中文摘要：

本文旨在探讨神经酰胺（ceramide,Cer）在酒精诱导神经细胞增殖及新生神经元形成过程中的作用及机制。因为Cer主要的代谢途径是经神经鞘磷脂合成酶（sphingomyelin synthase,SMS）作用转化成神经鞘磷脂（sphingomyelin,SM）,所以我们用SMS2基因敲除（sphingomyelin synthase2knockout,SMS2/）和野生型（wild type,WT）雌性小鼠建立孕期酒精暴露模型,将模型孕鼠所生仔鼠作为研究对象。用酶学法检测仔鼠SM水平,利用免疫荧光染色法观察各组仔鼠齿状回神经细胞增殖、新生神经元及蛋白激酶Cα（protein kinase Cα,PKCα）在脑组织中的表达,免疫印迹技术检测出生后7天（postnatal day7,P7）仔鼠海马组织PKCα蛋白的相对表达量。结果显示,由于SMS2基因的缺失,SMS2/P0仔鼠血液SM水平明显低于WT仔鼠（P〈0.01）。无论SMS2/还是WT P0仔鼠,酒精暴露剂量依赖性地降低其血液SM水平降低。无论SMS2/仔鼠还是WT仔鼠,其齿状回神经细胞增殖的数量及新生神经元随年龄的增长而减少且一直持续到成年。在P0、P7、P14和P30时间点,与WT仔鼠相比,SMS2/仔鼠齿状回神经细胞增殖的数量及新生神经元较多（P〈0.05）;酒精暴露可剂量依赖性地增加两基因型仔鼠齿状回神经细胞增殖的数量及新生神经元。PKCα是神经酰胺-神经酰胺-1-磷酸（Cer-C1P）通路中重要的激活蛋白,其表达于胞膜及胞核,在海马、齿状回、脑皮质中均有表达;与WT P7仔鼠相比,SMS2/P7仔鼠海马组织PKCα蛋白的表达量较低（P〈0.05）;而酒精暴露可剂量依赖性地增加两基因组仔鼠海马组织PKCα蛋白的表达量。以上结果提示,孕期酒精暴露能够诱导神经细胞发生代偿性增殖及新生神经元的形成,Cer-C1P通路参与酒精诱导细胞增殖的过程;同时,PKCα是Cer-C1P通路中重要的激活蛋白,可上调C1P调控酒精诱导神经细胞增殖及新生神经元形成的作用。

英文摘要：

To investigate the role and mechanism of ceramide （Cer） regulation in alcohol-induced neuronal proliferation and the newborn neurons formation, we used sphingomyelin synthase 2 （predominant enzyme of Cer metabolism） knockout （SMS2-/-） and wild type （WT） female mice to establish the model of prenatal alcohol exposure. In 24 h after being given birth （postnatal day 0, P0）, the offspring of model mice received blood sphingomyelin （SM） measurement with enzymatic method. On P0, P7, P14 and P30, the proliferation of granule cells in the dentate gyrus and newborn neurons were investigated with immunofluorescent labeling. The expression of protein kinase Ca （PKCa） in the hippocampus was tested with Western blot analysis. The results showed that the SM level of blood in SMS2 pups was significantly lower than that in WT pups. No matter in SMS2-/- or WT mice, the prenatal alcohol exposure down-regulated the SM levels in pups with dose-dependency. In both SMS2 and WT pups, the number of proliferative neurons and newborn neurons in the dentate gyrus gradually decreased with the growing age. Compared with the WT pups, SMS2- pups showed significantly more proliferative neurons and newborn neurons in the dentate gyrus. Notably, prenatal alcohol exposure dose-dependently increased proliferative neurons and newborn neurons in the dentate gyrus in both WT and SMS2 pups. The hippocampal expression of PKCa protein in SMS2 mice was lower than that in WT mice, and prenatal alcohol exposure could up-regulate the PKCa protein expression in both WT and SMS2 mice with dose dependency. These results suggest that alcohol exposure during pregnancy can induce the compensatory neural cell proliferation and the production of newborn neurons in offspring, and the Cer-ceramide-1- phosphate （C1P） pathway is involved in alcohol-induced neural cell proliferation. The activation of PKCct may be a key step to start the Cer-C1P pathway and up-regulate the alcohol-induced neural cell proliferation and the newborn neurons