TDP-43与β-淀粉样前体蛋白胞内结构域相互作用并诱导P53依赖的细胞凋亡

发布时间：2018-01-08 09:30

本文关键词：TDP-43与β-淀粉样前体蛋白胞内结构域相互作用并诱导P53依赖的细胞凋亡　出处：《苏州大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：探求TDP-43(TAR DNA-binding protein43)与β-淀粉样前体蛋白胞内结构域（the intracellular domain of Amyloid Precursor Protein，AICD）之间的关系，从而揭示其在阿尔茨海默病病理机制中可能发挥的作用。方法： 1.运用免疫荧光染色方法在HEK293/COS7细胞系上检测TDP-43与AICD在细胞水平的是否存在共定位。2.在细胞系上转染克隆质粒TDP-43-V5、APP-FLAG，，48h后收集细胞提取蛋白，用免疫共沉淀（co-immunoprecipitations）的方法检测APP（β淀粉样蛋白前体蛋白）与TDP-43在蛋白水平是否结合。在野生型C57/BL6J成年小鼠的大脑组织上用免疫共沉淀方法证明内源性的TDP-43与APP在正常小鼠脑组织中是否存在结合关系。进一步在细胞系上转染质粒TDP-43-V5、AICD-FLAG，以明确TDP-43是否与AICD存在结合关系。3.用双荧光素酶报告基因测试方法检测TDP-43对AICD是否存在转录作用，进一步加入APP γ酶切位点的分泌酶抑制剂，观察这种转录作用是否有变化。用Real-Time PCR的方法检测这种转录作用对P53mRNA水平的影响。4.在分别转染了AICD，TDP-43，AICD+TDP-43的细胞系上计数Caspase-3阳性细胞，直接比较对细胞凋亡的作用。使用TUNEL法再次重复实验。结果： 1.免疫荧光染色，观察到在体外培养的HEK293/COS7细胞系上，TDP-43在细胞水平定位于胞核，AICD大部分位于胞核，且二者存在共定位关系。2.在细胞系上转染质粒，进一步证明了TDP-43与AICD在蛋白水平存在结合。在野生型C57/BL6J成年小鼠的大脑组织上检测到内源性的TDP-43与AICD存在结合。3.TDP-43对AICD有类似于Fe65样的转录激活作用，但是这种作用可以被加入的APPγ酶切位点的分泌酶抑制剂所阻断（***p0.001；NS：无意义）。TDP-43有类似与AICD的作用，增加P53mRNA的表达水平，从而促进TP53基因编码的P53蛋白的转录作用，且TDP-43可以增强AICD该种转录激活作用（*：p 0.05；**: p 0.01；***p 0.001）。4. TDP-43在诱导细胞凋亡方面有与AICD类似的作用，其增加Caspase-3阳性或TUNEL阳性细胞数，促进细胞凋亡；并且TDP-43可以增强AICD诱导细胞凋亡的作用（*：p 0.05；**：p 0.01；***：p 0.001）。结论： TDP-43与AICD共定位存在与细胞核中；TDP-43增强AICD的转录作用，以及ACID对P53的转录调控作用；TDP-43促进AICD介导的细胞凋亡。因此，通过我们的研究结果可以设想，TDP-43可能为阿尔茨海默病研究的新靶点。阿尔茨海默病相关病理组织的萎缩可能与TDP-43调控的APP信号通路有相关性。
[Abstract]:Objective: Study on the relationship between TDP-43(TAR DNA-binding protein 43 and 尾 -amyloid precursor protein (尾 -amyloid precursor protein). The intracellular domain of Amyloid Precursor Protein. The relationship between AICD, thus revealing its possible role in the pathological mechanism of Alzheimer's disease. Methods: 1. Immunofluorescence staining was used to detect the co-localization of TDP-43 and AICD in HEK293/COS7 cell line. 2. Transfection of cloned plasmid T. DP-43-V5. After 48 hours of APP-FLAGG, the protein extracted from the cells was collected. Detection of app (尾 -amyloid precursor protein) by co-immunoprecipitation with co-immunoprecipitation. The immunoprecipitation method was used to prove that endogenous TDP-43 and APP were present in the brain of normal C57 / BL6J adult mice by immunoprecipitation. Further transfection of plasmid TDP-43-V5 on cell line was carried out. AICD-FLAG. To determine the binding relationship between TDP-43 and AICD. 3. To detect whether TDP-43 has transcriptional effect on AICD by double luciferase reporter gene test. The secretory enzyme inhibitor of APP 纬 restriction site was added further. Real-Time PCR was used to detect the effect of transcription on p53 mRNA level. 4. AICD was transfected respectively. Caspase-3 positive cells were counted on the cell line of TDP-43 AICD TDP-43, and the effect on apoptosis was compared directly. TUNEL method was used to repeat the experiment. Results: 1. Immunofluorescence staining showed that TDP-43 was located mostly in the nucleus of HEK293/COS7 cell line at the cellular level. And there is a colocalization relationship between them. 2. Transfection plasmid on cell line. It was further demonstrated that TDP-43 and AICD bind to protein level. Endogenous TDP-43 and AICD knots were detected in the brain tissues of wild-type C57 / BL6J adult mice. The transcriptional activation of AICD by TDP-43 is similar to that of Fe65. However, this effect could be blocked by the addition of secretory enzyme inhibitor at APP 纬 site. NS.TDP-43 has similar effect with AICD and increases the expression level of p53 mRNA, thus promoting the transcription of p53 protein encoded by TP53 gene. TDP-43 could enhance the transcriptional activation of AICD. P0.01; TDP-43 has similar effect as AICD in inducing apoptosis, which increases the number of Caspase-3 positive cells or TUNEL positive cells. Promoting apoptosis; TDP-43 could enhance the apoptosis induced by AICD. P0.01; A: P 0.001. Conclusion: TDP-43 and AICD co-located in the nucleus; TDP-43 enhanced the transcription of AICD and the transcriptional regulation of p53 by ACID. TDP-43 promotes apoptosis mediated by AICD. Therefore, through our research results, we can imagine. TDP-43 may be a new target for the study of Alzheimer's disease. The atrophy of Alzheimer's disease-related tissues may be related to the APP signaling pathway regulated by TDP-43.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R749.16

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