论文部分内容阅读
背景:研究提示,烟碱对帕金森病小鼠具有神经保护效应,临床试验也观察到,帕金森病患者在吸烟过程中其震颤、僵直、运动减少等症状减轻,但其作用机制目前尚不明了。目的:观察烟碱对大鼠脑纹状体多巴胺D1,D2受体mRNA表达的影响,分析烟碱对帕金森病神经保护效应的作用机制。设计:完全随机分组设计,对照试验。单位:中南大学湘雅医院神经病学研究所。材料:选用健康雄性清洁级SD大鼠24只,10周龄,体质量180～200g。主要试剂及仪器:烟碱(美国Sigma公司)、反转录试剂盒(美国MBI公司)、聚合酶链反应热循环仪(美国Beckman)、全自动紫外分光光度计(美国BeckmanDu-70)、图像分析处理系统(美国StratageneEagleEyeⅡ型)。方法:实验于2001-07/2002-07在中南大学湘雅医院神经病学研究所实验室完成。①按随机抽签法将大鼠随机分为2组:对照组、烟碱组,每组12只。分别皮下注射生理盐水、烟碱溶液4mg/(kg·d),2次/d,共14d。注射药物后0.5h观察大鼠行为活动,包括定型活动、走动活动、攀爬行为、旋转行为、理毛行为、张口行为、呕吐行为等,观察时间为0.5h。②于末次注射药物后0.5h处死动物,快速分离纹状体,提取总RNA,采用美国MBI反转录试剂盒反转录cDNA,在特定条件下进行聚合酶链反应扩增,将电泳凝胶在EagleEyeⅡ图像处理系统上分析计算出多巴胺D1,D2受体及内对照β-actin吸光度(A值),检测大鼠脑纹状体多巴胺D1,D2受体mRNA的表达。③两样本均数比较采用t检验。主要观察指标:①大鼠行为学改变。②大鼠脑纹状体多巴胺D1,D2受体mRNA的表达。结果:大鼠24只均进入结果分析。①烟碱组大鼠于用药第3天,出现走动增多,易激惹,定型活动明显,并于第7～14天达到高峰;对照组则无明显变化。②大鼠脑纹状体所有标本总RNAA260/280均>1.8,经12g/L琼脂糖浆电泳显示无明显降解;通过反转录聚合酶链反应和设定的引物,得到多巴胺D1,D2受体及actin的扩增产物分别为350bp,399bp,218bp,与预计值一致。③在大鼠纹状体内,烟碱组多巴胺D1受体mRNA表达比对照组上升23%(分别为98.63±1.13,65.29±1.45,P<0.01),两组多巴胺D2受体mRNA的表达差异无显著性意义(P>0.01)。结论:烟碱可能通过上调大鼠纹状体多巴胺D1受体mRNA的表达而诱导大鼠行为改变。
BACKGROUND: Nicotine has a neuroprotective effect on Parkinson’s disease in mice. It has also been observed in clinical trials that Parkinson’s disease alleviates the symptoms of tremor, stiffness and decreased motion during smoking, but its mechanism of action is unknown It’s Objective: To observe the effects of nicotine on the expression of dopamine D1 and D2 receptors in striatum of rat brain and the mechanism of nicotine on the neuroprotective effect of Parkinson’s disease. Design: Complete randomized block design, controlled trial. Unit: Xiangya Hospital, Central South University Institute of Neurology. MATERIALS: Twenty-four healthy male SD rats were selected, aged 10 weeks, with a body weight of 180-200 g. The main reagents and instruments: nicotine (Sigma, USA), reverse transcription kit (MBI, USA), polymerase chain reaction thermocycler (Beckman, USA), automatic UV spectrophotometer (BeckmanDu-70, Analysis and Processing System (USA Stratagene EagleEye Ⅱ type). Methods: The experiment was performed in the laboratory of Institute of Neurology, Xiangya Hospital, Central South University from July to July 2002. ① randomly selected rats were randomly divided into 2 groups: control group, nicotine group, 12 in each group. Subcutaneous injection of saline, nicotine solution 4mg / (kg · d), 2 times / d, a total of 14d. The rats were observed behavioral activities 0.5h after injection of drugs, including stereotyping activities, walking activities, climbing behavior, rotation behavior, grooming behavior, mouth behavior, vomiting behavior, the observation time was 0.5h. The animals were sacrificed at 0.5h after the last injection of the drug and the striatum was rapidly separated and the total RNA was extracted. The cDNA was reverse transcribed using the MBI reverse transcription kit from the United States, and subjected to polymerase chain reaction amplification under specific conditions. The electrophoresis gel Dopamine D1, D2 receptors and β-actin absorbance (A value) were calculated and analyzed in EagleEye Ⅱ image processing system to detect dopamine D1, D2 receptor mRNA expression in rat brain. ③ two samples were compared using t test. MAIN OUTCOME MEASURES: ① behavioral changes in rats. ② The expression of dopamine D1, D2 receptor mRNA in rat brain striatum. Results: All 24 rats were involved in the result analysis. ① The rats in nicotine group showed increased ambulation, easy irritability and stereotyped activity on the third day after medication, and reached the peak on the 7th to 14th days. There was no significant change in the control group. ② The total RNAA260 / 280 in all samples of rat brain striatum were all> 1.8, which showed no significant degradation by 12g / L agarose gel electrophoresis; the dopamine D1 and D2 receptors were obtained by reverse transcription polymerase chain reaction and the set primers And actin amplification products were 350bp, 399bp, 218bp, consistent with the expected value. (3) In rat striatum, expression of dopamine D1 receptor mRNA in nicotine group increased by 23% (98.63 ± 1.13, 65.29 ± 1.45, P <0.01, respectively) compared with the control group. There was no significant difference in dopamine D2 receptor mRNA expression between the two groups Significant significance (P> 0.01). Conclusion: Nicotine may induce behavioral changes in rats by up-regulating the expression of dopamine D1 receptor mRNA in rat striatum.