慢性应激对大鼠海马NGF、NT-3表达的影响及应激停止后的变化
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摘要
:目的 探讨慢性应激对大鼠海马神经元NGF、NT-3蛋白表达的影响及其应激停止后的变化。方法 采用慢性强迫游泳制作动物模型。运用open-field法观察大鼠行为学的变化,运用免疫组织化学方法观察大鼠海马DG区、CA3区NGF、NT-3的表达。结果与对照组相比,实验组1大鼠海马CA3、DG区NGF、NT-3的平均灰度值显著增加(p<0.05和p<0.01),实验组2平均灰度值与对照组2相比同样增加(p<0.05和p<0.05)。结论慢性应激使大鼠海马NGF、NT-3表达降低,应激三十天后,其表达仍低于对照组。porsolt swim test
关键词 :慢性应激 海马 NGF、NT-3 强迫游泳porsolt swim test forcedswim
[ 中图分类号] R395 [ 文献标识码] A
porsolt swim test
Effects of chronic stress on the expression of NGF、NT-3 in hippocampusof the rats porsolt swimtest
Bi Bo, Jin Kuihe,Zhu Yuzhang, et al.
Department ofPsychiatry and Medical Psychology, The First Affiliated Hospital,China Medical University, Shenyang 110001
Abstract: Objective To observe the changes ofNGF、NT-3 expression inhippocampus of the rats with the forced-swimming and the changes of NGF、NT-3 after poststress. Methods The protocol was established with the forced-swimming as the chronic stress model. Open-field test was executed to measure the behaviors of the rats. Immunohistochemistry was used to observe the changes of NGF、NT-3 expression in the hippocampus. Results Compared to the control group,the expression of NGF、NT-3 in CA3 region of the hippocampus and dendate gyrus(DG)of the rats was decreased morphologically. With the computerized image analysis, the gray degree increased significantly and the changes showed the same results after the poststress. Conclusion Chronic stress decreased the expression of the NGF、NT-3 in CA3 region and DG in the hippocampus of the rats and the changes showed the same results after the poststress.
Key words: chronic stress,hippocampus, NGF、NT-3,forced-swimming,
porsolt swim test porsolt swimtest
神经生长因子(neuron growth factor NGF)是第一个被发现、也是 生物 学功能了解得最清楚的一个神经营养因子。NGF对皮质、海马、基底前脑的胆
碱能神经元有神经营养作用。 [1] 它和神经营养因子3(neurotrophin3, N T-3 )广泛地分布在海马、大脑皮层、中隔等中枢神经系统中,它们在神经保护、突触再生、神经损伤后功能重建等方面也发挥了重要作用。应激事件导致神经化学方面的改变可能参与了抑郁障碍的发生,此外,神经内分泌、神经递质和神经可塑性的改变在抑郁障碍的发生中也起到十分重要的作用。 [2] 本研究利用免疫组化的方法观察NGF、NT3在慢性应激以及应激过后在海马的表达变化,以了解NGF、NT 3 和抑郁障碍的关系。porsolt swim test
1 对象与方法porsolt swim test
1.1实验动物porsolt swimtest
成年Wistar雄性大鼠32只(由中国医科大学动物实验室提供),体重160-180g。将实验大鼠置于自然昼夜节律光照条件下,自由进食、饮水,每笼6-8只,控制室温于18~20℃。大鼠适应环境1周后,称重,并采用 Open-field(开场)实验测定行为,记录大鼠5min内的动作行为表现,包括中央格停留时间、水平穿越格数、竖立次数、修饰次数和粪便粒数。依体重和Open-field实验评分,将大鼠随机分为四组,每组8只,分别为对照组1、对照组2、实验组1和实验组2。各组间体重和Open-field实验评分均无显著性差异。对照组每笼喂养4只,实验组的大鼠于单笼喂养。porsolt swim test forcedswim
1.2 实验方法porsolt swimtest
将实验组1和实验组2的大鼠每天上午十点钟放入长宽50cm×60cm、水深25cm、水温恒定0℃的冰水玻璃缸中强迫游泳5min,每天给予刺激一次,共21天。对照组1与对照组2的大鼠放在笼中群养,不给任何刺激。第22天杀死对照组1和实验组1的大鼠, 实验组2停止强迫游泳和对照组2继续喂养,30天后,处死动物。各组大鼠在被处死前均采用 Open-field法测定大鼠的行为并称体重。处死方法:用戊巴比妥(50-60mg/kg)经腹腔麻醉,麻醉起效后将大鼠固定在灌流台上,剖开胸腔,经左心室快速灌注37ºC生理盐水(含适量肝素)150-200ml,继而灌注4%多聚甲醛溶液(用PBS配制,pH=4)200-300ml,持续30-40min后,取脑组织并修块。将脑修块置于4%多聚甲醛溶液中固定48小时,梯度酒精脱水,石蜡包埋后,冠状切片,片厚约7µm。porsolt swim test forcedswim
通过ABC免疫组化染色方法检测NGF、NT 3 蛋白的表达。切片经脱蜡、水化后入PBS(PH7.4),3%H2O210分钟内去除过氧化物酶,切片用微波进行抗原修复,然后进行免疫组织化学ABC法染色。用兔抗鼠NGF、NT 3 作为一抗,工作浓度为1:200。用 生物 素化的羊抗兔IgG作为二抗,工作浓度为1:200。最后加ABC复合物,工作浓度为1;100,最后加DAB显色液显色5-10分钟。porsolt swim test
每只大鼠取双侧海马CA3区DG区切片各两张,在计算机图像分析系统(Meta Morph/Ax70)上测量NGF、NT 3 平均光密度和平均目标灰度值(所测阳性细胞平均灰度),灰度单位分为256个等级,免疫反应的强弱与灰度值呈反比关系。
采用SPSS for Windows 10.0软件分析,数据以均数加减标准差(x - ±s)表示,组间检验采用t检验,p值取0.05。porsolt swim test
关键词 :慢性应激 海马 NGF、NT-3 强迫游泳porsolt swim test forcedswim
[ 中图分类号] R395 [ 文献标识码] A
porsolt swim test
Effects of chronic stress on the expression of NGF、NT-3 in hippocampusof the rats porsolt swimtest
Bi Bo, Jin Kuihe,Zhu Yuzhang, et al.
Department ofPsychiatry and Medical Psychology, The First Affiliated Hospital,China Medical University, Shenyang 110001
Abstract: Objective To observe the changes ofNGF、NT-3 expression inhippocampus of the rats with the forced-swimming and the changes of NGF、NT-3 after poststress. Methods The protocol was established with the forced-swimming as the chronic stress model. Open-field test was executed to measure the behaviors of the rats. Immunohistochemistry was used to observe the changes of NGF、NT-3 expression in the hippocampus. Results Compared to the control group,the expression of NGF、NT-3 in CA3 region of the hippocampus and dendate gyrus(DG)of the rats was decreased morphologically. With the computerized image analysis, the gray degree increased significantly and the changes showed the same results after the poststress. Conclusion Chronic stress decreased the expression of the NGF、NT-3 in CA3 region and DG in the hippocampus of the rats and the changes showed the same results after the poststress.
Key words: chronic stress,hippocampus, NGF、NT-3,forced-swimming,
porsolt swim test porsolt swimtest
神经生长因子(neuron growth factor NGF)是第一个被发现、也是 生物 学功能了解得最清楚的一个神经营养因子。NGF对皮质、海马、基底前脑的胆
碱能神经元有神经营养作用。 [1] 它和神经营养因子3(neurotrophin3, N T-3 )广泛地分布在海马、大脑皮层、中隔等中枢神经系统中,它们在神经保护、突触再生、神经损伤后功能重建等方面也发挥了重要作用。应激事件导致神经化学方面的改变可能参与了抑郁障碍的发生,此外,神经内分泌、神经递质和神经可塑性的改变在抑郁障碍的发生中也起到十分重要的作用。 [2] 本研究利用免疫组化的方法观察NGF、NT3在慢性应激以及应激过后在海马的表达变化,以了解NGF、NT 3 和抑郁障碍的关系。porsolt swim test
1 对象与方法porsolt swim test
1.1实验动物porsolt swimtest
成年Wistar雄性大鼠32只(由中国医科大学动物实验室提供),体重160-180g。将实验大鼠置于自然昼夜节律光照条件下,自由进食、饮水,每笼6-8只,控制室温于18~20℃。大鼠适应环境1周后,称重,并采用 Open-field(开场)实验测定行为,记录大鼠5min内的动作行为表现,包括中央格停留时间、水平穿越格数、竖立次数、修饰次数和粪便粒数。依体重和Open-field实验评分,将大鼠随机分为四组,每组8只,分别为对照组1、对照组2、实验组1和实验组2。各组间体重和Open-field实验评分均无显著性差异。对照组每笼喂养4只,实验组的大鼠于单笼喂养。porsolt swim test forcedswim
1.2 实验方法porsolt swimtest
将实验组1和实验组2的大鼠每天上午十点钟放入长宽50cm×60cm、水深25cm、水温恒定0℃的冰水玻璃缸中强迫游泳5min,每天给予刺激一次,共21天。对照组1与对照组2的大鼠放在笼中群养,不给任何刺激。第22天杀死对照组1和实验组1的大鼠, 实验组2停止强迫游泳和对照组2继续喂养,30天后,处死动物。各组大鼠在被处死前均采用 Open-field法测定大鼠的行为并称体重。处死方法:用戊巴比妥(50-60mg/kg)经腹腔麻醉,麻醉起效后将大鼠固定在灌流台上,剖开胸腔,经左心室快速灌注37ºC生理盐水(含适量肝素)150-200ml,继而灌注4%多聚甲醛溶液(用PBS配制,pH=4)200-300ml,持续30-40min后,取脑组织并修块。将脑修块置于4%多聚甲醛溶液中固定48小时,梯度酒精脱水,石蜡包埋后,冠状切片,片厚约7µm。porsolt swim test forcedswim
通过ABC免疫组化染色方法检测NGF、NT 3 蛋白的表达。切片经脱蜡、水化后入PBS(PH7.4),3%H2O210分钟内去除过氧化物酶,切片用微波进行抗原修复,然后进行免疫组织化学ABC法染色。用兔抗鼠NGF、NT 3 作为一抗,工作浓度为1:200。用 生物 素化的羊抗兔IgG作为二抗,工作浓度为1:200。最后加ABC复合物,工作浓度为1;100,最后加DAB显色液显色5-10分钟。porsolt swim test
每只大鼠取双侧海马CA3区DG区切片各两张,在计算机图像分析系统(Meta Morph/Ax70)上测量NGF、NT 3 平均光密度和平均目标灰度值(所测阳性细胞平均灰度),灰度单位分为256个等级,免疫反应的强弱与灰度值呈反比关系。
采用SPSS for Windows 10.0软件分析,数据以均数加减标准差(x - ±s)表示,组间检验采用t检验,p值取0.05。porsolt swim test