發(fā)布時間：2018-05-04 13:35

  本文選題：Numb + 代謝性谷氨酸受體1�。� 參考：《浙江大學(xué)》2016年博士論文

【摘要】：背景：Numb最初是在果蠅上發(fā)現(xiàn)的,它可以決定在發(fā)育過程中細胞的命運。體外實驗顯示,在哺乳動物中Numb可以促進神經(jīng)突起的生長,還可以和網(wǎng)格蛋白clathrin結(jié)合從而參與內(nèi)吞。不過它在成熟神經(jīng)元中有什么功能還不清楚。目的：探索Numb在成熟小鼠浦肯野細胞中的功能和作用機制。方法：首先我們利用Cre/Loxp條件性敲除技術(shù),使用L7-Cre專門在小腦浦肯野細胞敲除Numb基因；之后我們使用平衡木和加速轉(zhuǎn)輪實驗測試小鼠的運動協(xié)調(diào)能力；使用Western Blot和免疫組化方法來檢測各種蛋白的表達情況；利用RT-PCR和原位雜交技術(shù)方法檢測浦肯野細胞內(nèi)一些目的基因的表達情況；利用激光共聚焦顯微鏡觀察浦肯野細胞的形態(tài)結(jié)構(gòu)以及鈣成像；使用電生理技術(shù)檢測小腦浦肯野細胞的興奮性突觸后電流和興奮性突觸后電位的變化。結(jié)果：我們在成年小鼠的小腦浦肯野細胞中發(fā)現(xiàn)Numb有高水平的表達,使用條件性敲除技術(shù)敲除浦肯野細胞的Numb基因后,發(fā)現(xiàn)小腦細胞結(jié)構(gòu)仍正常,但是小鼠的運動的協(xié)調(diào)性受損了。研究證明Numb對浦肯野細胞中代謝型谷氨酸受體1(mGlu1)的內(nèi)化和回收利用起至關(guān)重要的作用。在條件性敲除Numb的小鼠,浦肯野細胞突觸部位mGlul顯著減少,而且平行纖維-浦肯野細胞突觸的長時程抑制也受損�；蚯贸∈笾屑觿┱T導(dǎo)的mGlul運輸也顯著受到抑制,但Numb的缺失沒有影響離子型谷氨酸受體亞基和mGlul相關(guān)蛋白的在突觸部位的表達。并且,在缺失Numb的浦肯野細胞中可誘導(dǎo)出mGlu1自身的短時程和長時程的可塑性現(xiàn)象。研究證明Numb是mGlu1組成性表達和轉(zhuǎn)運的一個調(diào)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q42

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