精选国家基金申请存在的问题和对策.pptVIP

国家基金申请存在的问题和对策;申请书需回答的问题;标 题; 神经元凋亡时SP1对BH3-only蛋白Bim的转录调控;摘 要;神经元凋亡时SP1对BH3-only蛋白Bim的转录调控;CaMKII磷酸化GSK-3对去极化介导的神经元存活的调控;立项依据;Fig.6 Mithramycin A, a SP1-DNA binding inhibitor, attenuates bim upreguation by activity deprivation in CGNs. (A) Mithramycin A attenuates the upregulation of BimEL induced by activity deprivation in a dose dependent manner. CGNs were placed in 25K or 5K media in the absence or presence of mithramycin A at the indicated concentrations for 6 h. BimEL expression was assessed by Western blot as described in Fig.1(A). Results shown are representative of four separate experiments. (B) Mithramycin A inhibits the increase in bim mRNA level in a dose-dependent manner. Neurons were treated as described in (A), bim and actin mRNAs were determined by RT-PCR, Results shown are representative of five separate experiments. (C) Mithramycin A suppresses the activation of bim promoter induced by activity deprivation. Neurons were co-transfected with bim-Luc and pEF-RL for 8 hr. Luciferase activities were measured as described in Fig.2, The experiments were repeated three times with duplicates for each treatment. The data represent ±S.E.M. of three experiments. ;参 考 文 献;研究内容、研究目标与拟解决的关键问题;要求：明确 具体 解决学术问题为目的 ; 研究内容 （围绕研究目标） 1.??证明bim启动子核心区的SP1结合序列是否介导了bim基因的上调 为了证实bim启动子核心区的SP1结合序列是否介导了bim基因的上调，将其中的SP1结合序列进行点突变，使其不能结合SP1；确定这一突变是否能够消除bim启动子的撤钾反应。 2.??分析SP1 是否能够与bim启动子核心区的SP1结合序列结合 应用EMSA (Electrophoretic Mobility Shift Assay)技术，证实bim启动子核心区的SP1 结合序列是否能够与SP1结合；进一步采用CHIP(Chromatin immunoprecipitation)方法，获得SP1与bim启动子核心区在细胞内结合的证据。 3.??从 bim 的mRNA 和蛋白质水平，观察负显性SP1是否能够抑制撤钾诱导的bim基因的上调 质粒转染CGNs的转染率只能达到0.1-1.0 %，腺病毒的感染率可达70－80%[1]。 因此，构建dnSP1的腺病毒载体Ad-dnSP1，感染CGNs才能达到可靠分析bim的mRNA和蛋白质表达水平改变的目的。 4. 分析SP1诱导Bim这一机制对神经元凋亡的调控作用 确立SP1转录激活bim这一机制后，进一步分析这一机制对神经元凋亡的调控作用。 ; 项目需求为前提 可靠性第一，先进性第二 参考文献 突出关键技术的描述 不主张使用流程图，建议开头：“有关方法、技术路线、实验手段、关键技术综述如下……” ;基本方法;负显性SP1腺病毒(Ad-dnSP1)载体的构建、扩增、纯化和保存;申请者简历;黎明涛 (Li Mingtao) 项目负责人，中山大学中山医学院药理教研室教授，博