【作者】 王付龙；

【导师】 梁华平； 刘昕；

【作者基本信息】 第三军医大学 ， 野战外科学， 2002， 硕士

【摘要】 核因子-κB(NF-κB)是一类重要的核转录激活因子，它的过度激活是多种疾病发生发展的关键环节。以NF-κB为靶点，筛选、研制其拮抗药物是目前十分活跃的研究领域之一。 建立灵敏、可靠地监测NF-κB的活性和判定其活化状态程度的方法，是准确抑制NF-κB的过度活化，进行评价NF-κB拮抗药物效果以及研究NF-κB相关信号转导途径所必需的前提条件。通常应用测定报告基因转录表达的方法来反映和监测转录因子及其顺式作用元件的功能。常用的报告基因系统有Cat、Luc、GUS、LacZ、SEAP等，然而这些系统存在一个共同的缺陷，即它们都是酶类报告基因，都需要相应的底物和辅助因子的参与才能够间接监测报告分子，因此检测步骤较繁，实验重复性欠佳，更重要的是不能原位直接观察报告分子的动态表达情况；而正是这一缺陷，对药物筛选鉴定的研究工作来说，其影响往往是致命的。 绿色荧光蛋白(GFP)基因是目前唯一的蛋白类新型体内分析报告基因。GFP无种属依赖性、不需外源性底物和辅助因子参与、不需设内对照、无毒性、经紫外和蓝光激发产生可量化的绿色荧光，因而具有直观原位、实时全程、分析简单的特征。但野生型GFP及增强型GFP等因半衰期长，稳定性强，对反映一过性转录表达调控的变化不甚理想。新近研制的不稳定型EGFP(d2EGFP)，因其半衰期短，蓄积性低，较之EGFP更能灵敏动态反映转录因子及其顺式元件的作用效果的改变，故应用d2EGFP来研究顺反式因子的作用关系，可以一定程度上克服上述报告基因的缺憾。 以d2EGFP作为报告分子来反映、监测NF-κB活性的研究尚处于起始阶段，构建带有d2EGFP、哺乳动物细胞筛选标记新霉素抗性基因和SV40基本启动子的载体，进而建立可诱导表达d2EGFP报告细胞株的研究尚未见文献报道。因此本研究拟在含新霉素抗性基因的载体上，以dZEGFP为报告基因建立较理想的NF－KB反应性报告系统，进行特异、灵敏、精确地反映和监测NFKB的活性，为判定NF七B的活化程度及其相关信号转导途径，并进一步为后续研制的新型、高效、专一的NF－KB a抗药物进行高重复、大批量筛选鉴定工作，奠定体外、体内实验的基础。 本研究为获得较理想的NF－KB反应性dZEGFP报告系统，对人工改造与天然存在的顺式元件h基序的特异性、增强性以及dZEGFP与EGFP的稳定性、灵敏性等进行了对照比较研究，并获得了NF－KB反应性dZEGFP报告细胞株。继之在筛选鉴定NF－KB桔抗药物的研究中进行实际应用，进一步证明了本系统的效用性。 本研究主要结果与结论如下： 1．采用 NFKB的顺式元件KB X 4作为增强于、SV40为基础启动子或含有。B X 2的 HIV启动子，分别与报道基因 dZEGFP和 EGFP构建在含有新霉素抗性基因的载体上，准确构建了三种NF－KB反应性GFP报告基因载体。 2．三种报告基因载体分别与p65载体瞬时共转染HEK293细胞，通过p65蛋白诱导调控三种报告基因载体dZEGFP和EGFP表达的时间效应和剂量效应，进行功能比较分析，证明了p4。B刁ZEGFP载体是较理想的NF－KB反应性GFP报告基因系统。 3．将p4。B河 载体稳定转染HEK293细胞，经G418筛选出22株克隆细胞，建立了NF－KB反应性dZEGFP报告细胞株HEK－dZEGFP。 4．应用 NF－KB TFD与 p65瞬时共转染 HEK－dZEGFP，1、Zms／L NF。BTFD组明显桔抗了p65蛋白诱导的dZEGFP表达。证明NF－h反应性dZEGFP报告系统具有实效性。更多还原

【Abstract】 NF-icB(nuclear factor kappa B) is an important transactivator, whose overactivation is a key point to cause and develop many kinds of diseases. So, it is one of very important and prospective study to detect and screen antagonistic drugs of NF-KB as a target.How to report and detect NF-KB activation and it’s activated degree reliably and sensitively is the prerequisite for inhibiting NF-kB overactivation accurately , evaluating it’s antagonistic drugs and researching signal transduction pathway related to NF-icB .Usually reporter gene system is applied to report the interaction between transcription factor and it’s cis-element by detecting the reporter gene expression, such as Cat, Luc, GUS, LacZ, SEAP, etc. However, those enzyme reporter gene systems have a common defect that the reporter molecules are detected by certain substrate and cofactor to react, so the course is complicated and the results are not easy to be repeated and even the reporter molecules cannot be observed dynamically in cells. Consequently, screening drugs is fatal due to the defects.Green fluorescent protein (GFP) gene which is unique, new protein reporter gene analysed in vivo has more advantageous characters than enzyme reporter gene analysed in vitro, such as direct observation in situ, real time and whole kinetics, simple analysis , and so on. But wild type GFP and EGFP are not ideal to detect the transient changes of gene expression regulation. Hopefully, the new product-d2EGFP (destabilized EGFP) is more sensitive to detect the changes of gene transcription regulation than EGFP due to the factthat d2EGFP has a short fluorescence half-life of 2h, and low accumalation in cells . This characters give important hints on d2EGFP is an ideal reporter gene to study and detect NF-KB activation accurately. However, this study is in the early stage. The vector containing neor gene and SV40 minimal promoter and the stable transfected clonal cell lines have been not reported until now.In order to establish an ideal NF-KB activation-responsive d2EGFP reporter system, this experiment compared the-.enhancer ability of the man-made KB motif with that of the wild one, the fluorescent stability and sensibility of d2EGFP with that of EGFP , cultured clonal cell lines, and applied in screening drugs.The main results and conclusions were as follows:1. Three types of NF-KB-responsive GFP reporter gene vectors were constructed successfully with NF-KB cis-element KBx4 as enhancer , SV40 as mininal promoter or HIV with KBx2 motif as promoter ,d2EGFP and EGFP as reporter gene ,neor gene as selective gene.2. The time and dose effects of d2EGFP and EGFP induced by p65 protein were analysed after p65 vector transient contransfected with the three vectors respectively. It was demonstrated that p4KB-d2EGFP was the best NF-KB-responsive GFP reporter gene system among the three vectors.3. Stablely transfected p4KB-d2EGFP into HEK 293 cells and harvested positive clonal cell lines by G418 selection . The NF-KB-responsive d2EGFP clonal cell line named HEK-d2EGFP was established successfully.4. With the contransfection of NF-KB TFD(transcription factor decoy) and p65 vector into HEK-d2EGFP cells , the results showed the groups of 1 mg/L and 2mg/L TFD could antagonized the d2EGFP expression induced by p65 protein significantly. It was demonstrated that NF-KB-responsive d2EGFP reporter system had the specificity and actual effect.5. It was suggested that the NF-KB-responsive d2EGFP reporter systemscould report and detect NF-KB activation and screen it’s antagonistic drugs in certain degree, and might lay the model foundations for studying relevant transcription factors’ activation.更多还原