【作者】 李凌云；

【导师】 梁中琴； 秦正红；

【作者基本信息】 苏州大学 ， 药理学， 2007， 硕士

【摘要】 目的:研究自噬/溶酶体途径在6-羟基多巴胺(6-OHDA)致多巴胺能神经细胞死亡中的作用,探讨自噬在帕金森病发病中的机制。方法:采用脑立体定位黑质致密部给药法,建立6-OHDA介导的帕金森病大鼠模型;采用6-OHDA处理人神经母细胞瘤株SH-SY5Y细胞建立PD细胞模型。利用透射电镜和MDC染色法观察DA神经细胞中自噬和溶酶体的激活,免疫荧光法和Western Blot法检测6-OHDA对DA神经细胞中自噬、溶酶体和凋亡相关蛋白表达的影响,以及抑制自噬和溶酶体对这些蛋白表达的影响。结果:透射电镜显示,6-OHDA给药后1.5h至24h可见大鼠黑质致密部神经元内溶酶体被激活,自噬小体形成,24h自噬和凋亡同时存在。MDC染色发现6-OHDA能诱导SH-SY5Y细胞产生自噬小泡。免疫荧光法显示模型大鼠与对照组相比,cathepsin L、cathepsin B、Lamp1阳性细胞增多,cathepsin L出现核异位表达。3-MA能增加TH阳性细胞数量,显著降低cathepsin L的表达,明显减少NF-κB p65核转位和TUNEL阳性细胞数。在PD细胞模型中,LC3表达量增加,Cathepsin L被激活,在细胞核内大量表达,几乎同一时程,NF-κB p65也大量核转位;自噬抑制剂3-MA、溶酶体碱化试剂NH4Cl能增加6-OHDA早期的细胞毒性,减轻晚期的细胞毒性。3-MA、NH4Cl和组织蛋白酶抑制剂Leupeptin能减少LC3表达量,Leupeptin能下调cathepsin L的表达。结论: 6-OHDA介导的多巴胺能神经细胞死亡与溶酶体的激活及自噬的发生有关。在损伤发生早期,自噬可作为一种保护机制抵御氧化应激对神经细胞造成的损伤,在后期自噬通过激活溶酶体,与NF-κB介导的神经元凋亡通路相关联,共同调控神经细胞死亡。更多还原

【Abstract】 Aim: To study the role of an autophagy/lysosomal pathway in 6-OHDA-induced death of dopaminergic cells in PD models.Methods: In this study, we established two PD models in vivo and in vitro. Rats model were produced with stereotaxic administration of 6-OHDA into unilateral substantia nigra par compact (SNpc). SH-SY5Y cells were administrated with 6-OHDA. Autophagosomes in SNpc were observed with transmission electronmicro-scopy (TEM). Autophagosomes in SH-SY5Y cells were observed by MDC staining. Proteins related to apoptosis and autophagy were determined with immuno-fluorescence (IF) and Western blot. Effects of autophagy/lysosome inhibitors on 6-OHDA-induced cathepsin L and NF-κB activation were also detected. TUNEL assay was applied to detect apoptosis.Results: TEM revealed that the formation of autophagosomes in the ipsilateral SNpc after injection of 6-OHDA. MDC staining also showed that autophagosomes appeared in SH-SY5Y cell induced by 6-OHDA. Immunofluorescence demonstrated that expression of lamp-1, cathepsin B, L and LC3 increased after 6-OHDA treatment. Pretreatment with inhibitors can potentiated the cytotoxicitiy of 6-OHDA in the earlier stage, while it attenuated 6-OHDA-induced death in the later period. Nuclear-location of Cathepsin L and NF-κB p65was induced by 6-OHDA. Pretreatment with inhibitors downregulated 6-OHDA-induced activation of LC3, cathepsin L and NF-κB, also reduced the number of TUNEL-positive cells.Conclusions: The increase of autophagosomes and the activation of lysosomes may be involved in the toxicity induced by 6-OHDA. Autophagy/lysosome pathway may have a crosstalk with apoptosis mediated by NF-κB. Autophagy can be protective in the earlier stage of 6-OHDA administration, while promote the death of dopaminergic cells in the later period by activating autophagy/lysosome-mediated death pathways.更多还原