脑组织， 作为对照组和实验组。RT-PCR法检测两组标本中TRAF6的表达；将扩增的TRAF6 基因编码区全长片段克隆到pcDNA3.1(+)，并转染至HEK293T细胞，Western blot 检测TRAF6的蛋白表达；双萤光素酶报告基因系统检测TRAF6对NFκB报告基因 活化的影响；ELISA检测TRAF6对星形胶质瘤细胞趋化因子CCL18产生的影响。 结果:与对照组比较，实验组TRAF6的表达明显升高，且差异具有显著性 （P0.05）；构建的TRAF6真核表达载体可在HEK293T细胞中表达；与对照组比 较，实验组TRAF6能增强星形胶质瘤细胞NFκB报告基因的活性，并能促进星形 胶质瘤细胞趋化因子CCL18的产生，且差异具有显著性（P0.05）。 结论:TRAF6在急性脑损伤脑组织中的表达明显升高；TRAF6能促进星形胶质 瘤细胞转录因子NFκB的激活，并促进星形胶质瘤细胞趋化因子CCL18的表达。 关键词:急性脑损伤；TRAF6；NFκB；CCL18 The Role of TRAF6 in Acute Craniocerebral Injury Abstract Objective:To searching for a novel key targets to regulate inflammation in traumatic brain injures, we examined the expressing level of TRAF6 in injured brain tissue, and determined the effect of TRAF6 on NF?B activation and the secretion of CCL18 in astrocytomas. Methods: 30 patients with severe traumatic brain injury (GCS9) were selected from affiliated hospital of Bin Zhou medical college from July, 2010 to September, 2010. The injured brain tissue and peripheral normal brain tissue with no function were selected as experimental group and control group respectively. The mRNA expression level of TRAF6 in traumatic brain was detected by RT-PCR. The whole coding sequence of TRAF6 was cloned into pcDNA3.1 (+) vector. The recombinant vector was confirmed by restriction enzyme digestion, colony PCR and sequencing, subsequently it was transfected into HEK293T cells to detect TRAF6 expression by western blot. The effect of TRAF6 on NF?B activities was determined by dual-luciferase reporter assay and the secretion of CCL18 was determined by ELISA. Results: High-level expression of TRAF6 was detected in injured brain tissue than the normal control. Transfected in HEK293T cells, TRAF6 could be obviously detected by western blot. Dual-luciferase assay sh