本文選題：Spastin + CRMP5��； 參考：《暨南大學(xué)》2017年博士論文

【摘要】：目的:神經(jīng)網(wǎng)絡(luò)的建立需神經(jīng)突起的生長(zhǎng)發(fā)育提供物質(zhì)基礎(chǔ),而神經(jīng)突起的生長(zhǎng)發(fā)育主要通過(guò)改變細(xì)胞骨架的運(yùn)動(dòng)進(jìn)行調(diào)控,細(xì)胞骨架的運(yùn)動(dòng)主要依靠微管與微絲間的不斷聚合和解聚,而微管與微絲間的互作機(jī)制仍未完全闡明。微管切割蛋白Spastin與微管結(jié)合蛋白CRMP5均參與神經(jīng)突起生長(zhǎng)過(guò)程,前期我們已證實(shí)Spastin與C RMP5能相互結(jié)合并明確相互結(jié)合位點(diǎn),本研究的主要目的在于進(jìn)一步明確Spastin協(xié)同CRMP5如何介導(dǎo)細(xì)胞骨架的運(yùn)動(dòng),調(diào)控神經(jīng)突起的生長(zhǎng)發(fā)育。材料與方法:首先通過(guò)免疫熒光染色,在共聚焦顯微鏡下觀察Spastin與CRMP5在Hela細(xì)胞和神經(jīng)元中共定位的情況;在Hela細(xì)胞中過(guò)表達(dá)Spastin~(M87V)及各截短片并結(jié)合共聚焦顯微鏡觀察微管的切割情況;培養(yǎng)原代神經(jīng)元,并利用磷酸鈣轉(zhuǎn)染方法導(dǎo)入Spastin~(M87V)及各截短片,觀察神經(jīng)元突起的變化情況;進(jìn)一步我們?cè)O(shè)計(jì)針對(duì)Spastin相對(duì)特異的干擾小片段si-Spastin導(dǎo)入神經(jīng)元中并觀察對(duì)其突起的影響情況;將Spastin~(M87V)及各截短片導(dǎo)入神經(jīng)元中并記錄m EPSC的變化情況;同樣,在原代神經(jīng)元中轉(zhuǎn)染CRMP5觀察神經(jīng)元突起的變化情況;我們進(jìn)一步在Hela細(xì)胞中共轉(zhuǎn)染Spastin~(M87V)和CRMP5,觀察微管切割的變化;同時(shí),在神經(jīng)元中共轉(zhuǎn)染Spastin~(M87V)和CRMP5,觀察神經(jīng)元突起的變化情況;通過(guò)設(shè)計(jì)不同的分組將質(zhì)粒導(dǎo)入神經(jīng)元中,分別觀察同時(shí)沉默Spastin與CRMP5以及沉默Spastin而過(guò)表達(dá)CRMP5或沉默CRMP5而過(guò)表達(dá)Spastin不同處理對(duì)神經(jīng)元突起的影響情況;在神經(jīng)元中進(jìn)一步共轉(zhuǎn)染Spastin~(M87V)和CRMP5,觀察m EPSC的變化情況。結(jié)果:1)免疫細(xì)胞化學(xué)結(jié)果示:Spastin與CRMP5共存于Hela細(xì)胞的胞質(zhì),神經(jīng)元的胞質(zhì)和突起中。2)培養(yǎng)Hela細(xì)胞并過(guò)表達(dá)Spastin~(M87V)、Spastin N190、Spastin△AAA、Spastin△N1、Spastin△N2基因:Spastin~(M87V)和Spastin△N1基因能將Hela細(xì)胞微管切割成小片段。3)培養(yǎng)原代海馬神經(jīng)元并過(guò)表達(dá)Spastin~(M87V)、Spastin N190、Spastin△AAA、Spastin△N1、Spastin△N2:Spastin~(M87V)、Spastin△N1、Spastin△N2可以明顯促進(jìn)海馬神經(jīng)元軸突、樹(shù)突側(cè)枝的形成和突起生長(zhǎng)。4)將GFP、Spastin~(M87V)、Spastin N190、Spastin△AAA、Spastin△N1、Spastin△N2導(dǎo)入神經(jīng)元中,用全細(xì)胞膜片鉗檢測(cè)結(jié)果示:Spastin~(M87V)、Spastin△N1、Spastin△N2組神經(jīng)元的m EPSC的頻率和幅度均較GFP+m C herry組增大。5)設(shè)計(jì)Spastin si-RNA干擾片段,與Spastin~(M87V)共導(dǎo)入293T細(xì)胞中,western-blot檢測(cè)其干擾效果明顯,導(dǎo)入神經(jīng)元,觀察到干擾Spastin對(duì)神經(jīng)突起的生長(zhǎng)有抑制作用。6)在原代海馬神經(jīng)元過(guò)表達(dá)CRMP5、CRMP5N471、CRMP5△471結(jié)果示:CRMP5、CRMP5△471可以明顯促進(jìn)海馬神經(jīng)元軸突、樹(shù)突側(cè)枝的形成和突起生長(zhǎng)。7)在Hela細(xì)胞共轉(zhuǎn)染GFP和m Cherry、Spastin~(M87V)和CRMP5、Spastin~(M87V)和m C herry、GFP和CRMP5結(jié)果示:導(dǎo)入CRMP5不影響Spastin~(M87V)對(duì)Hela細(xì)胞微管的切割效率。8)在原代海馬神經(jīng)元共過(guò)表達(dá)GFP和Mecherry、Spastin~(M87V)和CRMP5、Spastin~(M87V)和m Cherry、GFP和CRMP5結(jié)果示:Spastin~(M87V)和CRMP5組促進(jìn)海馬神經(jīng)元軸突、樹(shù)突側(cè)枝的形成和突起生長(zhǎng)更加明顯。9)在神經(jīng)元中分別導(dǎo)入NC、si-Spastin、si-CRMP5、si-Spastin+si-CRMP5、si-Spastin+C RMP5、Spastin+si-CRMP5結(jié)果示:干擾Spastin或C RMP5均抑制神經(jīng)元突起的生長(zhǎng),同時(shí)干擾兩者則抑制神經(jīng)元突起生長(zhǎng)更明顯,若干擾Spastin或CRMP5,同時(shí)過(guò)表達(dá)Spastin或CRMP5則能部分抵消抑制突起生長(zhǎng)作用。10)將GFP+m C herry、Spastin~(M87V)+CRMP5、Spastin~(M87V)+m C herry和CRMP5+GFP導(dǎo)入神經(jīng)元,用全細(xì)胞膜片鉗檢測(cè)結(jié)果示:Spastin~(M87V)+CRMP5、Spastin~(M87V)+m C herry、CRMP5+GFP組的m EPSC的頻率和幅度均較GFP+m C herry組增大。結(jié)論:1)Spastin與C RMP5在體內(nèi)存在相互共定位。2)Spastin~(M87V)對(duì)Hela細(xì)胞的微管的切割作用需有完整的微管結(jié)合區(qū)(MTBD區(qū))和AAA ATP催化區(qū)(AAA區(qū))。3)過(guò)表達(dá)CRMP5不影響Spastin~(M87V)對(duì)Hela細(xì)胞微管的切割效率。4)Spastin~(M87V)對(duì)神經(jīng)元突起生長(zhǎng)及分支形成的促進(jìn)作用與MTBD及AAA功能域密切相關(guān)。5)CRMP5對(duì)神經(jīng)元突起生長(zhǎng)及分支形成促進(jìn)作用的主要功能域在472-564氨基酸間。6)Spastin~(M87V)與CRMP5能協(xié)同調(diào)控神經(jīng)元突起生長(zhǎng)及分支的形成。
[Abstract]:Objective: the establishment of neural networks requires a material basis for the growth and development of neurites, and the growth and development of the neurites are regulated mainly by the movement of the cytoskeleton. The movement of the cytoskeleton depends on the continuous polymerization and disaggregation between microtubules and microfilaments, and the interaction mechanism between microtubules and microfilaments is still not fully elucidated. Microtubule cuts Spastin and microtubule binding protein CRMP5 are both involved in the process of neurite growth. In the early stage, we have confirmed that Spastin and C RMP5 can be combined with each other and define the binding sites. The main purpose of this study is to further clarify how Spastin and CRMP5 can mediate the transport of cytoskeleton and regulate the growth and development of neurites. Methods: Spastin and CRMP5 were observed by immunofluorescence staining under confocal microscopy in Hela cells and neurons. Spastin~ (M87V) and short films were overexpressed in Hela cells and the microtubule cutting was observed with confocal microscopy, the primary neurons were cultured and Spast was transfected into Spast by calcium phosphate transfection method. In~ (M87V) and the short clips to observe the changes in the neurite protuberance; further we design a relatively specific Spastin interfering small fragment of si-Spastin into the neuron and observe its influence on its protuberance; Spastin~ (M87V) and each short film are introduced into neurons and the changes of M EPSC are recorded; the same, in the primary neurons CRMP5 was used to observe the changes in the neurites of the neurons. We further transfected Spastin~ (M87V) and CRMP5 in Hela cells to observe the changes in microtubule cutting. At the same time, we transfected Spastin~ (M87V) and CRMP5 in the neurons to observe the changes in the neurite protuberance, and the plasmid was introduced into neurons by designing different groups. The effects of simultaneous silence of Spastin and CRMP5 and silence of Spastin on the expression of CRMP5 or silent CRMP5 and the effects of Spastin on the neuronal protuberance were observed; Spastin~ (M87V) and CRMP5 were further co transfected in neurons, and the changes in M EPSC were observed. Results: 1) the immunocytochemical results showed that Spastin was coexisting with the M. Spastin~ (M87V), Spastin N190, Spastin Delta AAA, Spastin Delta N1, Spastin Delta N2 genes are expressed in the cytoplasm of the cell, the cytoplasm of the neuron and the.2 in the protuberance. N Delta AAA, Spastin Delta N1, Spastin Delta N2:Spastin~ (M87V), Spastin Delta N1, Spastin Delta N2 can obviously promote hippocampus neuron axon, dendritic lateral branch formation and initiation growth.4). The frequency and amplitude of M EPSC of pastin Delta N1, Spastin Delta N2 group neurons are more than GFP+m C Herry group.5) designed Spastin si-RNA interference fragment. The overexpression of CRMP5, CRMP5N471, CRMP5 delta 471 shows that CRMP5, CRMP5 delta 471 can obviously promote the axon of hippocampal neurons, the formation of dendrites and the growth of.7) in the Hela cells co transfected with GFP and m Cherry. Hela cell microtubule cutting efficiency.8) in the primary hippocampal neurons, the expression of GFP and Mecherry, Spastin~ (M87V) and CRMP5, Spastin~ (M87V) and m Cherry, GFP, and m Cherry, the formation of the hippocampal neurons, the formation of the dendrites and the growth of the protuberance are more obvious. Si-CRMP5, si-Spastin+si-CRMP5, si-Spastin+C RMP5, Spastin+si-CRMP5 results show that interference of Spastin or C RMP5 all inhibit the growth of neuronal protuberance, while interference both inhibits the growth of neuron protuberances more clearly, if Spastin or CRMP5 is interfered with, while overexpression Spastin or CRMP5 can partially counteract the inhibition of protuberance growth Herry, Spastin~ (M87V) +CRMP5, Spastin~ (M87V) +m C Herry and CRMP5+GFP import neurons. 87V) cutting effect on microtubules of Hela cells requires a complete microtubule binding zone (MTBD region) and AAA ATP catalytic region (AAA region).3) over expression CRMP5 does not affect the efficiency of Spastin~ (M87V) cutting efficiency of Hela cell microtubules. The main functional domain of neurite protuberance growth and branching formation is.6 between 472-564 amino acids.) Spastin~ (M87V) and CRMP5 can regulate the growth of neurites and the formation of branches in synergy.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R338

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