Caspr与β-淀粉样前体蛋白在体外相互作用减少了β-淀粉样蛋白的产生

发布时间：2019-01-24 19:25

【摘要】：目的：研究Caspr（Contactin associated protein）与β-淀粉样前体蛋白（Amyloid PrecursorProtein，APP）之间的关系，探讨Caspr抑制β-淀粉样蛋白（amyloid-β，Aβ）产生的机制，揭示Caspr在阿尔茨海默病病理机制中可能发挥的作用。方法：利用免疫荧光技术（Immunofluorescence staining）观察Caspr在APP/PS1老年痴呆模型小鼠脑内的分布情况；通过免疫印迹法（western blot）观察Caspr在APP/PS1老年痴呆模型小鼠和同窝野生型小鼠脑内的蛋白表达情况；以稳定表达印第安纳突变的APP的HEK-293细胞(V717F，HEK-APP)和表达内源性APP的CHO细胞为研究对象，利用细胞转染技术将质粒PCMV-Caspr-myc及它的空载体转染进细胞中，通过免疫印迹法观察细胞内APP的蛋白表达情况，并利用实时定量PCR（Quantitativereal-time PCR，qPCR）的方法检测APP的mRNA表达情况；利用免疫荧光技术观察Caspr和APP在细胞中和C57BL/6J成年小鼠（雄性）大脑皮层中的共定位情况；用免疫共沉淀法（Co-Immunoprecipitation，Co-IP）研究Caspr和APP的相互作用关系；将质粒PCMV-Caspr-myc转染进HEK-APP细胞中，，利用酶联免疫吸附试验（ELISA）研究Caspr对APP的酶切产物Aβ（Aβ40和Aβ42）的影响；用MTT比色法检测转染Caspr后对细胞活性的影响。结果：免疫荧光法显示，在APP/PS1老年痴呆模型小鼠的大脑皮层中，Caspr高度聚集在老年斑周围；Western blot显示，与对照组相比，Caspr在APP/PS1老年痴呆模型小鼠的大脑皮层中表达增加；Western blot和qPCR结果表明，在HEK-APP和CHO细胞中，过表达Caspr使APP蛋白的表达量下降，但并不影响APP的mRNA水平；通过免疫荧光技术发现，不论是在小鼠大脑皮层还是细胞系中，Caspr和APP都共定位于细胞膜上，并且Co-IP证实它们是有相互作用的；ELISA结果显示，Caspr能抑制APP的酶切产物Aβ（Aβ40和Aβ42）的产生，但是Aβ42/Aβ40的比值不受影响；MTT比色法证实转染Caspr后细胞活性并没有受到明显影响。结论：在APP/PS1模型小鼠皮层中，Caspr表达增加并聚集在老年斑周围。Caspr与APP相互结合，Caspr通过转录后调控机制减少了APP蛋白的表达，并且抑制导致老年斑形成的Aβ的产生。由此，我们的研究发现Caspr参与了阿尔茨海默病病理机制，其有可能是治疗AD的一个新的药物靶点。
[Abstract]:Objective: to study the relationship between Caspr (Contactin associated protein) and 尾 -amyloid precursor protein (Amyloid PrecursorProtein,APP), and to explore the mechanism of Caspr inhibiting the production of 尾 -amyloid protein (A 尾). To reveal the role of Caspr in the pathogenesis of Alzheimer's disease. Methods: the distribution of Caspr in the brain of APP/PS1 senile dementia mice was observed by immunofluorescence technique (Immunofluorescence staining). The expression of Caspr protein in the brain of APP/PS1 senile dementia mice and the same litter wild type mice was observed by Western blotting (western blot). HEK-293 cells (V717FnHEK-APP) expressing Indiana mutant APP and CHO cells expressing endogenous APP were used to transfect plasmid PCMV-Caspr-myc and its empty vector into the cells by cell transfection technique. The expression of APP protein was observed by Western blot and mRNA expression of APP was detected by real-time quantitative PCR (Quantitativereal-time PCR,qPCR). The co-localization of Caspr and APP in cells and cerebral cortex of adult C57BL/6J mice (male) was observed by immunofluorescence technique, and the interaction between Caspr and APP was studied by immunoprecipitation (Co-Immunoprecipitation,Co-IP). Plasmid PCMV-Caspr-myc was transfected into HEK-APP cells. The effect of Caspr on A 尾 (A 尾 40 and A 尾 42) of APP was studied by (ELISA), and the effect of Caspr transfection on cell activity was detected by MTT colorimetry. Results: immunofluorescence assay showed that in the cerebral cortex of APP/PS1 dementia mice, Caspr was highly concentrated around senile plaque. Western blot showed that the expression of Caspr was increased in the cerebral cortex of APP/PS1 senile dementia mice compared with the control group. The results of Western blot and qPCR showed that overexpression of Caspr in HEK-APP and CHO cells decreased the expression of APP protein, but did not affect the mRNA level of APP. Immunofluorescence technique showed that both Caspr and APP were located on the membrane of mouse cerebral cortex and cell line, and Co-IP confirmed that they interact with each other. The results of ELISA showed that Caspr could inhibit the production of A 尾 (A 尾 40 and A 尾 42), but the ratio of A 尾 42 / A 尾 40 was not affected, and the cell activity was not affected by MTT colorimetry. Conclusion: in the cortex of APP/PS1 model mice, the expression of Caspr increases and aggregates around the senile plaque. Caspr binds to APP and Caspr reduces the expression of APP protein through the mechanism of posttranscriptional regulation. And inhibit the production of A 尾, which leads to the formation of senile plaque. Therefore, our study found that Caspr is involved in the pathogenesis of Alzheimer's disease and may be a new drug target for the treatment of AD.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

【相似文献】