本文選題：帕金森病 + 多巴胺能神經(jīng)元��； 參考：《第四軍醫(yī)大學(xué)》2011年博士論文

【摘要】：帕金森病是(Parkinson’ disease,PD)中樞神經(jīng)系統(tǒng)常見的進(jìn)行性、變性性疾病，黑質(zhì)多巴胺能(Dopaminergic,DAG)神經(jīng)元選擇性的凋亡目前被認(rèn)為是PD的主要病理變化，既往的重點(diǎn)主要集中在對DAG神經(jīng)元凋亡機(jī)制的研究和治療方面，而軸突變性被認(rèn)為是神經(jīng)元死亡后的伴隨產(chǎn)物，因此一直未受到重視。但近年來的研究發(fā)現(xiàn)，單純抑制神經(jīng)元凋亡并不能有效延緩PD的發(fā)生和發(fā)展，而DAG軸突變性可能是PD發(fā)病的一個重要原因和靶點(diǎn)。盡管現(xiàn)在已經(jīng)逐漸認(rèn)識到PD中軸突變性的重要性，但是對于軸突變性在PD中的作用和機(jī)制，目前還缺乏了解，因此對軸突變性在PD中的作用和機(jī)制的研究不僅可以為PD發(fā)病機(jī)制的理解帶來理論革新，還可為我們治療PD提供新的策略和靶點(diǎn)，具有重大的理論和現(xiàn)實(shí)意義。 第一部分：一個新的帕金森病多巴胺能神經(jīng)元軸突變性體外實(shí)驗(yàn)細(xì)胞模型的建立 目的：利用1-甲基-4-苯基吡啶離子(MPP~+)對小鼠胚胎中腦多巴胺能神經(jīng)元的毒性作用，建立一個新的PD多巴胺能神經(jīng)元軸突變性體外實(shí)驗(yàn)細(xì)胞模型。方法：采用C57BL/6小鼠胚胎(孕14d)中腦腹側(cè)組織進(jìn)行原代細(xì)胞培養(yǎng)，實(shí)驗(yàn)分為對照組和不同濃度(0.1、0.5、1.0、10.0μM) MPP~+藥物實(shí)驗(yàn)組，應(yīng)用酪氨酸羥化酶(TH)免疫熒光化學(xué)細(xì)胞染色方法對DAG神經(jīng)元損傷的形態(tài)學(xué)進(jìn)行觀察，對DAG神經(jīng)元數(shù)量、軸突數(shù)目和長度分別在2、4、6、8、12、24h共6個時間點(diǎn)進(jìn)行研究。TUNEL檢測細(xì)胞凋亡情況。結(jié)果：MPP~+作用于DAG神經(jīng)元后細(xì)胞數(shù)量減少，絕大多數(shù)表現(xiàn)為胞體存在，軸突的數(shù)目及長度明顯減少，網(wǎng)狀交織的軸突變得稀疏，表面不光滑，有的呈串珠樣腫脹或軸突中斷，少數(shù)表現(xiàn)為胞體空虛、丟失，僅有軸突存在。DAG神經(jīng)元細(xì)胞數(shù)量、軸突長度、軸突數(shù)目的減少，對MPP~+有時間-劑量依賴性。分析數(shù)據(jù)發(fā)現(xiàn)只有10.0μM MPP~+作用DAG神經(jīng)元24小時組在神經(jīng)元數(shù)量、軸突長度、軸突數(shù)目這三個方面與其余各組之間比較有顯著性差異(P0.05)。此時，對照組DAG神經(jīng)元的數(shù)量、軸突長度、軸突數(shù)目分別為(254±11)個/孔、(158.99±12.13)μm/個、(1.82±0.30)個，MPP~+實(shí)驗(yàn)組為(126±16)個/孔、(64.18±19.06) μm/個、(1.17±0.35)個。DAG神經(jīng)元數(shù)量減少了50.39%，每個DAG神經(jīng)元軸突長度減少了59.8%，每個DAG神經(jīng)元的軸突數(shù)目減少35.7%。TUNEL檢測10.0μM MPP~+作用DAG神經(jīng)元24小時后DAG神經(jīng)元以凋亡為主，占丟失細(xì)胞總數(shù)的90.7%。結(jié)論:0.1、0.5、1.0、10.0μM MPP~+均能引起DAG神經(jīng)元損傷，形態(tài)改變、數(shù)量減少、軸突長度變短、數(shù)目減少。而以10.0μM MPP~+作用多巴胺能神經(jīng)元24小時造模最為理想。 第二部分:帕金森病多巴胺能神經(jīng)元軸突變性指標(biāo)的檢測 目的：觀察淀粉樣前體蛋白(APP)和微管蛋白β-TubulinⅢ在MPP~+作用DAG神經(jīng)元后表達(dá)的變化情況，從軸漿運(yùn)輸障礙、微管出現(xiàn)解聚兩方面來驗(yàn)證MPP~+誘導(dǎo)的DAG神經(jīng)元軸突發(fā)生了變性。方法：分MPP~+實(shí)驗(yàn)組和對照組。細(xì)胞培養(yǎng)7天后，實(shí)驗(yàn)組應(yīng)用10.0μM MPP~+作用24h，對照組不予任何干預(yù)。TH-APP免疫熒光雙標(biāo)來檢測DAG神經(jīng)元APP的表達(dá)。Weston-blot檢測細(xì)胞β-TubulinⅢ的表達(dá)變化。MPP~+實(shí)驗(yàn)組使用含有洗脫劑的4%多聚甲醛固定，洗出游離的微管蛋白，檢測細(xì)胞聚合狀態(tài)的微管蛋白。對照組使用4%多聚甲醛固定，檢測細(xì)胞總的微管蛋白。結(jié)果：對照組DAG神經(jīng)元及軸突形態(tài)正常， APP呈低表達(dá)。MPP~+作用后DAG神經(jīng)元軸突出現(xiàn)片段化，APP表達(dá)升高，，在軸突中可見APP局部濃集。Weston-blot檢測細(xì)胞β-TubulinⅢ的表達(dá)，對照組平均灰度值比值為10.08±0.6，實(shí)驗(yàn)組為4.36±0.3，與對照組相比實(shí)驗(yàn)組下降了56.75%。結(jié)論：10.0μM MPP~+作用DAG神經(jīng)元24h，出現(xiàn)了軸漿運(yùn)輸障礙及軸突微管解聚現(xiàn)象，軸突發(fā)生了變性。 第三部分:帕金森病多巴胺能神經(jīng)元軸突變性相關(guān)信號通路的研究 目的：探討PI3-kinase/Akt/GSK-3β和Rho/ROCK信號通路可能參與了MPP~+誘導(dǎo)的DAG神經(jīng)元軸突變性。方法：在已建立的細(xì)胞模型中使用GSK-3β抑制劑LiCl和ROCK抑制劑Fasudil。LiCl作用濃度為5、10、20、30μM，F(xiàn)asudil作用濃度為25、50、100、150μM。研究分對照組和實(shí)驗(yàn)組。對照組細(xì)胞培養(yǎng)7天后，應(yīng)用10.0μMMPP~+作用24h。實(shí)驗(yàn)組細(xì)胞培養(yǎng)7天后，應(yīng)用10.0μM MPP~++不同濃度LiCl或10.0Μm MPP~++不同濃度Fasudil作用24h。TH免疫熒光染色觀察軸突長度、軸突數(shù)目；實(shí)驗(yàn)組將有抑制作用濃度的LiCl、Fasudil組使用洗脫劑和對照組使用洗脫劑洗去游離的微管蛋白，用Weston-blot檢測細(xì)胞聚合狀態(tài)β-TubulinⅢ的表達(dá)。結(jié)果：①對照組軸突長度為(88.45±17.12)μm，實(shí)驗(yàn)組5、10、20、30μM LiCl作用后軸突長度分別為(119.03±25.19)、(115.92±20.10)、(97.21±15.58)、(88.31±16.26)μm。5、10μMLiCl組與對照組相比有顯著性差異(P0.05)，20、30μM LiCl組與對照組相比無顯著性差異(P＞0.05)。而5和10μM LiCl組、20和30μM LiCl組間無顯著性差異(P＞0.05)。對照組軸突數(shù)目為(1.56±0.32)個，實(shí)驗(yàn)組5、10、20、30μM LiCl作用后軸突長度分別為(1.75±0.41)、(1.66±0.33)、(1.65±0.30)、(1.64±0.38)個，與對照組相比各濃度LiCl組無顯著性差異(P＞0.05)。②對照組軸突長度為(86.34±16.18)μm，實(shí)驗(yàn)組25、50、100、150μM Fasudil作用后軸突長度分別為(95.97±20.67)、(118.30±19.11)、(132.39±26.95)、（87.01±25.89）μm。50、100μM Fasudil組與對照組相比有顯著性差異(P0.05)，25、150μMFasudil組與對照組相比無顯著性差異(P＞0.05)。而50和100μMFasudil組、25和150μMFasudil組間無顯著性差異(P＞0.05)。對照組軸突數(shù)目為(1.55±0.29)個，實(shí)驗(yàn)組25、50、100、150μM Fasudil作用后軸突數(shù)目分別為(1.69±0.43)、(1.65±0.35)、(1.53±0.38)、(1.57±0.50)個，與對照組相比各濃度Fasudil組無顯著性差異(P＞0.05)。Weston-blot檢測細(xì)胞β-TubulinⅢ：③LiCl組實(shí)驗(yàn)結(jié)果顯示：平均灰度值比值5μM LiCl組為5.68±0.29，10μM LiCl組為5.35±0.22，對照組為3.56±0.14，實(shí)驗(yàn)組與對照組有顯著性差異(p＜0.05)，實(shí)驗(yàn)組之間無顯著性差異（P＞0.05）。5、10μM LiCl作用后聚合狀態(tài)β-TubulinⅢ的表達(dá)增高，其抑制了變性DAG軸突微管蛋白的解聚。④Fasudil組實(shí)驗(yàn)結(jié)果顯示：平均灰度值比值50μM Fasudil組為4.61±0.32，100μMFasudil組為4.45±0.21，對照組為2.76±0.16，實(shí)驗(yàn)組與對照組有顯著性差異(p＜0.05)，實(shí)驗(yàn)組之間無顯著性差異(P＞0.05)。50、100μM Fasudil作用后聚合狀態(tài)β-TubulinⅢ的表達(dá)增高，其抑制了變性DAG軸突微管蛋白的解聚。結(jié)論：PI3-kinase/Akt/GSK-3β和Rho/ROCK信號通路參與了MPP~+誘導(dǎo)的DAG神經(jīng)元軸突變性。 LiCl、Fasudil在合適的濃度可以發(fā)揮信號通路抑制作用而保護(hù)了變性的DAG神經(jīng)元軸突。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R742.5;R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 劉洪恩,孫曉川;β淀粉樣前體蛋白與彌漫性軸索損傷[J];創(chuàng)傷外科雜志;2005年01期

2 姚慶和,高國棟;肌苷對帕金森病小鼠模型的神經(jīng)保護(hù)作用[J];第四軍醫(yī)大學(xué)學(xué)報;2005年02期

本文編號：2055719