隆朋经神经细胞信号转导通路对基因转录调控的麻醉机制研究

Anaesthesia center mechanism research is the clinical anesthesia and Medication foundation, xylazine is the animal basic clinical anesthesia combination junk. in view of this,In this study, wistar rats were selected as the experiment animals. And rats were divided randomly into four groups: the control group, the low dose group, the medium dose group and the high dose group. Animals in the experiment groups were treated with the same volume xylazine parenteral solution with different doses, such as low dose, the medium dose, or the high dose. Rats in the control group were administered with the same volume of physiological saline as the xylazine. The tissue samples of cerebral cortex, cerebellum, midbrain, thalamus and hippocampus in rats were individually collected at 10, 30, 60, 90min after the administration of xylazine. Effect of xylazine on differences expression of CREB mediated by intraneuronal Ca2+ and cAMP signal transduction in rat were investigated. And RNAi technique was used to silence key regulatory proteins CaMKII, CaMKIV and PKC of Ca2+ and cAMP signal transduction system of nerve cells. So that cellular level mechanism of CREB, the endonuclear transcriptional control protein on the gene transcription may be analyzed. Analyze the ctivate mechanism of xylazine on the phosphorylation of intraneuronal CREB mediated byCa2＋and cAMP and identify that which second messengers were selected after the anesthetics, which produced a marked effect on the endonuclear protein CREB, going into the nerve cells. So that it could accordance with it to utilize the anaresthetics, and it would be the base of medicine study in future.

麻醉中枢机制的研究是临床麻醉研究及用药的基础，隆朋是动物临床麻醉药物的组方基础，基于此，实验以大鼠为实验动物，隆朋设为对照、低剂量、中剂量和高剂量组，每组大鼠给药后设10min、30min、60min、90min、120min5个时间点，分别断头取大脑皮层、小脑、中脑、丘脑及海马等组织，利用RNAi技术对神经细胞内Ca2＋和cAMP信号转导系统中的关键调节蛋白CaMKⅡ、CaMKⅣ和PKC进行RNA沉默，分析CaMKⅡ-RNAi、CaMKⅣ-RNAi和PKC-RNAi与隆朋作用对p-CREB表达差异影响，在此基础上体外培养神经细胞，全面分析隆朋通过Ca2＋和cAMP途径对神经细胞核内CREB蛋白的磷酸化激活过程及细胞核内转录调控蛋白CREB对基因转录条控的机制。明确隆朋作用中枢神经系统后的麻醉作用机制，为今后指导临床更好应用药提供依据，也是今后开展相关药物研究的基础。

隆朋作为多种复合麻醉剂的重要组成成分之一，研究其麻醉作用机理对研究高效、副作用低的复合麻醉剂十分重要，目前，对隆朋麻醉作用的神经信号通路的研究较少。本试验从分子水平对隆朋的麻醉机制进行探讨，为今后的临床麻醉提供合理的分子理论依据。.试验以成年Wister大鼠和PC12神经细胞为研究对象，在体试验腹腔注射70mg/kg隆朋，通过连续观察大鼠行为学变化基础上，进一步检测大鼠不同脑区在不同麻醉时期的NR1、NR2A、NR2B、GluR1、GluR2、ERK、PKC、PKA、CaMKII、p-CaMKII、CaMKIV、p-CaMKIV、CREB、p-CREB和BDNF的蛋白和基因水平的表达量， c-jun、c-fos和fos-b的mRNA表达量；离体细胞试验通过在培育的PC12神经细胞中加入隆朋，并且通过siRNA技术对ERK、PKA、CaMKII和CaMKIV进行靶向沉默，探究ERK、PKA、CaMKII和CaMKIV对下游CREB/BDNF通路以及IEGs（c-jun、c-fos和fos-b）的影响。探讨隆朋麻醉对大鼠不同脑区及神经元所产生的影响与PKA/CREB/BDNF信号通路的关系。.试验结果发现隆朋能影响各脑区Na＋-K＋-ATP酶和Ca2＋-Mg2＋-ATP酶的活性以及Ca2＋的进入和cAMP的含量；进而能够影响NR1、NR2A、NR2B、GluR1和GluR2的表达量，进而通过干预CREB信号通路中ERK、PKA和CaMKⅡ调节CREB的磷酸化，对神经细胞进行调控，使其处于麻醉状态。表明隆朋能够通过对离子通道、信号通路和基因调控的影响使机体产生麻醉效果。.课题研究结果为今后麻醉及麻醉相关机制研究提供基础，也为今后麻醉药物组合提供思路，课题研究成果及成果应用将开创动物临床麻醉新的模式，必将产生深远影响。