摘要
目的探讨孕期酒精暴露对仔鼠小脑普肯耶细胞自噬的影响。方法从妊娠母鼠第5天酒精灌胃至仔鼠出生,建立孕期酒精暴露动物模型,出生后的仔鼠分对照组和酒精组共计180只;采用透射电子显微镜、免疫荧光染色和免疫印迹技术观察P7、P14和P30仔鼠小脑普肯耶细胞自噬的发生。结果电子显微镜下,模型组仔鼠小脑普肯耶细胞内出现较多自噬体或自噬溶酶体样结构,细胞器损伤严重。光镜下,可见模型组普肯耶细胞结构紊乱、胞体变小、树突分支减少、树突棘密度下降等一般形态学改变,模型组P7、P14自噬细胞数(PAC)及自噬指数(PAI)明显高于对照组(P<0.001),高剂量酒精组明显高于低剂量组(P<0.001),P30各组间差别不明显(P>0.05);对照组间随着年龄的增长PAC及PAI值逐渐增高(F=58.29,P<0.001);Beclin-1免疫细胞化学染色结果与MAPLC3一致,两者在同一细胞中基本呈重叠表达。免疫印迹法检测各组间普肯耶细胞MAPLC3和Beclin-1蛋白的相对表达量与上述结果一致。结论自噬参与了小脑普肯耶细胞的正常发育,在一定时期内,随着年龄的增长,自噬作用增强;孕期酒精暴露能够导致仔鼠小脑普肯耶细胞自噬作用增强,且有剂量依赖性和长时程效应。
Objective To investigate the effect of ethanol exposure on the autophagy in cerebellar Purkinje cells. Methods With intubating pregnant mice ethauol daily from E5 until the pup＇ s birth, the animal models of prenatal alcohol exposure （PAE） were made. Totally 180 pups included control treatment and ethyl alcohoi（EtOH） treatment were used in the study. On the other hand, the autophagy in cerebellar Purkinje cells in PT, P14 and P30 was investigated with transmission electron microscopy, immunofluorescent labeling and Western blotting technique. Results With electron microscopy, a numerous lysosome and auto-phagosomes were found in the Purkinje cells of alcohol exposure mice, with organelle injuries as well. Under light microscope, Purkinje cells changed lots after ethanol exposure, such as irregular arrangement, reduced dendritic branches and dentritic spines. The positive autophagy cells （PAC） and positive autophagy index （PAI） in ethanol treatment groups were obviously higher than that in control at either P7 or P14 （ P 〈 0. 001 ） with dose dependency （ P 〈 0. 001 ）. In the meantime, PAC and PAI increased gradually with age increasing （ F = 58.29, P 〈 0. 001 ）. The co-expression of Beclin-1 and MAPLC3 in the same Purkinje cell suggested the Beclin-1 ＇ s regulation to the autophagy. Western blotting analysis supported the above results from immunocytochemistrys. Conclusion Autophagy is involved in the normal development and alcohol＇ s toxicological effects of eerebellar Purkinje cells. The prenatal ethanol exposure can induce the autophagy of Purkinje cells with does-dependency and long-term effects.
出处
《解剖学报》
CAS
CSCD
北大核心
2010年第6期796-804,共9页
Acta Anatomica Sinica
基金
国家自然科学基金资助项目(30670688
30771140和31070952)
河南省科技厅国际合作项目(0646630014)
河南大学重点自然科学基金资助项目(2009222D003)
河南大学自然科学基金资助(2008YBGG048)
关键词
胎儿酒精综合征
普肯耶细胞
自噬
透射电子显微镜
免疫荧光
免疫印迹法
小鼠
Fetal alcohol syndrome （FAS）
Purkinje cell
Autophagy
Transmission electron microscopy
Immunofluorescence
Western blotting
Mouse
