【作者】 郭轶夫；

【导师】 关水；

【作者基本信息】 大连理工大学 ， 生物医学工程， 2012， 硕士

【摘要】 神经退行性疾病(Neurodegenerative disease)是指中枢神经系统组织非正常退变引起的功能缺陷与衰退的一类疾病,病理上表现为脑和脊髓发生神经元变性、丢失。研究发现,机体内过量的自由基产生导致的氧化应激损伤在神经退行性疾病的发病过程中起着重要的作用。近年来,神经干细胞(Neural Stem Cells, NSCs)因其具有自我更新的能力和多向分化潜能,在中枢神经系统再生和重建方面引起广泛关注,但NSCs移植后存活率低和易于受到氧化应激损伤的缺陷,则成为其应用在神经退行性疾病临床治疗上的限制。吡咯喹啉醌(Pyrroloquinline quinone, PQQ)在自然界中广泛存在,作为氧化还原酶的辅基参与机体内的多种氧化还原反应,具有多种生物学功能,尤其是其强大的清除自由基能力和神经营养保护功能已经引起人们的注意。本文首先通过体外悬浮方式培养NSCs,同时采用H2O2和Glu造成细胞氧化应激,以构建NSCs药物损伤模型。结果表明,二者均能对NSCs产生毒性作用,其毒性作用呈时间依赖和浓度依赖；据此,本文分别采用0.35mM H2O2作用细胞24h建立体外NSCs直接氧化应激模型,15mMGlu作用细胞48h建立间接氧化应激损伤模型。其次利用CCK-8试剂盒、免疫荧光染色等方法考察PQQ对NSCs自发的增殖、凋亡和分化的影响。结果显示,一定浓度(0.003-3μM) PQQ对NSCs的生长具有促进作用,并存在着浓度依赖,但高浓度的PQQ(30gM或以上)对NSCs的增殖反而有抑制作用,PQQ对NSCs的自发性凋亡和分化均无显著性影响。通过建立的损伤模型,采用Hoechst33258-PI荧光染色、AV-PI双染检测凋亡以及检测细胞内酶活性等方法,考察PQQ的预处理和后处理对NSCs的神经保护作用。结果表明,在药物诱导之前预加PQQ能够明显提高细胞存活率,并存在浓度-效应关系,而PQQ后处理则无明显效果。PQQ预处理组细胞的形态上的染色质浓集或碎块现象减少,并且活细胞增多,折光度增强,状态明显出现好转；其早期/晚期凋亡细胞的比率均有所减少。对胞内抗氧化酶活力水平检测发现,PQQ的预处理能够显著提高SOD和CAT的活性,但GSH-Px的变化则不明显。说明PQQ对NSCs的氧化应激损伤具有保护作用,并且可能是通过提高胞内抗氧化酶的活力来实现的。总之,本研究结果表明,PQQ对体外培养的NSCs具有促增殖的作用,PQQ预处理对氧化应激导致的NSCs损伤具有明显的保护作用,为神经退行性疾病的治疗提供了新的手段。更多还原

【Abstract】 Neurodegenerative disease is a kind of disease which is caused by the abnormally degeneration of the central nervous system organization. The symptoms of the neurodegenerative disease are the degeneration and loss of neurons. Recent research found that idative stress which caused by he production of excessive free radicals in the organism has plays an important role in the process of neurodegenerative disease attacking. In recent years, Neural stem cells (NSCs) have raised a lot of concern in regeneration and reconstruction of the CNS due to its ability of renew and multi-lineage differentiation potential. But the low cell viability and weakness of vulnerable to oxidative stress injury, have limited the application of NSCs in the clinical treatment of neurodegenerative disease. Pyrroloquinline quinine (PQQ) exists widely in the nature. As the coenzyme of oxidoreductase, it takes part in many kinds of redox reactions, and has a variety of ability of biological functions. Especially its powerful free-radicals scavengine capacity and neuro-protection has been attracting attention.Firstly, NSCs in vitro suspension culture, use H2O2and Glu which induce oxidative stress to establish the injury model. The results show that both can make toxic action on NSCs, and its toxic effects are time-dependency and concentration-dependent. Accordingly, we use H2O20.35mM24h and Glu15mM48h, respectively dispose NSCs, built the direct and the indirect oxidative stress injury model.Secondly, we use CCK-8kit, immunofluorescence staining method to examine the effect of PQQ on the proliferation, apoptosis and differentiation of NSCs. The results show that certain concentrations (0.003-3μM) of PQQ can promot the growth of NSCs, and there is a concentration-dependent. But high concentrations (30μM or above) may have the inhibition on the proliferation of NSCs. PQQ has no significant impact on the spontaneous apoptosis and differation of NSCs.Finally, we investigate the neuroprotective effect of pretreatment and aftertreatment of PQQ on NSCs through the use of Hoechst33258-PI staining, AV-PI and the detection of intracellular enzyme activity. The results show that the treatment of PQQ before the drug-induced can significantly improve the cell viability and has the concentration-effect relationship. While the PQQ of aftertreatment had no effect. The morphology of cells in pretreatment of PQQ group has chromatin condensation of fragments, and the number of the living cells increased, the refractive index enhanced. The early/late apoptotic cells ratio also reduced. The activities of antioxidant enzymes in the intracellular level of detection of PQQ pretreatment can significantly increase the activity of SOD and CAT, but not significant changes in GSH-Px. PQQ has a protective effect of NSCs oxidative stress injury, and may be achieved by increasing the intracellular antioxidant enzyme activity.In summary, the results of this study shows that PQQ can promote the proliferation of NSCs, and the pretreatment of PQQ has a significant protective effect on NSCs damage caused by oxidative stress, provides a new means for the treatment of neurodegenerative diseases.更多还原