本文選題：磷酸二酯酶4D　切入點：咯利普蘭　出處：《中國科學(xué)技術(shù)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：腦神經(jīng)發(fā)育可塑性的分子機制一直是腦科學(xué)研究的熱點。對視皮層的研究工作開始的較早,其結(jié)構(gòu)也了解的比較透徹,因此,視覺可塑性是研究大腦神經(jīng)發(fā)育可塑性常用的經(jīng)典模型。眼優(yōu)勢可塑性(ocular dominance plasticity,ODP)是指關(guān)鍵期內(nèi)單眼剝奪(monocular deprivation,MD)引起的初級視皮層細胞對剝奪眼反應(yīng)的降低,而對非剝奪眼的反應(yīng)增加。環(huán)單磷酸腺苷(cyclic adenosine monophosphate,cAMP)-蛋白激酶A(protein kinase A,PKA)是重要的第二信使通路,有很多報道都已經(jīng)證明cAMP-PKA通路對ODP的表達至關(guān)重要,但其上下游的調(diào)控機制并不清楚。本文主要是研究調(diào)控cAMP-PKA通路的兩個重要分子,磷酸二酯酶4D(phosphodiesterase 4D,PDE4D)和 A 型激酶錨定蛋白(A-kinase anchoring proteins,AKAPs)如何影響 ODP。前人的研究已經(jīng)證明cAMP參與到關(guān)鍵期內(nèi)視覺可塑性的表達。PDE4是cAMP的主要水解蛋白,可以調(diào)控cAMP的時空分布。PDE4D是PDE4家族中的一個重要成員,有很多報道證明它通過特異性的水解cAMP從而影響突觸可塑性,但是卻幾乎沒有人研究它是否影響視覺可塑性。因此,我們首先在小鼠以及貓上研究了PDE4D在發(fā)育過程中如何參與調(diào)節(jié)視覺可塑性。在小鼠上,通過選取發(fā)育時程中不同時間點的雙眼區(qū)的腦組織進行蛋白質(zhì)印跡(western blot)測定,我們發(fā)現(xiàn)視皮層中的PDE4D的表達時程與ODP的表達時程相吻合。在發(fā)育早期PDE4D的蛋白水平較低,在關(guān)鍵期達到峰值,而關(guān)鍵期后水平下降。然而改變視覺經(jīng)驗,例如暗飼養(yǎng)(dark rearing,DR)或者MD都沒有影響PDE4D在視皮層中的蛋白含量,表明PDE4D的表達是不依賴于視覺經(jīng)驗的。對關(guān)鍵期內(nèi)MD的小鼠腹腔注射PDE4的選擇性抑制劑咯利普蘭(rolipram)之后,通過使用內(nèi)源光學(xué)信號成像的方法檢測到它并沒有影響小鼠的眼優(yōu)勢(ocular dominance,OD)的偏移。同時我們也在1個月和2個月的幼貓上進行了電生理的實驗,通過緩釋微泵向左側(cè)視皮層給藥(rolipram)或者對照二甲基亞砜(Dimethyl Sulphoxide,DMSO),兩天之后再進行右眼的MD,對于1個月的幼貓MD1天,而兩個月的幼貓MD4天,記錄結(jié)果顯示抑制劑rolipram同樣也沒有影響MD造成的OD的偏移。因此,根據(jù)這些結(jié)果,我們認為PDE4D與視覺系統(tǒng)發(fā)育相關(guān),而與視覺經(jīng)驗誘發(fā)的可塑性可能無關(guān)。其次,我們對PKA的錨定蛋白AKAPs進行了研究。之前已經(jīng)有研究報道過PKA的活性對ODP的表達起到重要作用。向視皮層施加PKA的抑制劑,或者敲除PKA RII亞基,都能影響關(guān)鍵期內(nèi)MD造成的OD的偏移。PKA有眾多的靶目標(biāo),越來越多的證據(jù)表明,AKAPs對于PKA在細胞不同區(qū)域發(fā)揮其特異的作用至關(guān)重要。在本研究中,首先用胞外電生理的方法測量了 AKAP150的基因敲除鼠的關(guān)鍵期內(nèi)的ODP。結(jié)果發(fā)現(xiàn),敲除AKAP150的小鼠MD4天后,OD的偏移程度小于單純MD的對照組,即阻斷了 ODP。為了排除基因敲除可能造成的副作用,又使用了抑制肽段St-Ht31(一種穿膜肽段,干擾PKA與AKAPs的結(jié)合)以及其對照肽段St-H31P來研究PKA的區(qū)域化在ODP中的作用。實驗動物主要是小鼠和貓,通過腦內(nèi)埋管或者緩釋微泵直接向視皮層給藥St-Ht31/St-H31P,然后MD4天之后,使用電生理胞外單細胞記錄的方法研究發(fā)現(xiàn),施加St-Ht31的小鼠,MD誘發(fā)的OD的偏移被部分阻斷,而St-Ht31P組與對照組的偏移程度一致,并且抑制肽段不影響視皮層神經(jīng)元的功能特性。這表明阻斷PKA與AKAPs的結(jié)合能部分抑制小鼠的ODP。但是施加St-Ht31的幼年貓,MD4天之后引起的OD偏移與St-Ht31P組和MD對照組卻沒有顯著差異,說明它沒有影響幼貓ODP的表達。這些結(jié)果表明,PKA信號通路通過與錨定蛋白的結(jié)合實現(xiàn)其特異性的區(qū)域化,對于小鼠ODP是至關(guān)重要�？赡苡捎谖锓N差異,St-Ht31未能影響貓的ODP。本文對ODP的研究有著重要的臨床意義。弱視就是在關(guān)鍵期內(nèi),視覺神經(jīng)系統(tǒng)發(fā)生障礙造成的一種疾病,它能嚴重影響人們生活的質(zhì)量,我國就有幾千萬的弱視患者。我們對視覺可塑性的研究有助于人們了解視覺發(fā)育的基本規(guī)律,進一步闡明其可塑性和關(guān)鍵點,并為弱視的發(fā)病機制及臨床治療提供理論基礎(chǔ)。
[Abstract]:The molecular mechanism of brain plasticity of neural development is always the research focus in brain science. The earlier research work of visual cortex began, its structure is also more thorough understanding, therefore, visual plasticity is the classic models used in the study on the plasticity of brain development. Ocular dominance plasticity (ocular dominance, plasticity, ODP) is the key during the period of monocular deprivation (monocular deprivation, MD) caused by the decrease of cells in primary visual cortex of the deprived eye responses, while the non deprived eye responses increased. Cyclic AMP (cyclic adenosine monophosphate, cAMP) - protein kinase A (protein kinase A, PKA) is an important second messenger pathways, there are a lot of reports the expression of cAMP-PKA have proved crucial pathway of ODP, but its downstream regulatory mechanism is not clear. This paper is to study the regulation of cAMP-PKA pathway two important points, phosphodiesterase 4D (pH Osphodiesterase 4D, PDE4D) and A kinase anchoring protein (A-kinase anchoring, proteins, AKAPs) to study effect of ODP. predecessors has been proved that cAMP involved in the expression of.PDE4 in the critical period of visual plasticity is mainly hydrolyzed protein cAMP, can regulate cAMP distribution of.PDE4D is an important member of the PDE4 family, have many reports show that it can affect synaptic plasticity by hydrolysis of cAMP specific, but almost no one to study whether it affects visual plasticity. Therefore, we first study the PDE4D in the process of development and how to participate in the regulation of visual plasticity in mice and cats. In mice, by Western blotting at different time point selection the development process of the binocular zone (Western blot) determination of brain tissue, we found expression in the visual cortex of the PDE4D expression and the time course of ODP in early development coincide. The protein level of PDE4D was low, and reached the peak in the critical period, and after the critical period decreased. However the change of visual experience, such as dark rearing (dark rearing, DR or MD) did not affect the content of protein in the visual cortex of PDE4D, indicating that PDE4D expression is not dependent on visual experience. Intraperitoneal injection in mice PDE4 on the key period of MD selective inhibitor rolipram (rolipram), through the detection method using optical intrinsic signal imaging that it does not affect mice ocular dominance (ocular dominance, OD). At the same time we also offset in 1 months and 2 months of cat electrophysiology through the experiment, sustained-release drug pump to the left visual cortex (rolipram) or control two dimethyl sulfoxide (Dimethyl Sulphoxide, DMSO), two days after the right MD, the cat MD1 day of the 1 month, and two months of cat MD4 days, record the results The offset shown is also no effect of MD inhibitor rolipram caused by OD. Therefore, according to these results, we believe that PDE4D is related to the visual system development and plasticity and visual experience may not be induced. Secondly, the PKA anchoring protein AKAPs was studied. Previous studies have reported that the activity of PKA the expression of ODP plays an important role in applying PKA to the inhibitor. The visual cortex, or knockdown of PKA RII subunit, can offset.PKA critical period effects caused by MD OD is the target of many, more and more evidence that AKAPs is vital to its specific PKA in different regions in the cell. In this study, firstly, using extracellular electrophysiological measurements of AKAP150 gene knockout mice in the critical period of the ODP. results showed that AKAP150 knockout mice after MD4 days, the control group OD less than MD offset degree, namely resistance In order to eliminate the broken ODP. knockout possible side effects, and the use of inhibitory peptide St-Ht31 (a transmembrane peptide, with the interference of PKA and AKAPs) and the control peptide St-H31P of PKA region in ODP. The effect of experimental animal is mainly through mice and cats, the brain tube or release micro pump directly to the visual cortex after administration of St-Ht31/St-H31P, then MD4 days, research methods of single cell recording using electrophysiological extracellular, applied St-Ht31 mice, MD induced OD migration was partially blocked, while the St-Ht31P group and the control group shift consistency, and inhibitory peptide section does not affect the functional properties of visual cortical neurons. This suggests that blocking the combination of PKA and AKAPs could partially inhibit mouse ODP. but St-Ht31 on the young cat, MD4 days after induced OD migration and St-Ht31P group and MD control group had no significant difference, indicating that it No effect on the expression of cat ODP. These results suggest that the PKA signaling pathway by binding to the anchor protein region realize its specificity, is essential for mouse ODP. May be due to species differences did not affect St-Ht31 have important clinical significance of cat ODP. the study of ODP is in the critical period of amblyopia. In a visual nervous system disorder caused by disease, it can seriously affect the quality of people's lives, there are tens of millions of amblyopia in China. We research on visual plasticity can help people understand the visual development of the basic law, to further elucidate the plasticity and key points, and provide a theoretical basis for the the mechanism of amblyopia and clinical treatment.

【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R777.44

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