本文選題：瘦素 + 帕金森病　； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2014年碩士論文

【摘要】：帕金森病（Parkinson’s diseases, PD）的病理變化為中腦黑質(zhì)區(qū)多巴胺能神經(jīng)元變性、死亡、缺失，黑質(zhì)-紋狀體多巴胺能系統(tǒng)功能減退，并在殘存神經(jīng)元呈現(xiàn)α-突觸核蛋白（α-synuclein）和泛素染色陽(yáng)性的胞漿包涵體體形成。因此尋找有效的神經(jīng)保護(hù)因子，對(duì)帕金森病的發(fā)病進(jìn)程進(jìn)行干預(yù)，進(jìn)而減輕神經(jīng)損傷、恢復(fù)神經(jīng)功能，這是目前研究關(guān)注的熱點(diǎn)。 瘦素（Leptin）作為一種能量代謝調(diào)節(jié)因子，在炎癥反應(yīng)和創(chuàng)傷修復(fù)方面也具有重要功能。最近有關(guān)其神經(jīng)保護(hù)作用的研究越來(lái)越多，但對(duì)其作用機(jī)制的研究還知之甚少，，尤其是在帕金森病的研究領(lǐng)域。為了解瘦素在帕金森病中的作用及其病理生理機(jī)制，本研究擬通過(guò)帕金森病患者的血樣分析，探討瘦素對(duì)帕金森病的關(guān)聯(lián)性，并建立6-OHDA誘導(dǎo)C57BL/6J小鼠的帕金森動(dòng)物模型，6-羥基多巴胺（6-hydroxydopamine,6-OHDA）和魚(yú)藤酮（Rotenone）誘導(dǎo)多巴胺能樣細(xì)胞系SH-SY5Y細(xì)胞的帕金森病細(xì)胞模型來(lái)探討瘦素對(duì)帕金森病神經(jīng)損傷的保護(hù)以及機(jī)制。 本課題包括以下三個(gè)部分工作: 第一部分帕金森病患者血清瘦素及其受體，以及氧化應(yīng)激的變化 目的：探討血清中的leptin和可溶性瘦素受體（Soluble leptin receptor, sObR）水平，以及氧化和抗氧化指標(biāo)與帕金森病的關(guān)系性；方法：1.檢測(cè)健康人群（年齡范圍平均）人的leptin和sObR水平；2.檢測(cè)PD患者和對(duì)照組（主要老年群體）中的leptin和sObR水平，檢測(cè)兩個(gè)群體中的丙二醛（Malondialdehyde, MDA）、超氧化物歧化酶（Superoxide dismutase, SOD）和還原型谷胱甘肽（Glutathione, GSH）的水平；3.分析leptin和sObR水平與帕金森病的相關(guān)性，以及與年齡的相關(guān)性，與男女性別的相關(guān)性；4.分析MDA、GSH以及SOD的水平與帕金森的相關(guān)性，以及和leptin與sObR的相關(guān)性。結(jié)果：1.隨著年齡的增加，leptin的水平有所增加但是在40歲左右穩(wěn)定，sObR變化不大；2.帕金森病患者相對(duì)于對(duì)照組（老年群體），MDA和sObR水平有所增加，但leptin、SOD和GSH水平有所下降（P0.05）；3.女性的leptin水平高于男性，sObR水平低于男性（P0.05）；4. GSH和SOD與leptin水平呈正相關(guān)，與sObR呈負(fù)相關(guān)；MDA與leptin水平呈負(fù)相關(guān)，與sObR呈正相關(guān)。結(jié)論：1.帕金森患者中，血清中氧化應(yīng)激水平高，leptin水平低，sObR水平高；2.保持較高的血清leptin水平，有降低患帕金森病風(fēng)險(xiǎn)的可能性。 第二部分瘦素對(duì)帕金森病動(dòng)物模型黑質(zhì)區(qū)域的保護(hù) 目的：在建立的PD動(dòng)物模型基礎(chǔ)上，觀察leptin對(duì)于黑質(zhì)區(qū)域損傷的保護(hù)，以及下游氧化應(yīng)激水平的變化。方法：①建立6-OHDA誘導(dǎo)的右側(cè)損傷的PD動(dòng)物模型，假手術(shù)組（對(duì)照組），模型組，模型+leptin組，模型+leptin+nanobody組，通過(guò)皮下注射阿撲嗎啡，通過(guò)向左側(cè)旋轉(zhuǎn)次數(shù)鑒定動(dòng)物模型是否建立成功；②免疫組化檢測(cè)各組中腦黑質(zhì)區(qū)多巴胺能神經(jīng)元特異性蛋白酪氨酸羥化酶的變化；③收集各組血清，檢測(cè)MDA、GSH和SOD的變化。結(jié)果：1.阿撲嗎啡皮下注射后，模型組相對(duì)于對(duì)照組，左側(cè)旋轉(zhuǎn)次數(shù)顯著增加，模型+leptin組旋轉(zhuǎn)次數(shù)減少，加入nanobody后，旋轉(zhuǎn)次數(shù)右顯著增加；2.模型組及模型+leptin+nanobody組右側(cè)黑質(zhì)區(qū)域多巴胺能相對(duì)于左側(cè)明顯減少，模型+leptin組右側(cè)黑質(zhì)區(qū)域多巴胺能相對(duì)于左側(cè)有所減少，但較模型組和加入nanobody組有所增加。3.各組血清中，模型組相對(duì)于對(duì)照組MDA有所增加，GSH和SOD有所減少，模型+leptin組相對(duì)于模型組和leptin+nanobody組，MDA有所減少，GSH和SOD有所增加。結(jié)論:1.外源性Leptin能夠減少PD的嚴(yán)重程度，能夠減少多巴胺能神經(jīng)元的丟失；2. Leptin在PD動(dòng)物模型中，可顯著降低血清中MDA水平，升高SOD和GSH水平,在阻斷脂質(zhì)過(guò)氧化,增加抗氧化性，維持機(jī)體氧化平衡的穩(wěn)定具有積極作用。 第三部分瘦素減輕6-羥基多巴胺和魚(yú)藤酮誘導(dǎo)的SH-SY5Y細(xì)胞損傷的機(jī)制研究 目的:通過(guò)6-OHDA和rotenone誘導(dǎo)SH-SY5Y的帕金森病細(xì)胞模型，對(duì)leptin的神經(jīng)保護(hù)作用及機(jī)制進(jìn)行探討。方法:6-OHDA和rotenone去誘導(dǎo)SH-SY5Y。①CCK-8和LDH分別檢測(cè)不同濃度6-OHDA和rotenone誘導(dǎo)SH-SY5Y細(xì)胞的存活率和損傷率;②CCK-8、LDH、臺(tái)盼藍(lán)和流式細(xì)胞術(shù)分別檢測(cè)leptin對(duì)于100μmol/L6-OHDA和5μmol/Lrotenone誘導(dǎo)SH-SY5Y細(xì)胞的存活、損傷、死亡以及凋亡的影響，以及其受體阻斷劑nanobody的影響。③熒光探針檢測(cè)，leptin對(duì)于6-OHDA和rotenone損傷導(dǎo)致ATP和ROS變化的影響，以及，免疫熒光和Westernblot檢測(cè)和cleaved caspase-3的變化。④Nanobody加入后，檢測(cè)相關(guān)細(xì)胞裂解液中的SOD、MDA和GSH這些氧化性和抗氧化性指標(biāo)的變化。⑤CCK-8檢測(cè)，AG490、nanobody、LY294002、compound C、U0126、ZnPP，對(duì)于leptin對(duì)于6-OHDA和rotenone損傷減輕的影響，并比較LiCl和CoPP對(duì)細(xì)胞的保護(hù)。⑥Leptin模型保護(hù)加入LY294002、compound C和U0126后，Western blot檢測(cè)凋亡相關(guān)蛋白Bcl-2、Bax，以及相關(guān)通路分子p-Akt，p-ERK1/2，p-AMPK，p-GSK3β，HO-1，Western blot及RT-PCR檢測(cè)帕金森相關(guān)蛋白，RT-PCR檢測(cè)α-synuclein的mRNA水平。Westernblot檢測(cè)LiCl和CoPP對(duì)于模型組Bcl-2、Bax以及HO-1的表達(dá)影響。結(jié)果:1. Leptin提高了SH-SY5Y細(xì)胞的存活率，降低了損傷率及凋亡率；2.相應(yīng)的阻斷劑加入后，leptin的保護(hù)作用均有所減弱；3. Leptin提高了ATP的增加，降低了ROS的水平。4. Nanobody加入，逆轉(zhuǎn)了leptin引起的MDA的降低，SOD和GSH的升高；5. Leptin提高了Bcl-2和HO-1的表達(dá)，降低了cleaved caspase-3、Bax和α-synuclein的表達(dá)，并且提高了p-Akt、p-ERK1/2、p-AMPK以及p-GSK3β的水平，阻斷劑的加入部分逆轉(zhuǎn)了leptin的影響，相應(yīng)蛋白的阻斷劑都降低了p-GSK3β的水平，并降低了leptin提高的Bcl-2和HO-1的表達(dá)，以及提高leptin降低的Bax水平。結(jié)論：1.在建立帕金森的細(xì)胞模型基礎(chǔ)上，leptin減輕了損傷；2. Leptin對(duì)于細(xì)胞模型的保護(hù)，分別通過(guò)Akt、ERK以及AMPK途徑，匯聚于GSK-3β，進(jìn)而參與下游的調(diào)節(jié)，其下游的調(diào)節(jié)保護(hù)，主要是提高HO-1降低ROS和MDA的水平，提高SOD和GSH的水平以及能量ATP的水平，并下調(diào)凋亡的水平，進(jìn)而降低損傷。
[Abstract]:The pathological changes of Parkinson's disease (Parkinson 's diseases, PD) are the degeneration, death, deletion, degeneration of the nigrostriatal dopaminergic system in the mesencephalic substantia nigra, and the formation of an alpha synuclein (alpha -synuclein) and a cytoplasmic inclusion body with positive ubiquitin staining in the remaining neurons. Protective factors, which intervene in the pathogenesis of Parkinson's disease, reduce nerve injury and restore nerve function, are the focus of current research.
Leptin (Leptin), as an energy metabolic regulator, also plays an important role in inflammatory response and wound repair. Recently, more and more studies have been made on its neuroprotective effects, but little is known about its mechanism of action, especially in the field of Parkinson's disease. To understand the role of leptin in Parkinson's disease and to understand the role of leptin in Parkinson's disease, Pathophysiological mechanism, this study intends to explore the relevance of leptin to Parkinson's disease by analysis of blood samples from patients with Parkinson's disease, and to establish a Parkinson animal model of C57BL/6J mice induced by 6-OHDA, 6- hydroxyl dopamine (6-hydroxydopamine, 6-OHDA) and rotenone (Rotenone) to induce the Parkinson disease of the dopaminergic cell line SH-SY5Y cells. Cell model is used to investigate the protection and mechanism of leptin on Parkinson's disease.
This topic includes the following three parts:
Part 1 Changes of serum leptin and its receptors and oxidative stress in patients with Parkinson's disease
Objective: To investigate the levels of leptin and Soluble leptin receptor (sObR) in serum and the relationship between oxidation and antioxidant indicators and Parkinson's disease. Methods: 1. to detect leptin and sObR levels in healthy people (average age range); and 2. to detect leptin and sObR in PD patients and control groups (the main elderly group). Levels, the levels of Malondialdehyde, MDA, Superoxide dismutase, SOD and Glutathione, GSH in two groups; 3. analysis of the correlation between leptin and sObR levels and Parkinson's disease, the correlation with age, the correlation with sex and sex; 4. analysis of MDA, GSH, and The correlation between the level of SOD and Parkinson, as well as the correlation with leptin and sObR. Results: 1. with the increase of age, the level of leptin increased but the level of sObR was stable at the age of 40. The levels of MDA and sObR in 2. Parkinson's patients were increased relative to the control group (old group), but leptin, SOD and GSH decreased (P). 0.05): the level of leptin in 3. women was higher than that of men, and the level of sObR was lower than that of men (P0.05); 4. GSH and SOD were positively correlated with leptin, and negatively correlated with sObR; MDA and leptin were negatively correlated with sObR. Conclusion: in 1. Parkinson patients, the level of oxidative stress was high, leptin was low, sObR level was high, and 2. remained high. Serum leptin level has the potential to reduce the risk of Parkinson's disease.
The second part of leptin protects the substantia nigra in Parkinson's disease animal models.
Objective: on the basis of the established PD animal model, we observed the protection of leptin in the damage of the substantia nigra and the changes in the level of oxidative stress in the lower reaches. Methods: (1) the establishment of a PD animal model of the right injury induced by 6-OHDA, the sham operation group (control group), the model group, the model +leptin group, the model +leptin+nanobody group, and the subcutaneous injection of opiapine. The changes of dopaminergic neuron specific tyrosine hydroxylase of dopaminergic neurons in the substantia nigra of each group were detected by immunohistochemistry. (3) the changes in MDA, GSH and SOD were detected in each group of serum. Results: after subcutaneous injection of 1. apomorphine, the model group was compared with the control group, left The number of lateral rotation increased significantly, and the number of rotation in model +leptin group decreased. After adding nanobody, the number of rotations increased significantly. 2. model group and model +leptin+nanobody group were significantly reduced to the left side of the right substantia nigra compared to the left side, and the dopamine in the right substantia nigra region of model +leptin group decreased compared with the left side, but the model group was more than the model group. And the addition of nanobody group increased the serum of.3. in each group, the MDA of model group was increased compared with the control group, GSH and SOD decreased. The MDA decreased in model +leptin group and leptin+nanobody group, and GSH and SOD increased. Conclusion: 1. exogenous Leptin can reduce the severity of PD, and can reduce dopaminergic neurons. The loss of 2. Leptin in the PD animal model can significantly reduce the level of MDA in the serum, increase the level of SOD and GSH, and play an active role in blocking lipid peroxidation, increasing antioxidant activity, and maintaining the stability of the body's oxidative balance.
The third part is the mechanism of leptin alleviated 6- hydroxydopamine and rotenone induced SH-SY5Y cell injury.
Objective: to induce the Parkinson's disease cell model of SH-SY5Y by 6-OHDA and rotenone, the neuroprotective effect and mechanism of leptin were discussed. Methods: 6-OHDA and rotenone were used to induce SH-SY5Y. (CCK-8 and LDH) to detect the survival rate and damage rate of SH-SY5Y cells with different concentrations of 6-OHDA and rotenone, respectively. The effects of leptin on the survival, damage, death and apoptosis of SH-SY5Y cells induced by 100 and 5 mu mol/Lrotenone, and the effect of its receptor blocker nanobody. (3) fluorescence probe detection, the effect of leptin on ATP and ROS changes caused by 6-OHDA and rotenone damage, as well as immunofluorescence and Westernblot detection And cleaved caspase-3 changes. (4) after Nanobody, the changes in the oxidative and antioxidant properties of SOD, MDA and GSH in the related cell lysates were detected. 5. CCK-8 detection, AG490, nanobody, LY294002, compound C. 6. After the Leptin model is protected by LY294002, compound C and U0126, Western blot is used to detect apoptosis related proteins Bcl-2, Bax, and the associated pathway molecules p-Akt, p-ERK1/2, p-AMPK, and the detection of Parkinson related proteins. 2, Bax and HO-1 expression. Results: 1. Leptin increased the survival rate of SH-SY5Y cells and reduced the rate of damage and apoptosis; 2. the protective effect of leptin was weakened after the addition of the corresponding blockers; 3. Leptin increased the increase of ATP, reduced the.4. Nanobody addition of ROS, and reversed the MDA reduction caused by leptin. The increase of SH; 5. Leptin increased the expression of Bcl-2 and HO-1, reduced the expression of cleaved Caspase-3, Bax and alpha -synuclein, and increased the level of p-Akt, p-ERK1/2, p-AMPK and p-GSK3 beta. The expression of Bcl-2 and HO-1, as well as the Bax level reduced by leptin. Conclusion: 1. on the basis of the establishment of Parkinson's cell model, leptin reduces the damage; 2. Leptin protects the cell model by Akt, ERK and AMPK pathway, which converges to GSK-3 beta, and then participates in the regulation of the lower reaches, and the downstream regulation and protection is mainly to improve HO-1. Reduce the level of ROS and MDA, increase the level of SOD and GSH, and the level of energy ATP, and down regulate the level of apoptosis, thereby reducing the injury.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R742.5

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