摘要
目的:分析褪黑素替代治疗对去松果体成年大鼠神经干细胞增殖及学习记忆的影响。方法:实验于2004-06/2005-03在广西医科大学基础医学院解剖学教研室完成。将40只成年健康雄性Sprague-Dawley大鼠随机分为5组:假手术组、去松果体组、褪黑素20μg/kg、200μg/kg和2mg/kg组。术后第2天开始进行干预,分别按20μg/kg、200μg/kg和2mg/kg剂量将褪黑素溶于0.5mL的50g/L乙醇-生理盐水,每天在固定时间(18:00)腹腔注射1次,连续给药21d。在相同条件下,每天给予假手术组或去松果体组的每只大鼠腹腔注射0.5mL的50g/L乙醇-生理盐水。用Morris水迷宫测定大鼠的学习记忆能力,用免疫组织化学方法检测齿状回颗粒细胞下区的增殖细胞核抗原阳性细胞的变化。结果:40只大鼠实验中无死亡,全部纳入结果分析。①去松果体组大鼠平均逃避潜伏期较假手术组明显延长,差异有显著性犤(32.02±2.54),(18.58±1.97)s,P<0.01犦。褪黑素20μg/kg组和200μg/kg组的平均逃避潜伏期较假手术组稍延长犤(23.29±2.74),(24.94±2.81),(18.58±1.97)s,P>0.05犦,但比去松果体组明显缩短,差异有显著性(P<0.01)。褪黑素2mg/kg组的平均逃避潜伏期(30.57±2.43)s比假手术组、褪黑素20μg/kg、200μg/kg组明显延长(P<0.01),并接近去松果体组的水平(P>0.05)。②去松果体组大鼠平均游泳距离明显大于假手术组,差异有显著性犤(826.24±32.28),(611.27±27.63)cm,P<0.01犦。褪黑素20μg/kg和200μg/kg组的平均距离均明显小于去松果体组犤(680.27±25.15),(692.96±26.42)cm,P<0.01犦,而褪黑素2mg/kg组大鼠的平均距离(804.81±34.22)cm均明显大于假手术组、褪黑素20μg/kg、200μg/kg组(P<0.01),并接近去松果体组的水平(P>0.05)。③齿状回颗粒细胞下区的增殖细胞核抗原免疫反应产物定位于细胞核,呈棕黄色。增殖细胞核抗原阳性细胞核呈圆形、椭圆形或梭形,各组大鼠的增殖细胞核抗原阳性细胞核的形态无明显差异。去松果体组增殖细胞核抗原阳性细胞数较假手术组,差异有显著性犤(58.50±7.73),(82.69±7.36)个/切片,P<0.01犦。褪黑素20μg/kg、200μg/kg组的增殖细胞核抗原阳性细胞犤(74.20±6.74),(72.00±6.77)个/切片犦分别较去松果体组升高37.6%和23.1%,差异有显著性(P<0.01),但仍未能达到假手术组的水平(P<0.01)。褪黑素2mg/kg组的增殖细胞核抗原阳性细胞数(61.23±6.61个/切片)分别比假手术组、褪黑素20μg/kg、200μg/kg组下降25.9%、17.5%及14.9%,组间差异有高度显著性(P<0.01),并接近去松果体组的水平(P>0.05)。结论:低剂量(20μg/kg,200μg/kg)的褪黑素替代治疗对去松果体成年齿状回颗粒细胞下区神经干细胞增殖及学习记忆能力均有相似的正性调节作用,而高剂量(2mg/kg)的褪黑素替代治疗对上述指标具有负性调节作用;去松果体或褪黑素替代治疗与否,神经干细胞的形态均无明显的改变,提示褪黑素的作用可能是通过神经干细胞上的相应受体介导的机制使增殖细胞核抗原表达上调,加速细胞周期进程,促进神经干细胞增殖和分裂。
AIM： To investigate the effects of melatonin replacement therapy on the proliferation of neural stem cells and the learning and memory of pinealectomized adult rats. METHODS： The experiment was conducted at the Department of Anatomy, College of Basic Medical College, Guangxi Medical University from June 2004 to March 2005. Forty adult healthy male Sprague-Dawley rats were randomly divided into five groups with eight rats per group： sham-operation group, pinealcctomized ＋vehicle replacement group , pinealectomized＋melatonin replacement groups （2.0μg/kg, 200μg/kg and 2 mg/kg, body mass）. Intervention began on the second day after operation, and melatonin was dissolved in 0.5 mL 50 g/L ethanol-normal saline solution according to the dosage of 20 μg/kg, 200 μg/kg and 2 mg/kg, and then was intraperitoneally injected into the rats at the fixed time （18：00） once every day, for 21 successive days. At the same condition, 0.5 mL 50 g/L ethanol-normal saline solution was intraperitoneally injected into the rats in the sham-operation group and pinealectomized＋vehicle replacement group. The learning and memory ability of the rats was measured with Morris water Maze and the number of proliferating cell nuclear antigen immunoroactire （PCNA-IR） cells in the subgranular zone （SGZ） of dentate gyrus was counted with immunohistolocbemical method. RESULTS： Totally 40 rats survived in the experiment and all of them enterod the stage of result analysis. ① The average escape latency of the rats was obviously prolonged in the pinealectomized＋vehicle replacement group as compared with that of the sham-operation group, with significant difference [（32.02±2.545, （18.58±1.97） s,P 〈 0.01]. The average escape latency was slightly longer in the pinealectomized＋melatonin replacement groups （20 μg/kg, 200 μg/kg） than in the sham-operation group [（23.29±2.74）, （24.94±2.81）, （18.58±1.97） s, P 〉 0.05], but was obviously shorter than in the pinealectomized＋vehicle replacement group, with significant difference （P 〈 0.01）.The average of escape latency was obviously longer in the pinealeetomized＋melatonin replacement group （2 mg/kg） [（30.57±2.43） s ] than in the pinealectomized＋melatonin replacement groups （20μg/kg, 200 μg/kg） （P 〈 0.05）, but close to the level of pinealectomizod＋vehicle replacement group （P 〉 0.05）. ②The average swimming distance of the rats was obviously longer in the pinealeetomized＋vehiele replacement group than in the sham-operation group , with significant difference [（826.24±32.28）,（611.27±27.63） cm,P 〈 0.01].Tbe average swimming distance was remark- ably shorter in the pinealectomized＋melatonin replacement groups （20 μg/kg, 200μg/kg） [（680.27±25.15）, （692.96±26.42）cm,P 〈 0.01]than in the pinealectomized＋vehicle replacement group , while the average swimming distance was significantly longer in the pinealectomized＋melatonin replacement group （2 mg/kg） [（804.81±34.22） cm] than in the pinealectomized＋ melatonin replacement groups （20 ±g/kg, 200±g/kg）（P 〈 0.01）, and Close to the level of pinealectomized＋vehicle replacement group （P 〉 0,05）. ③ PCNA-IR cells in the SGZ of dentate gyrus were located in the nucleolus, presenting brown. The PCNA-IR positive nucleolus presented round, ellipse or shuttle-shape. There was no significant difference in the morphology of PGNA-IR positive nucleolus in each group. The number of PCNA-IR positive cells was smaller in the pinealectomized ＋vehicle replacement group than in the sham-operation group, with significant difference [（58.50±7.73）,（82.69±7.36）/slice, P 〈 0.01]. The number of PCNA-IR was increased by 37.6% and 23.1% in the pinealectomized＋melatonin replace- ment groups （20 μg/kg, 200 μg/kg） [（74.20±6.74）,（72.00±6.77）/slice] than in the pinealectomized＋vehicle replacement greup respectively, with significant difference （P 〈 0.01）, but still not reached the＇level of sham-operation group （P 〈 0.01）. The number of PCNA-IR was decreased by 25.9%,17.5% and 14.9% in the pinealectomized＋melatonin replacement group （2 mg/kg） （61.23±6.61 /slice）than in the sham-operation group, pinealectomized＋melatonin replacement group （20 μg/kg and 200 μg/kg）, respectively, close to the level of Pinealectomized ＋vehicle replacement group（P 〉 0.05）. CONCLUSION： Low doses （20 μg/kg and 200μg/kg） of melatonin replacement therapy or pinealectomized adult male ratscan greatly increase the proliferation rate of neural stem cells in SGZ, and elevate the abilities of learning and memory, while high dose （2 mg/kg） of melatonin replacement therapy can reverse the above indexes; There was no obvious change in cellular morphology of nerve stem cells whether pinealectomization or melatonin replacement therapy are performed or not, suggesting that the effects of melatonln may involve the mechanism of melatonin receptor-mediated signal transduction in neural stem cells, up-regulating the expression of PCNA and speeding up the progress of cell cycle as well as the proliferation and differentiation of neural stem cells, disease, which may be a potential strategy for hampering the worse advance of the digease.
出处
《中国临床康复》
CSCD
北大核心
2006年第9期27-30,i0001,共5页
Chinese Journal of Clinical Rehabilitation
