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酒精诱导小鼠海马齿状回神经细胞的增殖：神经酰胺的可能作用

邓同兴, 王志新, 高晓群, 石渊渊, 马战友, 金海啸, 邓锦波^*

河南大学神经生物学研究所，开封 475004；郑州大学基础医学院人体解剖学教研室，郑州 450052；漯河医学高等专科学校人体解剖学教研室，漯河 462002

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 synthase 2 knockout, 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 day 7, 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。

关键词： 神经酰胺; 神经细胞增殖; 神经鞘磷脂合成酶; 神经酰胺-1-磷酸

分类号：Q4

[Alcohol-induced proliferation of neurons in mouse hippocampal dentate gyrus: a possible role of ceramide.] [Article in Chinese]

DENG Tong-Xing, WANG Zhi-Xin, GAO Xiao-Qun, SHI Yuan-Yuan, MA Zhan-You, JIN Hai-Xiao, DENG Jin-Bo^*

Institute of Neurobiology of Henan University, Kaifeng 475004, China； Department of Anatomy, Basic Medical College, Zhengzhou University, Zhengzhou 450052, China； Department of Anatomy of Luohe Medical College, Luohe 462002, China

Abstract

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 Cα (PKCα) 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 PKCα protein in SMS2−/− mice was lower than that in WT mice, and prenatal alcohol exposure could up-regulate the PKCα 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 PKCα may be a key step to start the Cer-C1P pathway and up-regulate the alcohol-induced neural cell proliferation and the newborn neurons formation.

Key words: Ceramide; neural cell proliferation; sphingomyelin synthase 2; ceramide-1-phosphate

收稿日期：2011-03-04　　录用日期：2011-07-18

通讯作者：邓锦波　　E-mail: jinbo_deng@henu.edu.cn

引用本文：

邓同兴, 王志新, 高晓群, 石渊渊, 马战友, 金海啸, 邓锦波. 酒精诱导小鼠海马齿状回神经细胞的增殖：神经酰胺的可能作用[J]. 生理学报 2011; 63 (6): 479-490.

DENG Tong-Xing, WANG Zhi-Xin, GAO Xiao-Qun, SHI Yuan-Yuan, MA Zhan-You, JIN Hai-Xiao, DENG Jin-Bo. [Alcohol-induced proliferation of neurons in mouse hippocampal dentate gyrus: a possible role of ceramide.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (6): 479-490 (in Chinese with English abstract).