【作者】 李林静；

【导师】 谢俊霞；

【作者基本信息】 青岛大学 ， 神经生物学， 2007， 硕士

【摘要】 帕金森病(parkinson’s disease，PD)是一种常见的中枢神经系统退行性疾病，以肌强直、肢体震颤和运动减少等症状为主要临床表现。PD病理特征主要是中脑黑质致密带(substantia nigra pars compact，SNpc)多巴胺(dopamine，DA)能神经元死亡。PD的病因未完全明了，可能有多种因素参与，如遗传、环境因素、氧化应激、兴奋性毒素、自身免疫、黑质(substantia nigra，SN)、铁聚积和细胞凋亡等。近年来对PD中DA能神经元死亡机制的大量研究发现，细胞凋亡在PD发病中起着重要的作用，应用电镜技术可在DA能细胞中观察到染色质浓缩和凋亡小体等特征，能够抑制细胞凋亡的物质可以保护DA能神经元。Ghrelin是Kojima等1999年发现的一种脑-肠肽，由28个氨基酸残基组成，为生长激素促分泌素受体(growth hormone secretagogue receptor，GHS-R)的内源性配体，有刺激食欲，促进摄食，减少能量消耗和增加体重等作用。最近的研究表明Ghrelin能够刺激脂肪前体细胞、成骨细胞多种细胞的增殖并具有抑制凋亡的作用，GHS-R1a特异性拮抗剂D-Lvs~3-GHRP-6能阻断其抑制脂肪前体细胞和体外培养的下丘脑神经元凋亡的作用。由于Ghrelin的功能性受体GHS-R 1a mRNA在SN DA能神经元有较高表达，为研究Ghrelin是否能够保护SN DA能神经元并探索其作用的可能机制，本实验选择了MPTP制备小鼠PD模型，应用免疫荧光双标技术在蛋白水平上观察SNpc GHS-R 1a与酪氨酸羟化酶(tyrosine hydroxlase，TH)共存的情况，利用高效液相色谱电化学检测法(high performance liquid chromatography-electrochemical detection，HPLC-ECD)和免疫组织化学的方法观察Ghrelin对纹状体(striatum，Str)DA及其代谢产物二羟基苯乙酸(dihydroxyphenylace-tic acid，DOPAC)和高香草酸(homovanillic acid，HVA)的含量以及SNpc DA能神经元存活情况的影响；为了进一步研究Ghrelin的神经保护作用机制，我们采用半定量逆转录聚合酶链反应(semi-quantitive reverse transcription polymerase chain reaction，RT-PCR)和免疫组织化学法检测Ghrelin对SN TH、caspase-3、Bcl-2和Bax表达的影响；同时，我们应用GHS-R1a的特异性拮抗剂—D-Lys~3-GHRP-6观察其对Ghrelin神经保护作用的影响。结果如下：1．TH与GHS-R 1a在C57BL／6小鼠的SNpc共存。2．MPTP造成小鼠Str DA、DOPAC和HVA含量的减少以及DA代谢率的升高(P＜0.05)，较高剂量的Ghrelin(200ng，400ng)对于防止DA及其代谢产物的减少和抑制DA代谢率升高有明显的作用，可以有效减轻MPTP的毒性损伤作用。单独侧脑室注射Ghrelin对于小鼠Str DA及其代谢产物的含量及DA代谢率没有明显改变(P＞0.05)。应用GHS-R 1a的拮抗剂，D-Lys3-GHRP-6，可以阻断Ghrelin的上述作用。3．MPTP导致小鼠SN TH mRNA表达量降低(P＜0.05)，TH免疫阳性细胞数目减少(P＜0.05)。100ng、200 ng和400 ng Ghrelin可结抗MPTP造成的DA能神经元缺失及TH mRNA表达降低。Ghrelin组的TH免疫阳性细胞数及其mRNA表达量与Control组无统计学差别(P＞0.05)。Ghrelin受体GHS-R 1a的拮抗剂D-Lys~3-GHRP-6可以完全阻断Ghrelin对DA能神经元的保护作用。4．MPTP组小鼠SNpc的caspase-3免疫阳性细胞与Control组相比显著增加(P＜0.05)，且组织着色加深。Ghrelin对MPTP造成caspase-3活性增加的抑制具有剂量依赖性，50 ng与100ng不能减少caspase-3免疫阳性细胞的数量，200 ng和400 ng与50 ng与100 ng相比，具有明显的抑制作用(P＜0.05)。Ghrelin组SNpc的caspase-3免疫阳性细胞数目和Control组相比无明显差别(P＞0.05)。拮抗剂D-Lys~3-GHRP-6可以阻断Ghrelin下调caspase-3免疫阳性神经元的作用。5．MPTP PD模型小鼠与正常小鼠相比，SN Bcl-2 mRNA表达量及免疫阳性细胞数目减少(P＜0.05)。100 ng、200 ng和400 ng Ghrelin可以随剂量增加逐步逆转MPTP造成的Bcl-2 mRNA表达量和免疫阳性细胞减少的状况。Ghrelin对正常小鼠SN Bcl-2 mRNA和蛋白水平并无影响(P＞0.05)。GHS-R 1a的拮抗剂D-Lys~3-GHRP-6可以阻断Ghrelin对Bcl-2的上调作用。6．MPTP组小鼠SN Bax mRNA表达量显著高于正常小鼠(P＜0.05)，Ghrelin各个剂量组(50 ng、100 ng、200 ng和400 ng)对由MPTP造成的Bax mRNA的表达量增加都有抑制作用(P＜0.05)，该抑制作用随Ghrelin剂量的增加而增强。Ghrelin对Bax表达的抑制可以完全被GHS-R 1a拮抗剂D-Lys~3-GHRP-6阻断。Ghrelin组和Control组的表达量没有差别。7．Ghrelin可以使正常小鼠SN Bcl-2／Bax的比值变大(P＜0.05)，MPTP导致小鼠SN Bcl-2／Bax显著低于正常小鼠(P＜0.05)。50 ng Ghrelin即可缓解MPTP降低Bcl-2／Bax的状况(P＜0.05)，200 ng和400 ng Ghrelin可逆转Bcl-2／Bax比值的降低(P＜0.05)。D-Lys~3-GHRP-6组Bcl-2／Bax与MPTP组相比差别无统计学意义。上述结果说明，MPTP可引起SN TH蛋白和mRNA表达水平降低，Str DA及其代谢产物含量减少，DA代谢率升高；给予Ghrelin之后，可以明显减轻MPTP的神经毒性作用，增加SN TH mRNA的表达，减轻DA能神经元缺失的状况，升高Str DA及其代谢产物含量；该保护作用是通过增加Bcl-2 mRNA的表达和蛋白含量，抑制Bax mRNA的表达以及caspase-3的活性而实现的；Ghrelin的细胞保护作用由GHS-R 1a介导，其特异性拮抗剂D-Lys~3-GHRP-6能完全阻断Ghrelin的抗凋亡效果。本实验为Ghrelin保护细胞生存和抗凋亡的功能提供了新的佐证，也给研究开发新的防治PD的方法开辟了新的途径。更多还原

【Abstract】 Parkinson’s disease (PD) is a progressive neurodegenerative disorder associated withthe loss of dopaminergic neurons originating in the substantia nigra pars compacta (SNc)and terminating in the striatum (Str). This disorder is characterized symptomatically bybradykinesia, resting tremor and rigidity. The exact pathogenesis of PD has not beenrevealed yet. However, lots of researches suggests that multiple factors might be involved,such as heredity, environment, oxidative stress, excitotoxin, autoimmunity, ironaccumulation and cell apoptosis. Recent studies on the mechanism of the loss ofdopaminergic neurons support that apoptosis plays an important role in PD pathogenesis.Concentration of chromatin and apoptotic bodies could be observed in dopaminergicneurons by electron microscopy in the SN of PD patients. Agents that inhibit apoptosiscould prevent, or at least partly delay the progress of PD.Ghrelin, a novel 28-amino-acid brain-gut peptide, has been identified as anendogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R) byKojima in 1999. It is produced and secreted mainly from the stomach, and acts on thepituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, andappetite. Recent studies have reported that ghrelin stimulates proliferation and inhibitsapoptosis in several cell types, such as 3T3-L1 adipocytes and osteoblastic MC3T3-E1cells etc. D-lys~3-GHRP-6, the antagonist against GHS-R 1a, can eliminate the anti-apoptotic effect of ghrelin on 3T3-L1 adipocytes and primarily cultured hypothalamusneurons.In the present experiment, we investigated whether ghrelin could protectdopaminergic neurons. Using 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced mice model of PD, we detected the effects of ghrelin on the contents of dopamine(DA) and its metabolites in the Str by high performance liquid chromatography-electrochemical detection (HPLC-ECD), and the mRNA and protein expression of TH inthe SN respectively by semi-quantitive reverse transcriptase- polymerase chain reaction(RT-PCR) and immunohistochemistry. Furtherly, to explore the probable mechanism ofghrelin’s neuroprotective effect, we observed the colocalization of its full functionalreceptor—GHS-R 1a and TH in the SN by double immunofluorescent staining. Using RT-PCR and immunohistochemistry, we also detected the changes of Bcl-2, Bax and caspase-3 expression in the SN. Finally, we investigated the effect of D-Lys~3-GHRP-6—theantagonist of GHS-R 1a, on the indexes above.The results were as follows: 1. TH and GHS-R 1a colocalized in the SNpc of C57BL/6 mice.2. MPTP decreased the contents of DA, 3,4-dihydrophenyl acetic acid (DOPAC) andhomovanillic acid (HVA) in the Str (P＜0.05), and increased the striatal DA turnoverrate (P＜0.05). Pretreated with ghrelin (200 ng, 400 ng) significantly ameliorated thereductions of DA and its metabolites (P＜0.05), as well as the increase of striatal DAturnover rate induced by MPTP (P＜0.05). Ghrelin alone did not affect either striatalDA turnover rate or the contents of DA and its metabolisms (P＞0.05). D-Lys~3-GHRP-6 completely abolished the effects of ghrelin.3. MPTP reduced the number of TH immunoreactive neurons and TH mRNAexpression in the SN (P＜0.05). Pretreatment with 100 ng, 200 ng and 400 ng ghrelininhibited the reductions induced by MPTP (P＜0.05). Ghrelin alone did not changeTH mRNA expression and TH immunoreactive cell numbers in the SN (P＞0.05). Theneuroprotective effect of ghrelin could be thoroughly blocked by D-Lys~3-GHRP-6.4. Compared with the normal control mice, MPTP group exhibited a significant increaseof caspase-3 immunoreactive cell numbers in the SNpc (P＜0.05). Ghrelin inhibitedthe activity of caspase-3 dose-dependently. 50 ng and 100 ng ghrelin could not reducethe number of caspase-3 immunoreactive cells (P＞0.05). However, 200 ng and400 ng ghrelin showed a remarkable inhibition of caspase-3 activity compared withMPTP group (P＜0.05), which was stronger than lower doses. Ghrelin alone did notaffect caspase-3 immunoreactive cell numbers in the SNpc of nomal mice (P＞0.05).D-Lys~3-GHRP-6 could block ghrelin’s neuroprotective effect against MPTP.5. The mRNA and protein expression of Bcl-2 in the SN decreased after injections ofMPTP (P＜0.05). 100 ng, 200 ng and 400 ng ghrelin increased SN Bcl-2immunoreactive cell numbers and mRNA expression of PD mice (P＜0.05). And theincreases of Bcl-2 mRNA and protein were propotional with the doses of ghrelin.Ghrelin alone did not change Bcl-2 mRNA expression and Bcl-2 immunoreactive cellnumbers in the SNpc (P＞0.05). The up-regulation of Bcl-2 expression in MPTP miceinduced by ghrelin was inhibited by D-Lys~3-GHRP-6.6. MPTP induced a remarkable increase of Bax mRNA expression in the SN (P＜0.01).All doses of ghrelin (50 ng, 100 ng, 200 ng and 400 ng) could down-regulate BaxmRNA expression in the SN of MPTP mice (P＜0.05). The inhibitive effect paralleledthe dose of ghrelin. The expression of Bax mRNA of normal mice was not affectedby ghrelin (P＞0.05). D-Lys~3-GHRP-6 thoroughly blocked the effect of ghrelin onBax mRNA expression.7. The ratio of SN Bcl-2/Bax increased in the Ghrelin group (P＜0.05). MPTP induced areduction of Bcl-2/Bax (P＜0.05). Pretreated with 50 ng and 100 ng ghrelin inhibited the reduction of Bcl-2/Bax induced by MPTP (P＜0.05). 200 ng and 400 ng ghrelineven reversed this reduction (P＜0.05). Blocking GHS-R 1a with D-Lys~3- GHRP-6abolished the effect of ghrelin.In conclusion, ghrelin inhibited the reduction of TH mRNA and protein expressionin the SN induced by MPTP, as well as the contents of DA and its metabolists in the Str.The probable mechanism of this protective effect might be anti-apoptosis by enhancingBcl-2 expression, reducing Bax expression and inhibiting caspase-3 activity. Theneuroprotective effect was mediated by GHS-R 1a. Our data provided new evidences forthe cell protective effect of ghrelin, and offered a new way possible to prevent and treatPD.更多还原