内吞衔接蛋白Numb在成熟小脑浦肯野细胞中功能的研究

发布时间：2018-05-04 13:35

本文选题：Numb + 代谢性谷氨酸受体1　；参考：《浙江大学》2016年博士论文

【摘要】：背景：Numb最初是在果蝇上发现的,它可以决定在发育过程中细胞的命运。体外实验显示,在哺乳动物中Numb可以促进神经突起的生长,还可以和网格蛋白clathrin结合从而参与内吞。不过它在成熟神经元中有什么功能还不清楚。目的：探索Numb在成熟小鼠浦肯野细胞中的功能和作用机制。方法：首先我们利用Cre/Loxp条件性敲除技术,使用L7-Cre专门在小脑浦肯野细胞敲除Numb基因；之后我们使用平衡木和加速转轮实验测试小鼠的运动协调能力；使用Western Blot和免疫组化方法来检测各种蛋白的表达情况；利用RT-PCR和原位杂交技术方法检测浦肯野细胞内一些目的基因的表达情况；利用激光共聚焦显微镜观察浦肯野细胞的形态结构以及钙成像；使用电生理技术检测小脑浦肯野细胞的兴奋性突触后电流和兴奋性突触后电位的变化。结果：我们在成年小鼠的小脑浦肯野细胞中发现Numb有高水平的表达,使用条件性敲除技术敲除浦肯野细胞的Numb基因后,发现小脑细胞结构仍正常,但是小鼠的运动的协调性受损了。研究证明Numb对浦肯野细胞中代谢型谷氨酸受体1(mGlu1)的内化和回收利用起至关重要的作用。在条件性敲除Numb的小鼠,浦肯野细胞突触部位mGlul显著减少,而且平行纤维-浦肯野细胞突触的长时程抑制也受损。基因敲除小鼠中激动剂诱导的mGlul运输也显著受到抑制,但Numb的缺失没有影响离子型谷氨酸受体亚基和mGlul相关蛋白的在突触部位的表达。并且,在缺失Numb的浦肯野细胞中可诱导出mGlu1自身的短时程和长时程的可塑性现象。研究证明Numb是mGlu1组成性表达和转运的一个调控因子。
[Abstract]:Background: * numb was first found in Drosophila, which determines the fate of cells during development. In vitro experiments showed that Numb could promote the growth of neurite in mammalian cells and bind to griddle protein clathrin to participate in endocytosis. But what it does in mature neurons is unclear. Objective: to explore the function and mechanism of Numb in mature mouse Purkinje cells. Methods: firstly, we used Cre/Loxp conditional knockout technique to use L7-Cre to knockout Numb gene in cerebellar Purkinje cells, then we used balance beam and acceleration wheel test to test the motor coordination ability of mice. Western Blot and immunohistochemistry were used to detect the expression of various proteins, RT-PCR and in situ hybridization were used to detect the expression of some target genes in Purkinje cells. The morphological structure and calcium imaging of Purkinje cells were observed by laser confocal microscopy, and the changes of excitatory postsynaptic currents and excitatory postsynaptic potentials in cerebellar Purkinje cells were detected by electrophysiological techniques. Results: we found a high level of Numb expression in the cerebellar Purkinje cells of adult mice. After knockout the Numb gene of Purkinje cells by conditional knockout technique, we found that the structure of cerebellar cells was still normal. But the coordination of the mice's movements was impaired. It has been demonstrated that Numb plays an important role in the internalization and recycling of metabolic glutamate receptor 1 mGlu1 in Purkinje cells. In conditioned knockout Numb mice, mGlul at synaptic site of Purkinje cells decreased significantly, and the long-term inhibition of parallel fiber-Purkinje synapses was also impaired. MGlul transport induced by agonists in knockout mice was also significantly inhibited, but the deletion of Numb did not affect the expression of ionic glutamate receptor subunits and mGlul related proteins at synaptic sites. Furthermore, the short and long term plasticity of mGlu1 itself can be induced in Purkinje cells without Numb. It has been proved that Numb is a regulatory factor for the constitutive expression and transport of mGlu1.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q42

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