帕金森病多巴胺能神经元轴突变性新的体外实验细胞模型的建立及机制研究

发布时间：2018-06-23 04:14

本文选题：帕金森病 + 多巴胺能神经元　；参考：《第四军医大学》2011年博士论文

【摘要】：帕金森病是(Parkinson’ disease,PD)中枢神经系统常见的进行性、变性性疾病，黑质多巴胺能(Dopaminergic,DAG)神经元选择性的凋亡目前被认为是PD的主要病理变化，既往的重点主要集中在对DAG神经元凋亡机制的研究和治疗方面，而轴突变性被认为是神经元死亡后的伴随产物，因此一直未受到重视。但近年来的研究发现，单纯抑制神经元凋亡并不能有效延缓PD的发生和发展，而DAG轴突变性可能是PD发病的一个重要原因和靶点。尽管现在已经逐渐认识到PD中轴突变性的重要性，但是对于轴突变性在PD中的作用和机制，目前还缺乏了解，因此对轴突变性在PD中的作用和机制的研究不仅可以为PD发病机制的理解带来理论革新，还可为我们治疗PD提供新的策略和靶点，具有重大的理论和现实意义。第一部分：一个新的帕金森病多巴胺能神经元轴突变性体外实验细胞模型的建立目的：利用1-甲基-4-苯基吡啶离子(MPP~+)对小鼠胚胎中脑多巴胺能神经元的毒性作用，建立一个新的PD多巴胺能神经元轴突变性体外实验细胞模型。方法：采用C57BL/6小鼠胚胎(孕14d)中脑腹侧组织进行原代细胞培养，实验分为对照组和不同浓度(0.1、0.5、1.0、10.0μM) MPP~+药物实验组，应用酪氨酸羟化酶(TH)免疫荧光化学细胞染色方法对DAG神经元损伤的形态学进行观察，对DAG神经元数量、轴突数目和长度分别在2、4、6、8、12、24h共6个时间点进行研究。TUNEL检测细胞凋亡情况。结果：MPP~+作用于DAG神经元后细胞数量减少，绝大多数表现为胞体存在，轴突的数目及长度明显减少，网状交织的轴突变得稀疏，表面不光滑，有的呈串珠样肿胀或轴突中断，少数表现为胞体空虚、丢失，仅有轴突存在。DAG神经元细胞数量、轴突长度、轴突数目的减少，对MPP~+有时间-剂量依赖性。分析数据发现只有10.0μM MPP~+作用DAG神经元24小时组在神经元数量、轴突长度、轴突数目这三个方面与其余各组之间比较有显著性差异(P0.05)。此时，对照组DAG神经元的数量、轴突长度、轴突数目分别为(254±11)个/孔、(158.99±12.13)μm/个、(1.82±0.30)个，MPP~+实验组为(126±16)个/孔、(64.18±19.06) μm/个、(1.17±0.35)个。DAG神经元数量减少了50.39%，每个DAG神经元轴突长度减少了59.8%，每个DAG神经元的轴突数目减少35.7%。TUNEL检测10.0μM MPP~+作用DAG神经元24小时后DAG神经元以凋亡为主，占丢失细胞总数的90.7%。结论:0.1、0.5、1.0、10.0μM MPP~+均能引起DAG神经元损伤，形态改变、数量减少、轴突长度变短、数目减少。而以10.0μM MPP~+作用多巴胺能神经元24小时造模最为理想。第二部分:帕金森病多巴胺能神经元轴突变性指标的检测目的：观察淀粉样前体蛋白(APP)和微管蛋白β-TubulinⅢ在MPP~+作用DAG神经元后表达的变化情况，从轴浆运输障碍、微管出现解聚两方面来验证MPP~+诱导的DAG神经元轴突发生了变性。方法：分MPP~+实验组和对照组。细胞培养7天后，实验组应用10.0μM MPP~+作用24h，对照组不予任何干预。TH-APP免疫荧光双标来检测DAG神经元APP的表达。Weston-blot检测细胞β-TubulinⅢ的表达变化。MPP~+实验组使用含有洗脱剂的4%多聚甲醛固定，洗出游离的微管蛋白，检测细胞聚合状态的微管蛋白。对照组使用4%多聚甲醛固定，检测细胞总的微管蛋白。结果：对照组DAG神经元及轴突形态正常， APP呈低表达。MPP~+作用后DAG神经元轴突出现片段化，APP表达升高，，在轴突中可见APP局部浓集。Weston-blot检测细胞β-TubulinⅢ的表达，对照组平均灰度值比值为10.08±0.6，实验组为4.36±0.3，与对照组相比实验组下降了56.75%。结论：10.0μM MPP~+作用DAG神经元24h，出现了轴浆运输障碍及轴突微管解聚现象，轴突发生了变性。第三部分:帕金森病多巴胺能神经元轴突变性相关信号通路的研究目的：探讨PI3-kinase/Akt/GSK-3β和Rho/ROCK信号通路可能参与了MPP~+诱导的DAG神经元轴突变性。方法：在已建立的细胞模型中使用GSK-3β抑制剂LiCl和ROCK抑制剂Fasudil。LiCl作用浓度为5、10、20、30μM，Fasudil作用浓度为25、50、100、150μM。研究分对照组和实验组。对照组细胞培养7天后，应用10.0μMMPP~+作用24h。实验组细胞培养7天后，应用10.0μM MPP~++不同浓度LiCl或10.0Μm MPP~++不同浓度Fasudil作用24h。TH免疫荧光染色观察轴突长度、轴突数目；实验组将有抑制作用浓度的LiCl、Fasudil组使用洗脱剂和对照组使用洗脱剂洗去游离的微管蛋白，用Weston-blot检测细胞聚合状态β-TubulinⅢ的表达。结果：①对照组轴突长度为(88.45±17.12)μm，实验组5、10、20、30μM LiCl作用后轴突长度分别为(119.03±25.19)、(115.92±20.10)、(97.21±15.58)、(88.31±16.26)μm。5、10μMLiCl组与对照组相比有显著性差异(P0.05)，20、30μM LiCl组与对照组相比无显著性差异(P＞0.05)。而5和10μM LiCl组、20和30μM LiCl组间无显著性差异(P＞0.05)。对照组轴突数目为(1.56±0.32)个，实验组5、10、20、30μM LiCl作用后轴突长度分别为(1.75±0.41)、(1.66±0.33)、(1.65±0.30)、(1.64±0.38)个，与对照组相比各浓度LiCl组无显著性差异(P＞0.05)。②对照组轴突长度为(86.34±16.18)μm，实验组25、50、100、150μM Fasudil作用后轴突长度分别为(95.97±20.67)、(118.30±19.11)、(132.39±26.95)、（87.01±25.89）μm。50、100μM Fasudil组与对照组相比有显著性差异(P0.05)，25、150μMFasudil组与对照组相比无显著性差异(P＞0.05)。而50和100μMFasudil组、25和150μMFasudil组间无显著性差异(P＞0.05)。对照组轴突数目为(1.55±0.29)个，实验组25、50、100、150μM Fasudil作用后轴突数目分别为(1.69±0.43)、(1.65±0.35)、(1.53±0.38)、(1.57±0.50)个，与对照组相比各浓度Fasudil组无显著性差异(P＞0.05)。Weston-blot检测细胞β-TubulinⅢ：③LiCl组实验结果显示：平均灰度值比值5μM LiCl组为5.68±0.29，10μM LiCl组为5.35±0.22，对照组为3.56±0.14，实验组与对照组有显著性差异(p＜0.05)，实验组之间无显著性差异（P＞0.05）。5、10μM LiCl作用后聚合状态β-TubulinⅢ的表达增高，其抑制了变性DAG轴突微管蛋白的解聚。④Fasudil组实验结果显示：平均灰度值比值50μM Fasudil组为4.61±0.32，100μMFasudil组为4.45±0.21，对照组为2.76±0.16，实验组与对照组有显著性差异(p＜0.05)，实验组之间无显著性差异(P＞0.05)。50、100μM Fasudil作用后聚合状态β-TubulinⅢ的表达增高，其抑制了变性DAG轴突微管蛋白的解聚。结论：PI3-kinase/Akt/GSK-3β和Rho/ROCK信号通路参与了MPP~+诱导的DAG神经元轴突变性。 LiCl、Fasudil在合适的浓度可以发挥信号通路抑制作用而保护了变性的DAG神经元轴突。
[Abstract]:Parkinson's disease is a common progressive disease in the central nervous system of (Parkinson 'disease, PD), denatured disease, and selective apoptosis of Dopaminergic (Dopaminergic, DAG) neurons is considered to be the main pathological change of PD. The previous focus is mainly on the study and treatment of the apoptotic mechanism of the DAG deity, and the axonal degeneration is It is considered to be an accompanying product of neuron death, so it has not been paid attention to, but recent studies have found that simple inhibition of neuronal apoptosis can not effectively delay the occurrence and development of PD, and DAG axon degeneration may be an important cause and target for the pathogenesis of PD, although the importance of axonal degeneration in PD has been gradually recognized, However, the role and mechanism of axon degeneration in PD is still lack of understanding. Therefore, the study of the role and mechanism of axon degeneration in PD can not only bring theoretical innovation for the understanding of the pathogenesis of PD, but also provide new strategies and targets for the treatment of PD, which is of great theoretical and practical significance.
Part one: establishment of a new Parkinson cell model of axonal degeneration of dopaminergic neurons in vitro
Objective: to establish a new experimental cell model for the axonal degeneration of PD dopaminergic neurons by using 1- methyl -4- phenyl pyridine (MPP~+) on the mouse embryonic mesencephalon neurons. Methods: the primary cell culture of the C57BL/6 mouse embryo (pregnant 14d) was woven into the ventral ventral group, and the experimental group was divided into the control group and the control group. With the same concentration (0.1,0.5,1.0,10.0 mu M) MPP~+ drug experiment group, the morphology of DAG neuron injury was observed by the method of tyrosine hydroxylase (TH) immunofluorescent chemical cell staining. The number of DAG neurons, the number of axons and the length of the axon were studied at a total of 6 time points in 2,4,6,8,12,24h, and the results were as follows: MPP The number and the length of the axon decreased, the number and length of the axon decreased obviously, the axon became sparse, the surface was not smooth, and some of the beads like swelling or axons were interrupted. The number of the axons was empty and lost, only the axon existed in the number of.DAG neurons and axon length. The number of axons was reduced and MPP~+ had time and dose dependence. The analysis data found that the number of neurons, the length of axon and the number of axons in the 24 hour group of DAG neurons with only 10 mu MPP~+ were significantly different from those of the other groups (P0.05). At this time, the number of DAG neurons in the control group, the length of axon, and the number of axons. (254 + 11) / holes, (158.99 + 12.13) mu m/, (1.82 + 0.30), MPP~+ experimental group (126 + 16) / hole, (64.18 + 19.06) mu m/, (1.17 + 0.35).DAG neurons reduced 50.39%, the axon length of each DAG neuron decreased by 59.8%, and the number of axon of each DAG neuron decreased 35.7%.TUNEL detection DAG God DAG God mu M MPP~+ action DAG God After 24 hours, DAG neurons were mainly apoptosis, which accounted for the 90.7%. of the total number of lost cells. 0.1,0.5,1.0,10.0 mu M MPP~+ could cause damage to DAG neurons, the morphological changes, the decrease of the number, the shorter axon length, and the decrease in the number of neurons, and the most ideal model of the 10 mu M MPP~+ as dopaminergic neurons for 24 hours.
The second part: detection of dopaminergic neuron axonal degeneration in Parkinson's disease.
Objective: To observe the changes in the expression of amyloid precursor protein (APP) and microtubulin beta -Tubulin III after MPP~+ action in DAG neurons. From the two aspects of the axonal transport barrier and the depolymerization of microtubule, the MPP~+ induced DAG neuron axons were denatured. Methods: the MPP~+ group and the control group were divided into the MPP~+ group and the control group. After 7 days of cell culture, the experimental group should The effect of 10 micron M MPP~+ on 24h, the control group did not interfere with the.TH-APP immunofluorescence double standard to detect the expression of APP in DAG neurons, the expression of.Weston-blot detected cell beta -Tubulin III, the.MPP~+ experimental group was fixed with 4% polyformaldehyde containing eluant, washed out free microtubule protein, and detected the microtubule protein in the cell polymerization state. The group was fixed with 4% polyformaldehyde to detect the total microtubule protein of the cells. Results: the DAG neurons and axons in the control group were normal. The axons of DAG neurons were fragmented and the expression of APP increased after the APP showed low expression of.MPP~+. In the axon, the expression of APP local concentration.Weston-blot detection cell beta -Tubulin III was found, and the average gray value of the control group was observed. The ratio was 10.08 + 0.6 and the experimental group was 4.36 + 0.3. Compared with the control group, the experimental group decreased 56.75%. conclusion: 10 mu M MPP~+ acted DAG neurons 24h, and the axonal transport obstacle and axon microtubule disaggregation appeared, and the axon was denatured.
The third part: the signal pathway of dopaminergic neuron axonal degeneration in Parkinson's disease.
Objective: To investigate the possible involvement of PI3-kinase/Akt/GSK-3 beta and Rho/ROCK signaling pathways in MPP~+ induced DAG neuron axonal degeneration. Methods: the concentration of Fasudil.LiCl in the established cell model was 5,10,20,30 micron M with the GSK-3 beta inhibitor LiCl and the ROCK inhibitor Fasudil.LiCl, and the Fasudil action concentration was studied in the control group and the reality. In the control group, 7 days after cell culture, the cell culture of 24h. experimental group was cultured for 7 days with 10 MMPP~+. The axon length and the number of axon were observed with different concentrations of LiCl or 10 m MPP~++ of M MPP~++ with different concentrations of LiCl or 10 MPP~++ m MPP~++ in the experimental group. The experimental group would have the LiCl of inhibitory concentration and the Fasudil group using eluant and the eluant. The control group used eluent to wash away free microtubule protein, and the expression of cell polymerization state beta -Tubulin III was detected by Weston-blot. Results: (1) the axon length of the control group was (88.45 + 17.12) mu m, and the axon length of the experimental group was (119.03 + 25.19), (115.92 + 20.10), (97.21 + 15.58), (88.31 + 16.26) mu m.5,10 mu MLiC. There was significant difference in the l group compared with the control group (P0.05), and there was no significant difference between the 20,30 and M LiCl groups (P > 0.05). There was no significant difference between the 5 and 10 M LiCl groups, 20 and 30 micron LiCl groups (P > 0.05). The axon number of the control group was (1.56 + 0.32), and the axon length of the experimental group was (1.75 + 0.41), respectively (1.75 +. 0.41), respectively. 0.33), (1.65 + 0.30), (1.64 + 0.38), compared with the control group, there was no significant difference in the LiCl group (P > 0.05). The axon length of the control group was (86.34 + 16.18) mu m, and the axon length of the experimental group 25,50100150 mu M Fasudil was (95.97 + 20.67), (118.30 + 19.11), (132.39 + 0.30), and (0.05) m.50100 mu M Fasudil group and the pair. Compared with the control group, there was no significant difference (P0.05) compared with the control group (P > 0.05), but there was no significant difference between the 50 and 100 MFasudil groups, 25 and 150 MFasudil groups (P > 0.05). The number of axons in the control group was (1.55 + 0.29), and the number of axons after 25,50100150 mu M in the experimental group was (1.69 + 0.43) (1.69 + 0.43), respectively. 1.65 + 0.35), (1.53 + 0.38), (1.57 + 0.50), compared with the control group, there was no significant difference in the concentration of Fasudil group (P > 0.05).Weston-blot detection cells beta -Tubulin III: the results of LiCl group showed that the average gray value ratio 5 mu LiCl group was 5.68 + 0.29,10 mu M LiCl group 5.35 + 0.22, the control group was 3.56 + 0.14, the experimental group and the control group Significant difference (P < 0.05), there was no significant difference between the experimental group (P > 0.05) and the expression of the polymerization state beta -Tubulin III increased after the action of.5,10 mu M LiCl, and it inhibited the depolymerization of the denatured DAG axon microtubule protein. 4. The results of the Fasudil group experiment showed that the average gray value ratio 50 mu M Fasudil group was 4.61 + 0.32100 micron MFasudil group of 4.45 + 0.21. The group was 2.76 + 0.16. There was a significant difference between the experimental group and the control group (P < 0.05). There was no significant difference between the experimental group (P > 0.05) and the expression of the polymerization state beta -Tubulin III was increased after the action of.50100 mu M Fasudil, and it inhibited the depolymerization of the denatured DAG axon microtubule protein. Conclusion: PI3-kinase/Akt/GSK-3 beta and Rho/ROCK signaling pathway participated in MPP~+. The axonal degeneration of DAG neurons was induced. LiCl and Fasudil could inhibit the degeneration of DAG neuron axons by inhibiting signaling pathway at suitable concentration.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R742.5;R-332

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