Mash-1基因过表达对脑创伤后C57BL/6成年雄性小鼠神经细胞增殖、分化及学习、记忆功能的影响

The effect of Mash-1 gene overexpression on neural cell proliferation,differentiation and learning and -memory ability in C57BL/6 adult male mice after brain trauma

目的观察Mash-1基因过表达对脑创伤后C57BL/6成年雄性小鼠神经细胞增殖、分化及学习、记忆功能的影响。方法将160只健康成年雄性C57BL/6小鼠随机分为假手术组、单纯创伤组、阴性对照组和过表达组。利用重组腺病毒Ad5-mMash-1进行基因转染。于脑创伤前1 d和脑创伤后1 d、3 d、7 d、14 d采取RT-PCR检测Mash-1 mRNA水平,Western blotting检测Mash-1蛋白表达,水迷宫评价学习和记忆功能。采用免疫荧光法检测脑创伤后3 d和7 d神经细胞增殖情况。结果与假手术组比较,单纯创伤组与阴性对照组脑创伤后1 d、3 d、7 d和14 d Mash-1 mRNA相对表达量显著降低(P <0. 05～0. 01),过表达组脑创伤后1 d、3 d、7 d和14 d Mash-1 mRNA相对表达量显著升高(均P <0. 01)。过表达组脑创伤后1 d、3 d、7 d和14 d的Mash-1 mRNA相对表达量显著高于单纯创伤组与阴性对照组(均P <0. 05)。与假手术组比较,单纯创伤组脑创伤后1 d、7 d、14 d,阴性对照组和过表达组脑创伤后1 d、3 d、7 d、14 d时Mash-1蛋白相对表达量显著升高(P <0. 05～0. 01),单纯创伤组脑创伤后3 d Mash-1蛋白相对表达量显著降低(P <0. 05)。过表达组脑创伤后1 d、3 d、7 d、14 d的Mash-1蛋白相对表达量显著高于单纯创伤组及阴性对照组(均P <0. 05)。与假手术组比较,单纯创伤组脑创伤后3 d、7 d的Brd U阳性细胞数,脑创伤后3 d DCX阳性细胞数显著减少(P <0. 05～0. 01),过表达组显著增多(均P <0. 05)。过表达组脑创伤后3 d、7 d的Brd U阳性细胞数,脑创伤后3 d DCX阳性细胞数显著高于单纯创伤组(均P <0. 05)。单纯创伤组、阴性对照组和过表达组的脑创伤后1 d、3 d、7 d、14 d的逃避潜伏期差异无统计学意义(均P> 0. 05)。与假手术组比较,单纯创伤组、阴性对照组和过表达组脑创伤后1 d、3 d、7 d、14 d的逃避潜伏期均显著延长(均P <0. 05)。结论 Mash-1基因过表达增加脑创伤后成年C57BL/6小鼠海马齿状回和大脑皮质的神经细胞增殖和分化,但对其学习和记忆功能无影响。

Objective To investigate the effects of Mash-1 gene overexpression on neural cell proliferation,differentiation and learning and memory ability in C57BL/6 adult male mice after brain trauma.Methods One hundred and sixty healthy adult male C57BL/6 mice were randomly divided into sham operation group,simple trauma group,negative control group and overexpression group.Gene transfection using recombinant adenovirus Ad5-mMash-1.Detection of Mash-1 mRNA level by RT-PCR at 1 d before TBI and 1 d,3 d,7 d,14 d after traumatic brain injury(TBI).Western blotting was used to detect the expression of Mash-1 protein.The learning and memory ability was evaluated by means of water maze.The proliferation of nerve cells in dentate gyrus and cerebral cortex of hippocampus at 3 d and 7 d after TBI were detected by immunofluorescence.Results Compared with those in sham operation group,the relative expression of Mash-1 mRNA in simple trauma group and negative control group at 1 d,3 d,7 d,14 d after TBI were significantly lower(P<0.05-0.01),and the relative expression of Mash-1 mRNA in overexpression group at 1 d,3 d,7 d,14 d after TBI were significantly higher(all P<0.01).The relative expression of Mash-1 mRNA in overexpression group at 1 d,3 d,7 d,1 4 d after TBI were significantly higher than those in simple trauma group and negative control group(all P<0.05).Compared with those in sham operation group,expression of Mash-1 protein in simple trauma group at 1 d,7 d,14 d after TBI and negative control group and overexpression group at 1 d,3 d,7 d,14 d after TBI(P<0.05-0.01),expression of Mash-1 protein in simple trauma group at 3 d after TBI(P<0.05).The expression of Mash-1 protein in overexpression group at 1 d,3 d,7 d,14 d after TBI were significantly higher than those in simple trauma group and negative control group(all P<0.05).Compared with those in sham operation group,the number of BrdU positive cells in simple trauma group at 3 d,7 d after TBI and the number of DCX positive cells at 3 d after TBI were significantly decreased(P<0.05-0.01),and they were significantly increased in overexpression group(all P<0.05).The number of BrdU positive cells at 3 d,7 d after TBI and the number of DCX positive cells at 3 d after TBI in overexpression group were significantly increased than those in simple trauma group(all P<0.05).There was no statistical difference of escape latency between simple trauma group,negative control group and overexpression group at 1 d,3 d,7 d,14 d after TBI(all P>0.05).Compared with those in sham operation group,escape latency in simple trauma group,negative control group and overexpression group at 1 d,3 d,7 d,14 d after TBI were significantly increased(all P<0.05).Conclusion Overexpression of Mash-1 gene increases neuronal proliferation and differentiation in dentate gyrus and cortex of adult C57BL/6 mice after traumatic brain injury,but it has no effect on learning and memory ability.