眼镜蛇神经毒镇痛小肽片段的作用机制研究

发布时间：2018-04-27 06:08

本文选题：蛇毒神经毒素 + 镇痛活性中心　；参考：《昆明理工大学》2017年硕士论文

【摘要】：疼痛是各种疾病中最为常见的信号之一,给患者带来极大的痛苦,严重影响他们的工作和生活。目前,临床用于疼痛治疗的药物(如吗啡、可待因、芬太尼、哌替啶、舒林酸等)几乎均具有成瘾性、剂量依赖性、毒性等不良效应。因此天然类镇痛药物给疼痛患者带来了希望。在这类药物的研究中,蛇毒一直处于研究的热点。研究表明蛇毒神经毒素的镇痛活性良好,且具有无成瘾性、无剂量依赖性、镇痛时效长等优点,故蛇毒神经毒素是开发副作用小、无成瘾性等镇痛药物的潜在理想原料,但对其具体的作用机制尚未明确。以往的研究一般认为蛇毒的镇痛效应是由其毒性引起的,但近些年有研究表明蛇毒神经毒素的镇痛效应是通过中枢系统起效,与经外周神经系统起作用的毒性机制截然不同。另外,有研究表明口服蛇毒神经毒素具有镇痛效应,而且发现其中一些低神经毒性多肽具有显著的镇痛活性,故我们推测其镇痛活性中心与毒性中心可能是两个独立的组分,且镇痛组分不易被胃蛋白酶所分解。本文首先对文献中部分具有镇痛效应的蛇毒神经毒素的LD50和镇痛起效剂量的相关性作了分析,进一步确认两者间并非完全一致,表明蛇毒镇痛活性中心与毒性中心确有可能是两个独立区域。课题组前期的研究结果表明,来自中华眼镜蛇的短链神经毒蛋白short neurotoxin C/coborotoxin c(CBTc)具有相对很弱的神经毒性和显著的口服镇痛活性,意味着该神经毒素具有独立的、镇痛效应很强的活性中心,且该镇痛活性中心耐胃蛋白酶水解。鉴于此,对CBTc的氨基酸序列和结构进行分析,根据胃蛋白酶水解位点及其在三级结构上所处的位置,设计了具有10个氨基酸的10肽(KDHRGTRIER),用热板法、醋酸扭体法、福尔马林注射法验证其确有镇痛活性,其LD50为8.4 g/kg,95%可信限为7.3-9.7g/kg,表明小肽的毒性很低或无毒性,进而验证了蛇毒镇痛活性中心与毒性中心可能是两个独立组分的推测。根据长链神经毒素也具有镇痛活性,故对10肽作相应改造,期望有更高的镇痛活性和更长的镇痛时效:包括增加Loop C前端氨基酸,预期能更全面结合靶蛋白;末端增加2个Cys,预期能自然氧化形成二硫键,能增加稳定性和活性。经热板法和醋酸扭体法测定改造肽的镇痛活性,结果显示16肽的镇痛活性明显高于13肽和15肽,且均高于10肽,表明设计的肽达到了预期的目的,也验证了我们的假设。5-TAMRA标记10肽在无疼痛模型、右腿疼痛模型、左腿疼痛模型的成像结果显示,我们可初步推断镇痛小肽在灌胃和腹腔注射两种给药方式中的镇痛机制具有差异性:镇痛小肽灌胃给药具有镇痛活性可能与胃肠道的特殊受体或离子通道相关;在腹腔给药后起到镇痛效应可能与背根神经节密切相关;标记小肽的荧光富集于疼痛部位,初步推测该镇痛小肽可能是主动镇痛。
[Abstract]:Pain is one of the most common signs of various diseases, which brings great suffering to patients and seriously affects their work and life. At present, drugs used in pain therapy (such as morphine, codeine, fentanyl, pethidine, sulinic acid, etc.) are almost addictive, dose-dependent, toxic and other adverse effects. Therefore, natural analgesic drugs bring hope to patients with pain. In the research of this kind of drugs, snake venom has been in the research hot spot all the time. The study shows that the neurotoxin of snake venom has good analgesic activity and has the advantages of no addiction, no dose dependence and long analgesic effect. Therefore, snake venom neurotoxin is a potential ideal material for the development of analgesic drugs such as less side effect and no addiction. However, the specific mechanism of its action has not yet been clarified. Previous studies have generally considered that the analgesic effect of snake venom is caused by its toxicity, but in recent years, some studies have shown that the analgesic effect of snake venom neurotoxin works through the central system, which is different from the toxic mechanism acting through the peripheral nervous system. In addition, some studies have shown that oral snake venom neurotoxin has analgesic effect, and found that some of the low neurotoxic peptides have significant analgesic activity, so we speculate that its analgesic active center and toxicity center may be two independent components. The analgesic component is not easily decomposed by pepsin. In this paper, the correlation between LD50 and analgesic dose of some snake venom neurotoxins with analgesic effect in the literature was analyzed, and it was further confirmed that the two were not completely consistent with each other. It is suggested that the analgesic active center and toxicity center of snake venom may be two independent regions. The results of our previous study showed that the short chain neurotoxin (short neurotoxin C/coborotoxin ctc) from Cobra chinensis has relatively weak neurotoxicity and significant oral analgesic activity, which means that the neurotoxin is independent. The analgesic effect of the active center is very strong, and the analgesic active center is resistant to pepsin hydrolysis. In view of this, the amino acid sequence and structure of CBTc were analyzed. According to the site of pepsin hydrolysis and its position in tertiary structure, a 10-peptide KDHRGTRIERA with 10 amino acids was designed by hot plate method and acetic acid writhing method. Formalin injection confirmed its analgesic activity, and its LD50 was 8.4g / kg ~ 95% confidence limit of 7.3-9.7g / kg, indicating that the toxicity of small peptide was very low or non-toxic, which proved that the analgesic active center and toxicity center of snake venom might be two independent components. According to the long chain neurotoxin also has analgesic activity, it is expected to have higher analgesic activity and longer analgesic time to modify the 10 peptide, including increasing the amino acid in front of Loop C, and expecting more comprehensive binding to target protein; The addition of 2 Cyss to the terminal is expected to naturally oxidize to form disulfide bonds and to increase stability and activity. The analgesic activity of modified peptide was determined by hot plate method and acetic acid writhing method. The results showed that the analgesic activity of 16 peptide was significantly higher than that of 13 peptide and 15 peptide, and both of them were higher than 10 peptide, which indicated that the designed peptide had achieved the desired purpose. We also tested our hypothesis. 5-TAMRA labeled 10 peptide in pain free model, right leg pain model, left leg pain model imaging results showed, We can infer preliminarily that the analgesic mechanism of analgesic small peptide in intragastric administration and intraperitoneal injection is different: the analgesic activity of analgesic small peptide may be related to the special receptor or ion channel in gastrointestinal tract; The analgesic effect after intraperitoneal administration may be closely related to the dorsal root ganglion, and the fluorescence of the labeled peptide is concentrated in the pain site, suggesting that the analgesic small peptide may be active analgesia.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

【参考文献】