神经鞘磷脂合成酶2基因敲除小鼠海马神经细胞自噬现象  

Autophagy in hippocampal neurons of sphingomyelin synthase 2 knockout mice

作　　者：曹瑞萍[1,2] 鲁广秀[2] 张俊士[2] 文曙光[1] 臧建峰[1] 邓锦波[1] 

机构地区：[1]河南大学神经生物学研究所,河南开封475004 [2]河南大学淮河医院神经内科,河南开封475000

出　　处：《解剖学报》2012年第3期299-305,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(31070952;30771140);河南大学自然科学基金资助项目(2008YBGG048)

摘　　要：目的利用神经鞘磷脂合成酶2(SMS2)基因敲除小鼠探讨神经酰胺对海马神经细胞自噬现象的影响。方法将SMS2杂合子(SMS2+/-)小鼠采用杂交、回交、互交的方法进行繁殖,用酚-氯仿提取法提取小鼠基因组DNA,PCR扩增目的基因,琼脂糖凝胶电泳对小鼠基因型作出鉴定,建立纯合子(SMS2-/-)小鼠模型;采用透射电子显微镜、免疫荧光染色及Western blotting技术观察生后第7天(P7)、P14和P30 SMS2-/-小鼠海马CA1区神经细胞自噬的发生(每种方法每个时间点8只小鼠)。结果 1.SMS2-/-小鼠海马CA1区神经细胞自噬现象:电子显微镜下,模型组海马神经元内出现较多自噬体或自噬溶酶体样结构。光镜下,模型组P7、P14和P30 CA1区神经元自噬细胞数较对照组高(P<0.01);2.自噬相关蛋白Beclin-1对于自噬的调节作用:Beclin-1在模型组与对照组间的表达规律与微管相关蛋白1轻链3(MAPLC3)基本一致,P7、P14、P30模型组Beclin-1阳性细胞数明显多于对照组(P<0.01)。Beclin-1与MAPLC3两者在同一细胞中基本呈重叠表达;3.Western blotting检测各组海马CA1区神经细胞MAPLC3蛋白的相对表达量与上述结果一致。结论 SMS2-/-小鼠海马神经细胞内神经酰胺增高,神经酰胺不仅促进细胞凋亡,同时促进自噬,造成小鼠海马神经细胞自噬现象增强。Objective To investigate the influence of ceramide on the autophagy of the hippoeampal neurons of sphingomyelin synthase 2 knockout （SMS2-/-） mice. Methods SMS2＋/- mice were bred by varied methods of hybridism, backcross and cross. The genomie DNA of these mice was achieved by the phenol chloroform extraction method. Then the target DNA was amplified by PCR method andidentified by agarose gel electrophoresis to establish the SMS2-/- mice model. The autophagy of hippocampal neurons in P7, P14 and P30 in CA1 area of the SMS2 -/- mice was investigated by transmission electron microscopy, immunofluorescent labeling method and Western blotting technique with 8 mice per time group. Results 1. The autophagy phenomenon of hippocampal neurons of SMS2-/- mice： With the electron microscopy, numerous lysosome and auto-phagosomes were found in the hippocampal neurons in SMS2-/- mice model. Under the light microscope, the number of autophagy cells of P7, P14 and P30 in the CA1 area of the model were obviously higher than that of control group （P 〈 0.01 ） ; 2. Accommodation of Beclin-1 on autophagy phenomenon ： The difference of the expression of Beclin-1 between model and control group was consistent with microtubule-associated protein 1 light chain 3 （MAPLC3）. The number of positive cells of Beclin-1 in model group of P7, P14, P30 were obviously higher than that in control group （P 〈0. 01 ）. The co-expression of Beclin-1 and MAPLC3 was found in the same hippocampal neurons; 3. The result of Western blotting analysis was consistent with that from immunocytochemistrys. Conclusion The number of ceramide in neurons of SMS2 -/mice increases which not only promotes the apoptosis, but also promotes the autophagy of the neurons.

关 键 词：神经鞘磷脂合成酶2 自噬 海马神经细胞 神经酰胺 免疫荧光 免疫印迹法 小鼠 

分 类 号：Q189[生物学—神经生物学]