睡眠肽DSIP的人工合成及应用初步研究

发布时间：2018-04-19 17:58

本文选题：人工合成肽 + 鼻腔给药　；参考：《桂林医学院》2012年硕士论文

【摘要】：目的：人工合成目的肽DSIP并分析纯化,研究其促眠活性,并进一步研究经鼻腔给药的促眠作用,同时探索毒副作用,从而为治疗失眠症提供一种人工合成因而不受来源限制、质量易于控制,在失眠症的治疗中取得较好效果、给药方便并免除不良副作用,可迅速推广应用的高效外源性促眠药物。方法：利用生物信息学对已知结构的天然存在睡眠肽进行分析,选择其α同分异构体并采用全化学合成的固相多肽合成法合成DSIP,通过高效液相色谱和质谱法进行纯化分析及鉴定,确定合成的多肽即为设计需要的目的肽并分析其纯度。进行分组动物实验研究目的肽的促眠效果：用大鼠初步判定此人工合成的目的肽DSIP是否具有促眠作用；确认其效用后第一步用新西兰大耳白兔判定目的肽经静脉给药是否如文献报道的有诱导新西兰大耳白兔睡眠的作用；第二步研究鼻腔给药并比较不同剂量DSIP的促眠效果,初步探索出目的肽鼻腔给药的适宜剂量。最后通过病理学实验检测人工合成的睡眠肽DSIP在大中剂量中长期给药后对脑组织是否有毒性。结果：1.成功合成具有生物活性的睡眠肽DSIP,经高效液相法纯化后达到所需要的95%以上纯度,质谱法与HPLC法确认合成的是目的肽；2.安装新西兰大耳白兔脑电极后兔子仍然存活状况良好,给药前后均成功引导出新西兰大耳白兔的脑电图,记录数据得到有效分析；3.分析脑电图并进行统计学检验后,鼻腔给药组、静脉给药组新西兰大耳白兔均与生理盐水组新西兰大耳白兔总睡眠时间、慢波睡眠时间统计学有显著性差异,且给药组慢波睡眠时间长、总睡眠时间长；4.分析脑电图并进行统计学检验后,鼻腔给药15nmol/kg组与30nmol/kg组及45nmol/kg组新西兰大耳白兔总睡眠时间、慢波睡眠时间统计学有显著性差异,鼻腔给药30nmol/kg组、及45nmol/kg组新西兰大耳白兔与静注剂量30nmol/kg组新西兰大耳白兔总睡眠时间、慢波睡眠时间统计学无显著性差异；5.中长期大中剂量DSIP静脉给药后光镜下对照组和给药组的脑组织切片经HE染色经肉眼观察无明显差异。结论：1.人工合成的睡眠肽DSIP通过鼻腔给药有促眠作用；2.人工合成的睡眠肽DSIP通过鼻腔给药可得到静注相近的促眠作用,初步认为30nmol/kg为新西兰大耳白兔鼻腔给药适宜剂量；3.人工合成的睡眠肽DSIP中长期大中剂量给药对脑组织细胞无明显损伤。
[Abstract]:Objective: to synthesize the objective peptide DSIP, analyze and purify it, study its activity of promoting sleep, further study the effect of nasal administration, and explore the side effects, so as to provide an artificial synthesis for the treatment of insomnia, so as not to be limited by the source.The quality is easy to control, and good effect has been obtained in the treatment of insomnia. It is convenient to give drugs and avoid adverse side effects. It can be used quickly to popularize the highly effective exogenous sleep-promoting drugs.Methods: the natural sleep peptides of known structures were analyzed by bioinformatics.The 伪 -isomer was selected and synthesized by solid-phase polypeptide synthesis. The synthesized peptide was purified and identified by HPLC and MS. The synthesized peptide was determined to be the target peptide for design and its purity was analyzed.To study the effect of the objective peptide on promoting sleep: the rat was used to determine whether the synthetic peptide DSIP had the effect of promoting sleep.In the first step, New Zealand Big ear White Rabbit was used to determine whether intravenous administration of the target peptide could induce sleep in New Zealand Big ear White Rabbit as reported in the literature, and the second step was to study nasal administration and compare the effects of different doses of DSIP on sleep promotion in New Zealand Big ear White Rabbit.Objective: to explore the appropriate dose for nasal administration of objective peptide.Finally, the toxicity of synthetic sleep peptide DSIP to brain tissue was detected by pathological experiments.The result is 1: 1.The sleep peptide DSIPs with biological activity were successfully synthesized and purified by high performance liquid chromatography (HPLC) to achieve the desired purity of more than 95%. It was confirmed by mass spectrometry and HPLC that the synthesized peptide was the target peptide 2.The rabbit survived well after installing the brain electrode of New Zealand big ear white rabbit. The EEG of New Zealand big ear white rabbit was successfully guided before and after administration, and the recorded data were analyzed effectively.After electroencephalogram analysis and statistical test, there were significant differences in the total sleep time and slow wave sleep time between the nasal administration group and intravenous administration group and normal saline group.The slow wave sleep time was long and the total sleep time was 4.After analysis of EEG and statistical test, there were significant differences in total sleep time and slow wave sleep time between 15nmol/kg group, 30nmol/kg group and 45nmol/kg group.There was no significant difference in total sleep time and slow wave sleep time between 45nmol/kg group and 30nmol/kg group.There was no significant difference in brain sections between the control group and the administration group by HE staining under light microscope after medium and long term intravenous administration of DSIP.Conclusion 1.Synthetic sleep peptide DSIP can promote sleep by nasal administration.Synthetic sleep peptide DSIP can get similar sleep promoting effect by nasal administration. It is preliminarily considered that 30nmol/kg is the appropriate dose for nasal administration of New Zealand big ear white rabbits.The synthetic sleep peptide DSIP was given in medium and long term dose without obvious damage to brain tissue cells.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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