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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 prenatal alcohol exposure on the autophagy in Purkinje cells of the offspring. Methods The pregnant rats were sacrificed on the 5th day of gestation until their birth. The animal models of alcohol exposure during pregnancy were established. The postnatal rats were divided into the control group and the alcohol group for a total of 180 animals. Transmission electron microscopy, immunofluorescence staining and immunoblotting To observe the P7, P14 and P30 offspring Cerebellar Purkinje cell autophagy. Results Under the electron microscope, there were more autophagosomes or autolysosome-like structures in the cerebellar Purkinje cells in the model group, and the organelles were damaged severely. Under the light microscope, we can see that the general morphological changes of the Pukeye cells in the model group were disorganized, cell bodies became smaller, dendritic branches decreased, dendritic spine density decreased. The number of autophagic cells (P < (P <0.001). The levels of PAI in the high-dose alcohol group were significantly higher than those in the low-dose alcohol group (P <0.001), while those in the P30 group were not significantly different (P> 0.05) The values of PAC and PAI increased gradually (F = 58.29, P <0.001). The results of Beclin-1 immunocytochemistry were consistent with those of MAPLC3. The relative expression of MAPLC3 and Beclin-1 protein in Perkenye cells in each group by Western blot was consistent with the above results. Conclusions Autophagy is involved in the normal development of Purkinje cells. During a certain period of time, autophagy is enhanced with age. Alcohol exposure during pregnancy can lead to increased autophagy in Purkinje cells of pups, Dependency and long-term effects.