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  本文選題：蛇毒神經(jīng)毒素 + 鎮(zhèn)痛活性中心�。� 參考：《昆明理工大學(xué)》2017年碩士論文

【摘要】：疼痛是各種疾病中最為常見的信號之一,給患者帶來極大的痛苦,嚴(yán)重影響他們的工作和生活。目前,臨床用于疼痛治療的藥物(如嗎啡、可待因、芬太尼、哌替啶、舒林酸等)幾乎均具有成癮性、劑量依賴性、毒性等不良效應(yīng)。因此天然類鎮(zhèn)痛藥物給疼痛患者帶來了希望。在這類藥物的研究中,蛇毒一直處于研究的熱點。研究表明蛇毒神經(jīng)毒素的鎮(zhèn)痛活性良好,且具有無成癮性、無劑量依賴性、鎮(zhèn)痛時效長等優(yōu)點,故蛇毒神經(jīng)毒素是開發(fā)副作用小、無成癮性等鎮(zhèn)痛藥物的潛在理想原料,但對其具體的作用機制尚未明確。以往的研究一般認(rèn)為蛇毒的鎮(zhèn)痛效應(yīng)是由其毒性引起的,但近些年有研究表明蛇毒神經(jīng)毒素的鎮(zhèn)痛效應(yīng)是通過中樞系統(tǒng)起效,與經(jīng)外周神經(jīng)系統(tǒng)起作用的毒性機制截然不同。另外,有研究表明口服蛇毒神經(jīng)毒素具有鎮(zhèn)痛效應(yīng),而且發(fā)現(xiàn)其中一些低神經(jīng)毒性多肽具有顯著的鎮(zhèn)痛活性,故我們推測其鎮(zhèn)痛活性中心與毒性中心可能是兩個獨立的組分,且鎮(zhèn)痛組分不易被胃蛋白酶所分解。本文首先對文獻中部分具有鎮(zhèn)痛效應(yīng)的蛇毒神經(jīng)毒素的LD50和鎮(zhèn)痛起效劑量的相關(guān)性作了分析,進一步確認(rèn)兩者間并非完全一致,表明蛇毒鎮(zhèn)痛活性中心與毒性中心確有可能是兩個獨立區(qū)域。課題組前期的研究結(jié)果表明,來自中華眼鏡蛇的短鏈神經(jīng)毒蛋白short neurotoxin C/coborotoxin c(CBTc)具有相對很弱的神經(jīng)毒性和顯著的口服鎮(zhèn)痛活性,意味著該神經(jīng)毒素具有獨立的、鎮(zhèn)痛效應(yīng)很強的活性中心,且該鎮(zhèn)痛活性中心耐胃蛋白酶水解。鑒于此,對CBTc的氨基酸序列和結(jié)構(gòu)進行分析,根據(jù)胃蛋白酶水解位點及其在三級結(jié)構(gòu)上所處的位置,設(shè)計了具有10個氨基酸的10肽(KDHRGTRIER),用熱板法、醋酸扭體法、福爾馬林注射法驗證其確有鎮(zhèn)痛活性,其LD50為8.4 g/kg,95%可信限為7.3-9.7g/kg,表明小肽的毒性很低或無毒性,進而驗證了蛇毒鎮(zhèn)痛活性中心與毒性中心可能是兩個獨立組分的推測。根據(jù)長鏈神經(jīng)毒素也具有鎮(zhèn)痛活性,故對10肽作相應(yīng)改造,期望有更高的鎮(zhèn)痛活性和更長的鎮(zhèn)痛時效:包括增加Loop C前端氨基酸,預(yù)期能更全面結(jié)合靶蛋白;末端增加2個Cys,預(yù)期能自然氧化形成二硫鍵,能增加穩(wěn)定性和活性。經(jīng)熱板法和醋酸扭體法測定改造肽的鎮(zhèn)痛活性,結(jié)果顯示16肽的鎮(zhèn)痛活性明顯高于13肽和15肽,且均高于10肽,表明設(shè)計的肽達到了預(yù)期的目的,也驗證了我們的假設(shè)。5-TAMRA標(biāo)記10肽在無疼痛模型、右腿疼痛模型、左腿疼痛模型的成像結(jié)果顯示,我們可初步推斷鎮(zhèn)痛小肽在灌胃和腹腔注射兩種給藥方式中的鎮(zhèn)痛機制具有差異性:鎮(zhèn)痛小肽灌胃給藥具有鎮(zhèn)痛活性可能與胃腸道的特殊受體或離子通道相關(guān);在腹腔給藥后起到鎮(zhèn)痛效應(yīng)可能與背根神經(jīng)節(jié)密切相關(guān);標(biāo)記小肽的熒光富集于疼痛部位,初步推測該鎮(zhèn)痛小肽可能是主動鎮(zhèn)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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R91

【參考文獻】

相關(guān)期刊論文 前1條

1 Bo-wenCHEN;RongHAN;PaulFREID;LaurenceNRAYMOND;;A long-form α-neurotoxin from cobra venom produces potent opioid-independent analgesia[J];Acta Pharmacologica Sinica;2006年04期

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本文編號：1809510