APC-Cdh1在机械拉伸损伤后少突胶质前体细胞增殖活化中的作用及机制研究

发布时间：2018-05-02 07:12

  本文选题：细胞末期促进复合物 + 少突胶质前体细胞　； 参考：《山西医科大学》2017年硕士论文

【摘要】：研究背景:随着现代交通和建筑工业的快速发展,脊髓损伤(spinal cord injury,SCI)的发病率逐年增加,并成为青年人致残的主要原因之一。目前脊髓损伤与修复的机制还不清楚,临床上诊疗效果不能满足需求。少突胶质细胞是中枢神经系统内唯一形成髓鞘的细胞,其与神经元一样对损伤刺激非常敏感。近年来研究认为,脊髓损伤后少突胶质细胞凋亡导致幸存轴突脱髓鞘,是影响神经元轴突再生、修复的病理生理基础。因此,研究如何有效抑制凋亡以及促进损伤后少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)增殖活化是减轻脊髓损伤的一个重要环节。研究显示,细胞周期末期促进复合物(anaphase promoting complex,APC)及其调节亚基Cdh1在中枢神经系统生长、发育及损伤修复中具有重要的作用。最近的研究已证实Cdh1参与缺血性脑损伤后神经元凋亡和星形胶质细胞反应性增殖的病理生理过程,但APC-Cdh1在脊髓损伤后少突胶质前体细胞增殖活化中的作用,尚不清楚。本研究拟培养大鼠原代OPCs并建立体外细胞机械拉伸损伤模型,探究机械损伤后OPCs增殖活化是否与APC-Cdh1活性有关。其次,利用腺病毒介导的RNA干扰技术,探讨下调Cdh1对损伤后OPCs增殖活性的影响。最后,通过对干预前后Cdh1下游底物Id2和skp2的研究,探索Cdh1调控机械损伤后OPCs增殖活化的可能机制。本实验从细胞周期调控的角度出发,旨在研究APC-Cdh1在机械损伤后脊髓少突胶质前体细胞增殖活化中的作用及机制,从而为选择APC-Cdh1作为脊髓保护新靶点提供实验依据。第一部分大鼠脊髓源性少突胶质前体细胞体外机械拉伸损伤模型的建立与评价目的:利用FX-4000T?柔性基底加载系统建立体外大鼠脊髓源性少突胶质前体细胞机械拉伸损伤模型。方法:取新生48h内SD乳鼠脊髓,在原代混合胶质细胞培养的基础上利用改良的振摇法获得纯化的少突胶质前体细胞,并采用其特异性标志物A2B5进行免疫荧光细胞鉴定。利用FX-4000T?柔性基底加载系统建立少突胶质前体细胞机械拉伸损伤模型,将纯化培养3d的少突胶质前体细胞随机分为A组(对照组)、B组(拉伸幅度5%)、C组(拉伸幅度10%)、D组(拉伸幅度15%),拉伸2h后于倒置显微镜下观察各组细胞形态;MTT法检测细胞存活率;双染流式细胞术检测细胞凋亡情况。结果:体外成功培育少突胶质前体细胞且A2B5阳性细胞约占90%。B组拉伸幅度对细胞形态及存活没有明显影响,基本不构成损伤。C组和D组细胞存活率较A组显著降低(P0.05),凋亡率也明显升高(P0.05),并出现明显病理性形态改变;但D组有明显的细胞脱落现象且细胞存活率降到50%以下,不利于后续实验进行。结论:改良的振摇法是体外培养大鼠脊髓少突胶质前体细胞的可靠方法,利用FX-4000T?柔性基底加载系统以10%为拉伸强度可建立少突胶质前体细胞机械拉伸损伤模型。第二部分APC-Cdh1在机械拉伸损伤后少突胶质前体细胞增殖活化中的作用及机制目的:探讨APC-Cdh1在机械拉伸损伤后少突胶质前体细胞增殖活化中的作用及其可能的机制。方法:将体外纯化培养3d的少突胶质前体细胞随机分组。前期实验分为4组,分别为正常对照组(未进行拉伸),Stretch-2h组(拉伸2h),Stretch-6h组(拉伸6h),Stretch-12h组(拉伸12h);后期实验加入RNA干扰因素,分为4组,分别为正常对照组(未进行腺病毒感染和拉伸),Stretch组(未进行腺病毒感染但拉伸12h),Ad-Control-Stretch组(空载体腺病毒感染后拉伸12h),Ad-Cdh1-Stretch组(Cdh1-sh RNA腺病毒感染后拉伸12h)。所有组别均于拉伸后继续静态培养48h收集细胞。采用MTT比色法和流式细胞术周期分析综合测定细胞增殖情况;采用实时荧光定量PCR和Western blot检测Cdh1及其下游底物Skp2和Id2 m RNA及蛋白的表达水平。结果:1.机械拉伸2h即可发现增殖活性开始降低并且随着拉伸时间的延长而不断下降,以Stretch-12h组下降最为明显;Western blot结果显示:Stretch-2h组、Stretch-6h组、Stretch-12h组Cdh1蛋白表达水平均明显高于对照组,且表达具有时间递增趋势,尤以Stretch-12h组上调最为明显(P0.05)。2.RNA干扰后,Ad-Cdh1-Stretch组Cdh1蛋白表达明显低于Ad-Control-Stretch组(P0.05);而Ad-Cdh1-Stretch组细胞增殖活性显著高于Ad-Control-Stretch组(P0.05)。3.Cdh1的下游底物Skp2和Id2表达随着Cdh1人为干预的变化而变化,且趋势与Cdh1相反:Skp2和Id2在细胞机械损伤后被下调,而腺病毒介导的Cdh1 RNA干扰能上调损伤后Skp2和Id2的表达。结论:1.机械拉伸损伤引起OPCs增殖活性下降同时导致细胞内Cdh1高表达,下调细胞内Cdh1表达能显著促进损伤后OPCs的增殖活化,提示Cdh1蛋白表达水平与损伤后OPCs增殖活化有关。2.APC-Cdh1参与机械损伤后OPCs增殖活化的调控,其机制可能是通过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.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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