Analyses on the pathogenesis of hydrocephalus associated with L1CAM-mutation using in utero shRNA silencing

为了解L1CAM突变相关脑积水的发病机制，采用L1CAM-shRNA宫内电穿孔技术对L1CAM进行沉默。与阴性对照shRNA(ShNC)相比，L1-shRNA可下调培养的神经细胞(Neuro2a)中L1CAM基因和蛋白的表达。将L1-shRNA导入胚胎13.5天(E13.5天)培养的小鼠前脑皮质神经元，可引起神经元的形态改变，如短突起、多突起和丰富的棘细胞出现频率增加。与shNC组相比，L1-shRNA组突起总长度缩短，分枝数目增加。在胚胎13.5天时，将L1-shRNA电穿孔到小鼠前脑室带内。转染后2~4d，L1-shRNA处理组和shNC处理组大鼠脑内神经元分化和迁移模式无明显差异。

L1cam-silencing using in utero electroporation of shRNA for L1cam (L1-shRNA) was performed in order to gain insight into the pathogenesis of hydrocephalus associated with L1cam mutation. Transfection of L1-shRNA induced down-regulation of the expression of both L1cam mRNA and protein in cultured neuronal cells (Neuro2a) as compared to those transfected by negative control shRNA (shNC). When transfected to the cortical neurons that were cultured from mouse forebrain at embryonal 13.5 days (E13.5), L1-shRNA induced morphological changes, such as increased frequency of neurons with short and multiple neurites and abundant spines. L1-shRNA resulted in decreased length of total neurites and increased numbers of arborization as compared to those transfected by shNC. In utero electroporation of L1-shRNA was performed into the ventricular zone of murine forebrain at E13.5 days. On 2 to 4 days after transfection, the pattern of neuronal differentiation and migration showed no significant differences between L1-shRNA- and shNC-treated brains.