摘要
目的 观察饮水型慢性氟中毒子代大鼠学习记忆能力及大脑胆碱能毒蕈碱受体（Muscarinic acetylcholine receptors,mAChRs,简称M受体）mRNA和蛋白表达,探讨慢性氟中毒子代大鼠中枢神经系统损伤发生机制.方法 健康纯系SD大鼠40只,按体质量采用随机数字表法分为2组,每组20只,雌雄各半.对照组：自由饮用自来水（含氟量＜ 0.5 mg/L）;染氟组：自由饮用加入氟化钠（NaF）的自来水（含氟量为50.0 mg/L）.2组大鼠给予标准饲料（含氟量为6.2 mg/kg）喂养.6个月后2组大鼠分别交配,每组新生仔鼠由母乳喂养.采集出生第1、7、14、21、28天仔鼠脑组织（各时间点每组10只）,采用实时荧光定量PCR及蛋白印迹法检测M1 、M3受体mRNA及蛋白表达水平;采用Morris水迷宫方法检测出生第28天仔鼠行为学变化,并分析第28天仔鼠学习记忆能力与M1、M3受体蛋白表达的相关性.结果 染氟组第28天仔鼠逃避潜伏期时间[（35.61±9.00）s]较对照组显著延长[（8.46±3.09）s,P＜0.05],空间探索次数及逗留平台象限时间[（5.00±2.90）次,（16.66±2.79）s]较对照组明显减少[（15.17±3.66）次,（22.51±2.66）s,P均＜0.05].第1、7、14、21、28天染氟组仔鼠脑组织M1、M3受体mRNA、蛋白表达水平均低于对照组[对照组M1 mRNA：（100.00± 11.00）％、（100.00±17.57）％、（100.00±9.14）％、（100.00±7.52）％、（100.00±15.78）％;染氟组M1 mRNA：（20.47±8.07）％、（14.00±4.53）％、（16.57±7.62）％、（25.56±12.78）％、（16.27±4.82）％;对照组M3 mRNA：（100.00±16.30）％、（100.00±14.40）％、（100.00±7.20）％、（100.00±14.31）％、（100.00±13.16）％;染氟组M3 mRNA：（29.17±8.00）％、（12.77±2.22）％、（26.40±7.20）％、（15.74±3.55）％、（28.14±7.53）％;对照组M1蛋白：（100.00±2.24）％、（100.00±8.30）％、（100.00±4.61）％、（100.00±13.78）％、（100.00±11.72）％;染氟组M1蛋白：（20.47±8.07）％、（14.00±4.53）％、（16.57±7.62）％、（25.56±12.78）％、（16.27±4.82）％;对照组M3蛋白：（100.00±16.30）％、（100.00±14.40）％、（100.00±7.20）％、（100.00±14.31）％、（100.00±13.16）％;染氟组M3蛋白：（29.17±8.00）％、（12.77±2.22）％、（26.40±7.20）％、（15.74±3.55）％、（28.14±7.53）％,P均＜0.05或＜0.01].相关分析结果发现,第28天仔鼠逃避潜伏期时间与M1、M3蛋白表达水平呈负相关关系（r=-0.827、-0.742,P均＜0.05）,空间探索次数与M1、M3蛋白表达水平呈正相关关系（r=0.843、0.806,P均＜0.05）.结论 不同阶段的慢性氟中毒大鼠所生仔鼠脑组织M受体及蛋白表达水平明显不同程度降低,这可能是氟中毒引起动物学习记忆能力降低的机制之一.
Objective To observe the changes of learning and memory ability and detect the expression of muscarinic acetylcholine receptor （mAChR,M receptor） at mRNA and protein levels in brains of offspring rats with chronic fluorosis,and to reveal the mechanism of the central nervous system damage.Methods Forty healthy SD rats were divided into two groups （20 in each group,half male and half female） by random number table according to body weight.In the control group,the rats were fed with drinking water containing no more than 0.5 mg/L fluoride;in the fluoride group,the rats were fed with high dose of sodium fluoride in drinking water （50.0 mg/L fluoride）.Each group was fed with normal diet （6.2 mg/kg fluoride）.After exposed to fluoride for 6 months,each group was mated,and brains of newborn offspring rats aged 1,7,14,21 and 28 days were taken,and expression of M1 and M3 receptors at mRNA and protein levels were analyzed by real-time PCR and Western blotting,respectively.Behavioral changes were measured by Morris water maze test at the 28 days after birth.The correlations between protein levels of M1 and M3 receptors and the ability of learning and memory at the 28 days after birth were analyzed.Results In fluoride group of the offspring rats at 28 days after birth,the escape latency time [（35.61 ± 9.00）s] was significantly longer than that in control group [（8.46 ± 3.09）s,P 〈 0.05],while the numbers of crossing the platforms and the time of staying the platforms [（5.00 ± 2.90）times,（16.66 ± 2.79）s] were significantly decreased as compared to that of control group [（15.17 ± 3.66）times,（22.51 ± 2.66）s,all P 〈 0.05].Furthermore,the mRNA expression and the protein levels of M1 and M3 receptors in rat brain at each phase in fluoride group were significantly decreased as compared to controls [M1 mRNA in control groups：（100.00 ± 11.00）%,（100.00 ± 17.57）%,（100.00 ± 9.14）%,（100.00 ± 7.52）%,（100.00 ± 15.78）%;M1 mRNA in fluoride groups：（20.47 ± 8.07）%,（14.00 ± 4.53）%,（16.57 ± 7.62）%,（25.56 ± 12.78）%,（16.27 ± 4.82）%;M3 mRNA in control groups：（100.00 ± 16.30）%,（100.00 ± 14.40）%,（100.00 ± 7.20）%,（100.00 ± 14.31）%,（100.00 ± 13.16）%;M3 mRNA in fluoride groups：（29.17 ± 8.00）%,（12.77 ± 2.22）%,（26.40 ± 7.20）%,（15.74 ± 3.55）%,（28.14 ± 7.53）%;M1 protein in control groups：（100.00 ± 2.24）%,（100.00 ± 8.30）%,（100.00 ± 4.61）%,（100.00 ± 13.78）%,（100.00 ± 11.72）%;M1 protein in fluoride groups：（20.47 ± 8.07）%,（14.00 ± 4.53）%,（16.57 ± 7.62）%,（25.56 ± 12.78）%,（16.27 ± 4.82）%;M3 protein in control groups：（100.00 ± 16.30）%,（100.00 ± 14.40）%,（100.00 ± 7.20）%,（100.00 ± 14.31）%,（100.00 ± 13.16）%;M3 protein in fluoride groups：（29.17 ± 8.00）%,（12.77 ± 2.22）%,（26.40 ± 7.20）%,（15.74 ± 3.55）%,（28.14 ± 7.53）%,P 〈 0.05 or 〈 0.01].The escape latency and M1,M3 receptors protein levels were negatively correlated （r =-0.827,-0.742,all P 〈 0.05）,and the number of space exploration and M1,M3 receptors protein levels were positively correlated （r =0.843,0.806,all P 〈 0.05）.Conclusion The expression of M receptor at protein and mRNA levels in offspring rat brains of different ages are significantly declined,which might be one of the mechanism of the decreased ability of learning and memory induced by fluoride toxicity.
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2015年第5期326-330,共5页
Chinese Journal of Endemiology
基金
国家自然科学基金（81160335）
科技部国际合作重大项目（2013BA105803）
贵州省科技厅国际合作项目（黔科合外G字[2011]7014号）
关键词
氟化物
脑
胆碱能毒蕈碱受体
学习
记忆
大鼠
Fluorides
Brain
Muscarinic acetylcholine receptors
Learning Memory
,Rats
