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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 effect of ceramide on autophagy in hippocampal neurons by using sphingomyelin synthetase 2 (SMS2) knockout mice. Methods The SMS2 +/- mice were propagated by hybridization, backcrossing and reciprocal crosses. The genomic DNA of mice was extracted by phenol - chloroform extraction and the target genes were amplified by PCR. The results of electrophoresis on agarose gel electrophoresis (SMS2 - / -) mouse model was established. Transmission electron microscopy, immunofluorescence staining and Western blotting were used to detect the expression of Smad4 in P7, P14 and P30 SMS2 - / - mice Autophagy of hippocampal CA1 region neurons (8 mice per time point for each method). Autophagy of neurons in hippocampal CA1 region ofSMS2 - / - mice: Under the electron microscope, more autophagosomes or autolysosome-like structures appeared in hippocampal neurons of model group. The number of autophagic cells in the P7, P14 and P30 CA1 neurons in the model group was significantly higher than that in the control group (P <0.01) under light microscope.2. Regulatory effect of Beclin-1 on autophagy: Beclin- The expression of Beclin-1 in P7, P14 and P30 model groups was significantly higher than that in control group (P <0.01). Beclin-1 and MAPLC3 were almost overlapped in the same cells.3.Western blotting detected the relative expression of MAPLC3 in hippocampal CA1 neurons in each group was consistent with the above results. Conclusions Ceramide is increased in hippocampal neurons of SMS2 - / - mice. Ceramide not only promotes apoptosis but also promotes autophagy, resulting in increased autophagy in hippocampal neurons of mice.