本文選題：細(xì)胞末期促進(jìn)復(fù)合物 + 少突膠質(zhì)前體細(xì)胞��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景:隨著現(xiàn)代交通和建筑工業(yè)的快速發(fā)展,脊髓損傷(spinal cord injury,SCI)的發(fā)病率逐年增加,并成為青年人致殘的主要原因之一。目前脊髓損傷與修復(fù)的機制還不清楚,臨床上診療效果不能滿足需求。少突膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)內(nèi)唯一形成髓鞘的細(xì)胞,其與神經(jīng)元一樣對損傷刺激非常敏感。近年來研究認(rèn)為,脊髓損傷后少突膠質(zhì)細(xì)胞凋亡導(dǎo)致幸存軸突脫髓鞘,是影響神經(jīng)元軸突再生、修復(fù)的病理生理基礎(chǔ)。因此,研究如何有效抑制凋亡以及促進(jìn)損傷后少突膠質(zhì)前體細(xì)胞(oligodendrocyte precursor cells,OPCs)增殖活化是減輕脊髓損傷的一個重要環(huán)節(jié)。研究顯示,細(xì)胞周期末期促進(jìn)復(fù)合物(anaphase promoting complex,APC)及其調(diào)節(jié)亞基Cdh1在中樞神經(jīng)系統(tǒng)生長、發(fā)育及損傷修復(fù)中具有重要的作用。最近的研究已證實Cdh1參與缺血性腦損傷后神經(jīng)元凋亡和星形膠質(zhì)細(xì)胞反應(yīng)性增殖的病理生理過程,但APC-Cdh1在脊髓損傷后少突膠質(zhì)前體細(xì)胞增殖活化中的作用,尚不清楚。本研究擬培養(yǎng)大鼠原代OPCs并建立體外細(xì)胞機械拉伸損傷模型,探究機械損傷后OPCs增殖活化是否與APC-Cdh1活性有關(guān)。其次,利用腺病毒介導(dǎo)的RNA干擾技術(shù),探討下調(diào)Cdh1對損傷后OPCs增殖活性的影響。最后,通過對干預(yù)前后Cdh1下游底物Id2和skp2的研究,探索Cdh1調(diào)控機械損傷后OPCs增殖活化的可能機制。本實驗從細(xì)胞周期調(diào)控的角度出發(fā),旨在研究APC-Cdh1在機械損傷后脊髓少突膠質(zhì)前體細(xì)胞增殖活化中的作用及機制,從而為選擇APC-Cdh1作為脊髓保護(hù)新靶點提供實驗依據(jù)。第一部分大鼠脊髓源性少突膠質(zhì)前體細(xì)胞體外機械拉伸損傷模型的建立與評價目的:利用FX-4000T?柔性基底加載系統(tǒng)建立體外大鼠脊髓源性少突膠質(zhì)前體細(xì)胞機械拉伸損傷模型。方法:取新生48h內(nèi)SD乳鼠脊髓,在原代混合膠質(zhì)細(xì)胞培養(yǎng)的基礎(chǔ)上利用改良的振搖法獲得純化的少突膠質(zhì)前體細(xì)胞,并采用其特異性標(biāo)志物A2B5進(jìn)行免疫熒光細(xì)胞鑒定。利用FX-4000T?柔性基底加載系統(tǒng)建立少突膠質(zhì)前體細(xì)胞機械拉伸損傷模型,將純化培養(yǎng)3d的少突膠質(zhì)前體細(xì)胞隨機分為A組(對照組)、B組(拉伸幅度5%)、C組(拉伸幅度10%)、D組(拉伸幅度15%),拉伸2h后于倒置顯微鏡下觀察各組細(xì)胞形態(tài);MTT法檢測細(xì)胞存活率;雙染流式細(xì)胞術(shù)檢測細(xì)胞凋亡情況。結(jié)果:體外成功培育少突膠質(zhì)前體細(xì)胞且A2B5陽性細(xì)胞約占90%。B組拉伸幅度對細(xì)胞形態(tài)及存活沒有明顯影響,基本不構(gòu)成損傷。C組和D組細(xì)胞存活率較A組顯著降低(P0.05),凋亡率也明顯升高(P0.05),并出現(xiàn)明顯病理性形態(tài)改變;但D組有明顯的細(xì)胞脫落現(xiàn)象且細(xì)胞存活率降到50%以下,不利于后續(xù)實驗進(jìn)行。結(jié)論:改良的振搖法是體外培養(yǎng)大鼠脊髓少突膠質(zhì)前體細(xì)胞的可靠方法,利用FX-4000T?柔性基底加載系統(tǒng)以10%為拉伸強度可建立少突膠質(zhì)前體細(xì)胞機械拉伸損傷模型。第二部分APC-Cdh1在機械拉伸損傷后少突膠質(zhì)前體細(xì)胞增殖活化中的作用及機制目的:探討APC-Cdh1在機械拉伸損傷后少突膠質(zhì)前體細(xì)胞增殖活化中的作用及其可能的機制。方法:將體外純化培養(yǎng)3d的少突膠質(zhì)前體細(xì)胞隨機分組。前期實驗分為4組,分別為正常對照組(未進(jìn)行拉伸),Stretch-2h組(拉伸2h),Stretch-6h組(拉伸6h),Stretch-12h組(拉伸12h);后期實驗加入RNA干擾因素,分為4組,分別為正常對照組(未進(jìn)行腺病毒感染和拉伸),Stretch組(未進(jìn)行腺病毒感染但拉伸12h),Ad-Control-Stretch組(空載體腺病毒感染后拉伸12h),Ad-Cdh1-Stretch組(Cdh1-sh RNA腺病毒感染后拉伸12h)。所有組別均于拉伸后繼續(xù)靜態(tài)培養(yǎng)48h收集細(xì)胞。采用MTT比色法和流式細(xì)胞術(shù)周期分析綜合測定細(xì)胞增殖情況;采用實時熒光定量PCR和Western blot檢測Cdh1及其下游底物Skp2和Id2 m RNA及蛋白的表達(dá)水平。結(jié)果:1.機械拉伸2h即可發(fā)現(xiàn)增殖活性開始降低并且隨著拉伸時間的延長而不斷下降,以Stretch-12h組下降最為明顯;Western blot結(jié)果顯示:Stretch-2h組、Stretch-6h組、Stretch-12h組Cdh1蛋白表達(dá)水平均明顯高于對照組,且表達(dá)具有時間遞增趨勢,尤以Stretch-12h組上調(diào)最為明顯(P0.05)。2.RNA干擾后,Ad-Cdh1-Stretch組Cdh1蛋白表達(dá)明顯低于Ad-Control-Stretch組(P0.05);而Ad-Cdh1-Stretch組細(xì)胞增殖活性顯著高于Ad-Control-Stretch組(P0.05)。3.Cdh1的下游底物Skp2和Id2表達(dá)隨著Cdh1人為干預(yù)的變化而變化,且趨勢與Cdh1相反:Skp2和Id2在細(xì)胞機械損傷后被下調(diào),而腺病毒介導(dǎo)的Cdh1 RNA干擾能上調(diào)損傷后Skp2和Id2的表達(dá)。結(jié)論:1.機械拉伸損傷引起OPCs增殖活性下降同時導(dǎo)致細(xì)胞內(nèi)Cdh1高表達(dá),下調(diào)細(xì)胞內(nèi)Cdh1表達(dá)能顯著促進(jìn)損傷后OPCs的增殖活化,提示Cdh1蛋白表達(dá)水平與損傷后OPCs增殖活化有關(guān)。2.APC-Cdh1參與機械損傷后OPCs增殖活化的調(diào)控,其機制可能是通過泛素化降解下游底物Skp2和Id2。
[Abstract]:Background: with the rapid development of modern transportation and construction industry, the incidence of spinal cord injury (SCI) is increasing year by year, and it has become one of the main reasons for the disability of young people. The mechanism of spinal cord injury and repair is unclear and the clinical diagnosis and treatment effect can not meet the needs. Oligodendrocytes are central nervous system. The only myelin forming cells in the inside are very sensitive to damage stimulation like neurons. In recent years, studies have shown that the apoptosis of oligodendrocytes after spinal cord injury leads to the survival of the myelinated axon demyelination, which is the pathophysiological basis for the regeneration of axons and the pathophysiological basis of repair. Therefore, the study of how to effectively inhibit apoptosis and promote oligodendrocytes after injury The proliferation and activation of oligodendrocyte precursor cells (OPCs) is an important link in reducing spinal cord injury. Studies have shown that the late cell cycle promoting complex (anaphase promoting complex, APC) and its regulatory subunit Cdh1 play an important role in the growth, development and damage repair of the central nervous system. Recent studies have shown that It is proved that Cdh1 is involved in the pathophysiological process of neuronal apoptosis and astrocyte reactive proliferation after ischemic brain injury, but the role of APC-Cdh1 in the proliferation and activation of oligodendrocyte precursor cells after spinal cord injury is not clear. This study intends to cultivate primary OPCs in rats and establish a mechanical tensile damage model in vitro to explore the mechanical damage. Whether the proliferation and activation of post OPCs is related to the activity of APC-Cdh1. Secondly, the effect of Cdh1 on OPCs proliferation activity after injury is explored by adenovirus mediated RNA interference. Finally, the possible mechanism of Cdh1 regulation of OPCs proliferation activation after mechanical injury is explored by the study of Id2 and Skp2 of the downstream substrate of Cdh1 before and after intervention. This experiment is from the cell cycle. The purpose of the study is to study the role and mechanism of APC-Cdh1 in the proliferation and activation of oligodendrocyte precursor cells after mechanical injury, and to provide experimental basis for the selection of APC-Cdh1 as a new target for spinal cord protection. The establishment and evaluation of the mechanical tensile damage model in vitro of the spinal oligodendrocyte precursor cells in part 1 of the rat Objective: to establish the mechanical tensile damage model of the spinal cord derived oligodendrocyte precursor cells in vitro by using the FX-4000T? Flexible substrate loading system. Methods: the spinal cord of SD milk rat was taken from the newborn 48h, and the purified oligodendrocyte precursor cells were obtained by the modified vibration method on the basis of the culture of the primary mixed glial cells, and the specific markers were used. A2B5 was used to identify the immunofluorescent cells. The mechanical tensile damage model of oligodendrocyte precursor cells was established by FX-4000T flexible substrate loading system. The oligodendrocyte precursor cells were randomly divided into A group (control group), B group (tensile amplitude 5%), C group (tensile amplitude 10%), D group (tensile amplitude 15%), and inverted microscope after 2h in inverted microscope. The cell morphology of each group was observed under the MTT method, and the cell apoptosis was detected by double dye flow cytometry. Results: the oligodendrocyte precursor cells were successfully cultured in vitro, and the A2B5 positive cells accounted for no obvious influence on the cell morphology and survival in the 90%.B group, and the survival rate of the cells in the group.C and the D group was less than that of the A group. Decrease (P0.05), the rate of apoptosis also increased significantly (P0.05), and there were obvious pathological changes. But there was obvious cell loss in D group and the cell survival rate decreased to less than 50%. Conclusion: the improved vibration shaking method is a reliable method for the cultivation of oligodendrocyte precursor cells in the rat spinal cord in vitro, and the use of FX-4000T? Flexibility The mechanical tensile damage model of oligodendrocyte precursor cells can be established by the base loading system with 10% tensile strength. The role and mechanism of the second part of APC-Cdh1 in the proliferation and activation of oligodendrocyte precursor cells after mechanical tensile damage: To explore the role of APC-Cdh1 in the proliferation and activation of oligodendrocyte precursor cells after mechanical tensile damage. Possible mechanism. Methods: the oligodendrocyte precursor cells purified and cultured in vitro were randomly divided into 4 groups, which were divided into 4 groups: normal control group (not stretching), Stretch-2h group (tensile 2H), Stretch-6h group (tensile 6h), Stretch-12h group (tensile 12h), and late experiment adding RNA interference factors, which were divided into normal control group (no normal control group). Adenovirus infection and stretching), group Stretch (without adenovirus infection but stretching 12h), group Ad-Control-Stretch (12h after adenovirus infection), Ad-Cdh1-Stretch group (Cdh1-sh RNA adenovirus stretching after infection). All groups continue to statically cultivate 48h collecting cells after stretching. MTT colorimetric method and flow cytometry are used. The cell proliferation was measured by periodic analysis, and the expression level of Cdh1 and its downstream substrate Skp2 and Id2 m RNA and protein were detected by real time fluorescence quantitative PCR and Western blot. Results: 1. mechanical tensile 2H can find that the proliferation activity begins to decrease and decreases with the extension time, which is most obvious in Stretch-12h group; W The results of estern blot showed that the expression level of Cdh1 protein in group Stretch-2h, Stretch-6h and Stretch-12h was significantly higher than that of the control group, and the expression had a trend of increasing time. The Cdh1 protein table in the Ad-Cdh1-Stretch group was significantly lower than that of the Ad-Control-Stretch group after the Stretch-12h group was up to the most obvious (P0.05).2.RNA interference. The cell proliferation activity of CH group was significantly higher than that of the Ad-Control-Stretch group (P0.05).3.Cdh1, the downstream substrate Skp2 and Id2 expression changed with the change of Cdh1 human intervention, and the trend was opposite to Cdh1: Skp2 and Id2 were downregulated after the cell mechanical damage, and the Cdh1 RNA interference mediated by adenovirus could increase the expression after the injury. Conclusion: 1. Tensile damage causes the decrease of OPCs proliferation activity and the high expression of Cdh1 in cells, and the down-regulation of Cdh1 expression can significantly promote the proliferation and activation of OPCs after injury, suggesting that the expression level of Cdh1 protein is related to the regulation of.2.APC-Cdh1 in the proliferation and activation of OPCs after the damage of OPCs, and the mechanism may be through ubiquitination. Degradation of downstream substrates Skp2 and Id2.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.2

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相關(guān)期刊論文 前1條

1 馬祥;祁月紅;衛(wèi)建峰;李燕則;畢競;呂磊;郭永清;;大鼠脊髓源性少突膠質(zhì)前體細(xì)胞體外機械拉伸損傷模型的建立與評價[J];中國當(dāng)代醫(yī)藥;2016年36期

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本文編號：1832883