替来他明与曲马多对大鼠不同脑区LKB1-AMPK-mTOR信号传导通路的影响

发布时间：2018-03-22 08:14

本文选题：小型猪　切入点：替来他明　出处：《东北农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：小型猪复合麻醉剂(XFM)是课题组根据小型猪的生理特点和平衡麻醉理论,研制出的一种小型猪专用的复合麻醉剂,具有麻醉诱导时间短、麻醉维持时间适宜、副作用小的优点。替来他明和曲马多作为XFM的成份之一,均作用于动物的中枢神经系统,并且都具有确实的镇痛效果。最新的研究证明:LKB1-AMPK-m TOR信号转导通路中的关键蛋白参与了机体疼痛和神经元兴奋性的调节,推测LKB1-AMPK-m TOR信号转导通路可能参与了麻醉的镇痛调控。所以本实验通过给大鼠腹腔注射生理盐水、盐酸替来他明和盐酸曲马多,利用分子生物学技术检测药物注射后不同时间中枢神经系统(大脑皮层、海马、丘脑、小脑、脑干)中LKB1-AMPK-m TOR信号通路的关键蛋白基因的转录量和蛋白的磷酸化水平,进而探讨他们的镇痛机制,从而推测XFM的全麻镇痛机理。将54只SD大鼠随机分为对照组(n=6)、麻醉组(n=24)和镇痛组(n=24)。各组大鼠分别腹腔注射生理盐水、盐酸替来他明或盐酸曲马多后,对照组大鼠在注射生理盐水后10 min颈椎脱臼处死,麻醉组和镇痛组根据注射药物后10 min、20 min、40 min和60 min分为四个亚组(n=6),分别在相应时间点颈椎脱臼处死,并迅速采集大脑皮层、海马、丘脑、小脑和脑干部位,利用RT-PCR检测样品中LKB1 mRNA、AMPKα1 m RNA、AMPKα2 m RNA和4EBP1m RNA的相对转录量,利用Western-blot方法检测各样品p-LKB1/LKB1、p-AMPK/AMPK和p-4EBP1/4EBP1蛋白磷酸化水平。实验结果表明:(1)替来他明和曲马多能使LKB1 m RNA、AMPKα1 m RNA和AMPKα2 m RNA的转录量极显著性升高(p0.01),而抑制4EBP1 m RNA的转录量(p0.01)。(2)替来他明和曲马多能促进LKB1和AMPK的磷酸化水平(p0.01),相反抑制了4EBP1的磷酸化水平(p0.01)。(3)在大鼠的中枢神经系统中,替来他明和曲马多在海马、丘脑、小脑以及脑干区表现出高效的调节作用,且多为药物注射后的20 min和40 min。综上,实验得出结论:LKB1-AMPK-mTOR信号转导通路参与了替来他明和曲马多的麻醉、镇痛调节,而大鼠的海马、小脑、丘脑、脑干可能是LKB1-AMPK-m TOR信号通路参与麻醉与镇痛的靶位;通过激活LKB1-AMPK-mTOR信号通路作用于该药物相对应的靶蛋白上产生麻醉、镇痛作用可能是替来他明和曲马多的作用机制,这一研究结果对于深入揭示小型猪复合麻醉剂的镇痛和麻醉机理奠定了坚实的基础。
[Abstract]:The miniature pig compound anesthetic (XFM) is a special compound anesthetic for miniature pigs, which is developed by the research group according to the physiological characteristics and balanced anesthetic theory of miniature pigs. It has the advantages of short induction time and suitable maintenance time. The advantages of small side effects. Teletamine and tramadol, as one of the components of XFM, both act on the central nervous system of animals. Recent studies have shown that the key proteins in the TOR signal transduction pathway of 1: LKB1-AMPK-m participate in the regulation of pain and neuronal excitability. It is speculated that the LKB1-AMPK-m TOR signal transduction pathway may be involved in the analgesic regulation of anaesthesia. Therefore, in this experiment, the rats were injected intraperitoneally with normal saline, teletamine hydrochloride and tramadol hydrochloride. The transcription and phosphorylation of key protein genes of LKB1-AMPK-m TOR signaling pathway in central nervous system (cerebral cortex, hippocampus, thalamus, cerebellum, brainstem) at different time after drug injection were measured by molecular biological techniques. 54 SD rats were randomly divided into control group (n = 6), anesthesia group (n = 24) and analgesia group (n = 24). Rats in each group were given intraperitoneal injection of normal saline, teletamine hydrochloride or tramadol hydrochloride, respectively. The rats in the control group were killed 10 min after injection of normal saline. The anesthetic group and the analgesia group were divided into four subgroups according to the time of 10 minutes, 20 minutes, 40 min and 60 min after injection. The rats were killed at the corresponding time points, and the cerebral cortex was collected rapidly. In hippocampus, thalamus, cerebellum and brain stem, RT-PCR was used to detect the relative transcription of AMPK 伪 1 m RNA-AMPK 伪 2 m RNA and 4EBP1m RNA. The phosphorylation levels of p-LKB1 / AMPK and p-4EBP1/4EBP1 proteins in each sample were detected by Western-blot method. The results showed that teletamine and tramadol could significantly increase the transcription of LKB1 m RNAAMPK 伪 1 m RNA and AMPK 伪 2 m RNA and inhibit the transcription quantity of 4EBP1 m RNA. Lertamine and tramadol could promote the phosphorylation level of LKB1 and AMPK (p0.01), but inhibit the phosphorylation level of 4EBP1 (p0.01) in the central nervous system of rats. Teletamine and tramadol showed high regulatory effects in the hippocampus, thalamus, cerebellum and brain stem area, and most of them were 20 min and 40 mins after drug injection. It is concluded that the LKB1-AMPK-m TOR signal transduction pathway may be involved in the regulation of anesthesia and analgesia by teletamine and tramadol, while the hippocampus, cerebellum, thalamus and brain stem may be the targets of LKB1-AMPK-m TOR signaling pathway. By activating the LKB1-AMPK-mTOR signaling pathway to produce anesthesia on the corresponding target protein of the drug, the analgesic effect may be the mechanism of teletamine and tramadol. The results laid a solid foundation for further revealing the analgesic and anesthetic mechanism of miniature pig compound anesthetic.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.791

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