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

Abstract: Objective To investigate the influence of ceramide on the autophagy of the hippocampal neurons of sphingomyelin synthase 2 knockout (SMS2SUP>-/-/SUP> ) mice. MethodsSMS2SUP>+/-/SUP>mice were bred by varied methods of hybridism, backcross and cross. The genomic 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 SMS2SUP>-/-/SUP>mice model. The autophagy of hippocampal neurons in P7, P14 and P30 in CA1 area of the SMS2SUP>-/-/SUP>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 SMS2SUP>-/-/SUP>mice: With the electron microscopy, numerous lysosome and auto-phagosomes were found in the hippocampal neurons in SMS2SUP>-/-/SUP>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 (EM>P/EM> <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 (EM>P/EM> <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 SMS2SUP>-/-/SUP>mice increases which not only promotes the apoptosis, but also promotes the autophagy of the neurons.

Key words: Sphingomyelin synthase 2, Autophagy, Hippocampal neuron, Ceramide, Immunofluorescence, Western blotting, Mouse