本文選題：蜂毒明肽 + 姜黃素��； 參考：《西南大學》2015年碩士論文

【摘要】：脊髓損傷(spinal cord injury, SCI)是一種由機械創(chuàng)傷、炎癥及其他因素造成的脊髓病理損傷的疾病,是脊柱損傷中最嚴重的一種并發(fā)癥。脊髓損傷往往導致?lián)p傷節(jié)段以下肢體嚴重的功能障礙,因此患者不得不接受長期甚至終身的治療和護理。這不僅給患者本人帶來身體和心理的嚴重傷害,還給患者家庭及整個社會造成了巨大的經(jīng)濟負擔。盡管在脊髓損傷治療過程中,我們已經(jīng)在藥學和手術(shù)治療方面獲得了一些經(jīng)驗,但是目前臨床上對脊損損傷的治療與康復仍然沒有切實有效的治療方案和藥物,這對患者及其親屬乃至主治醫(yī)生而言都是一個巨大的困擾。目前臨床公認的用于治療脊髓損傷的藥物以甲基強的松龍(methylprednisolone, MP)為主。然而大量的臨床經(jīng)驗表明甲基強的松龍對脊髓損傷的治療效果有限,而其副作用卻非常明顯。此外,現(xiàn)在臨床上使用的大劑量甲基強的松龍沖擊療法也受到了質(zhì)疑。因為大劑量甲基強的松龍在治療脊髓損傷的同時也會引起嚴重的感染甚至可以導致死亡。因此在脊髓損傷治療方面我們亟需探索出更為有效的藥物和治療手段來滿足臨床需要。本研究的目的在于,構(gòu)建以蜂毒明肽(apamin)為導向分子的主動靶向性遞藥系統(tǒng)治療脊髓損傷,為中樞神經(jīng)系統(tǒng)相關(guān)疾病的治療提供理論依據(jù)和實驗基礎(chǔ)。本研究的內(nèi)容主要包括：蜂毒明肽修飾的聚合物膠束的制備及表征和靶向聚合物膠束的體內(nèi)評價兩個方面。首先以中樞神經(jīng)系統(tǒng)高親和性的蜂毒明肽作為靶向分子,以兩親性高分子材料聚乙二醇-二硬脂酰乙醇胺(PEG-DSPE)作為藥物載體材料,以具有抗炎、抗氧化藥效的脂溶性化合物姜黃素(CUR)作為模型藥物,選用以上三種臨床上正在使用或者正在進行臨床實驗的成分來構(gòu)建靶向膠束給藥系統(tǒng)apamin-PM-CUR。然后,建立小鼠脊髓損傷模型,并從行為學、電生理學及組織學三個方面對治療后造模小鼠恢復情況進行評價。最后對所構(gòu)建的靶向膠束給藥系統(tǒng)apamin-PM-CUR的安全性進行評估。具體實驗方案及結(jié)果如下。蜂毒明肽修飾的聚合物膠束的制備及表征。首先利用蜂毒明肽的活性氨基基團和NHS活化PEG-DSPE之間的酰胺反應(yīng)合成得到生物靶向材料apamin-PEG-DSPE,用核磁共振氫譜儀和生物質(zhì)譜儀對產(chǎn)物進行結(jié)構(gòu)鑒定和分子量測定。實驗結(jié)果表明通過該法可以成功合成聚合物膠束靶向材料apamin-PEG-DSPE。然后以脂溶性的姜黃素作為模型藥物,采用薄膜水化法制備apamin-PM-CUR口PM-CUR膠束。所制備膠束并測得膠束粒徑可達50nm,大小均一,分散良好,包封率為79.11%。通過X-射線光電子能譜(X-PS)分析和X-射線粉末衍射(X-RD)分析實驗可知,膠束表面確實有修飾的多肽存在,而且姜黃素在聚合物膠束中也完全包載。膠束在4℃放置3個月后,并未發(fā)現(xiàn)有姜黃素泄露,自身的粒徑和分散性也無明顯變化。體外釋放度實驗表明apamin-PM-CUR和PM-CUR在釋放度上無明顯區(qū)別,由此可見蜂毒明肽的修飾并不影響膠束的釋藥行為。蜂毒明肽膠束的體內(nèi)評價。首先,采用Allen's垂直重物打擊法建立小鼠脊髓損傷模型,通過BMS評分、MEP檢測以及組織學觀察結(jié)果證實造模成功。然后,在正式實驗中將造模小鼠分為apamin-PM-CUR組、PM-CUR、甲基強的松龍組和生理鹽水組連續(xù)給藥7天,從行為學(BMS評分)、電生理學(MEP)、組織學(H E、HRP染色)3個水平對各組造模小鼠恢復情況進行評估。實驗證明,PM-CUR組與生理鹽水組情況類似小鼠恢復情況并不好。該組小鼠存活率低,僅存的少數(shù)小鼠直到給藥后24周下肢仍然不能自由活動,MEP-N1潛伏期也大于10ms,說明該組小鼠神經(jīng)通路受阻。通過組織切片可知該組小鼠損傷組織部位炎癥嚴重,脊髓的結(jié)構(gòu)完整性和功能完全性并未得到恢復。相反的apamin-PM-CUR組小鼠恢復良好,恢復效果甚至優(yōu)于陽性對照MP組小鼠的狀況。該組小鼠在給藥后24周,小鼠存活率約為80%,而且下肢能夠較為協(xié)調(diào)地運動,神經(jīng)通路恢復良好,受損脊髓部位神經(jīng)元再生較多,組織空洞減少,神經(jīng)功能恢復較好。在安全性方面,靶向膠束中的apamin含量大約是apamin-PEG-DSPELD50的1/700,EEG實驗可以證明在低于1 00mg/kg的給藥劑量下,注射apamin-PEG-DSPE的小鼠腦電波和生理鹽水組小鼠腦電圖相似,振幅穩(wěn)定、波動平穩(wěn)。充分說明經(jīng)PEG-DSPE定點修飾的apamin具有很好的安全性。綜上所述,本文設(shè)計并制備了由生物多肽修飾的靶向遞藥系統(tǒng),借助主動靶向遞送策略,解決了藥物不能足量進入脊髓損傷部位的問題,并通過多種途徑,證明了該遞藥系統(tǒng)在體內(nèi)能夠表現(xiàn)出良好的藥效和較理想的治療指數(shù)。高效低毒是藥物研究的長遠目標,本研究工作可為脊髓損傷以及中樞神經(jīng)系統(tǒng)相關(guān)疾病藥物的研發(fā)提供實驗經(jīng)驗和理論基礎(chǔ)。
[Abstract]:Spinal cord injury (SCI) is a pathological injury of the spinal cord caused by mechanical trauma, inflammation, and other factors. It is the most serious complication of spinal injury. Spinal cord injury often leads to severe impairment of the following segment of the segment, so the patient has to receive long-term or even life-long treatment and care. This not only brings serious physical and psychological harm to the patient, but also gives a huge economic burden to the family and the whole society. Although we have gained some experience in pharmacy and surgical treatment in the process of spinal cord injury treatment, there is still no practical treatment and rehabilitation for spinal damage. Effective treatments and drugs are a great problem for patients and their relatives and even to the doctor. Currently, the most clinically recognized drugs for the treatment of spinal cord injury are methylprednisolone (MP). However, a large number of clinical experiences have shown that methylprednisolone has a therapeutic effect on spinal cord injury. It is limited, and its side effects are very obvious. In addition, large doses of methylprednisolone in clinical use are now questioned. Because large doses of methylprednisolone can cause severe infection and even lead to death in the treatment of spinal cord injury. Therefore, we need to explore more for the treatment of spinal cord injury. The purpose of this study is to provide a theoretical basis and experimental basis for the treatment of spinal cord related diseases by constructing an active targeted drug delivery system with apamin as a guide molecule and to provide theoretical basis and experimental basis for the treatment of central nervous system related diseases. The preparation and characterization of polymer micelles and the evaluation of the target polymer micelles in vivo two aspects. First, the high affinity of the central nervous system was used as a target molecule, and the two amphiphilic polymer material polyethylene glycol two stearyl ethanolamine (PEG-DSPE) was used as the drug carrier material, with the anti inflammatory and antioxidant effect of lipid solution. As a model drug, the sexual compound curcumin (CUR) is used to construct the targeted micellar drug delivery system (apamin-PM-CUR.) by using the three kinds of clinical components that are being used or undergoing clinical trials. Then, the model of mouse spinal cord injury is established and the recovery of the mice after treatment is obtained from three aspects of behavior, electrophysiology and histology. In the end, the safety of the targeted micellar drug delivery system apamin-PM-CUR was evaluated. The specific experimental scheme and results were as follows. The preparation and characterization of the polymer micelles modified by the bee venom peptide. First, the biological target material a was synthesized by the active amino group of the melittin and the amide reaction between the activated PEG-DSPE and the amido. Pamin-PEG-DSPE, the structure and molecular weight of the products were identified by nuclear magnetic resonance spectroscopy and biological mass spectrometry. The results showed that the polymer micelle targeted material apamin-PEG-DSPE. could be successfully synthesized by this method and then liposoluble curcumin was used as a model drug, and apamin-PM-CUR mouth PM-CUR micelles were prepared by the film hydration method. The micelles were prepared and the size of the micelles was up to 50nm, the size was uniform, and the encapsulation efficiency was good. The encapsulation efficiency was 79.11%. through X- ray photoelectron spectroscopy (X-PS) analysis and X- ray powder diffraction (X-RD) analysis experiments. It was found that the surface of the micelle had a modified polypeptide and Jiang Huang was completely loaded in the polymer micelles. The micelles were placed at 4 degrees centigrade. After month, no curcumin leakage was found, and there was no obvious change in its particle size and dispersity. In vitro release test showed that there was no obvious difference between apamin-PM-CUR and PM-CUR in the release degree. The mouse spinal cord injury model was established by the attack method. The model was successfully established by BMS score, MEP detection and histological observation. Then, the model mice were divided into apamin-PM-CUR group, PM-CUR, methylprednisolone group and saline group were given 7 days continuously, from BMS score, electrophysiology (MEP), histology (H E, HRP). 3 levels were used to evaluate the recovery of mice in each group. The experiment proved that the condition of the PM-CUR group and the saline group was similar to that of the mice. The survival rate of the mice in this group was low, and the only surviving mice were still unable to move freely until 24 weeks after the administration, and the latent period of the MEP-N1 was greater than that of 10ms. The tissue integrity and functional completeness of the spinal cord were not recovered by tissue section. In contrast, the mice in the apamin-PM-CUR group recovered well and were better than the positive control group MP mice. The mice had a survival rate of about 80% after 24 weeks of administration. The lower extremities can be more coordinated, the nerve pathway is well restored, the neurons in the damaged spinal cord are regenerated more, the tissue cavity is reduced, and the nerve function is recovered well. In the safety, the apamin content in the targeted micelle is about apamin-PEG-DSPELD50 1/700, and the EEG experiment can prove that the injection of apamin under the dosage of less than 1 00mg/kg can be proved to be an injection of apamin. The electroencephalogram (EEG) of mice in -PEG-DSPE was similar to that of the saline group in the normal saline group. The amplitude was stable and the fluctuation was stable. It was fully explained that the PEG-DSPE fixed-point modified apamin was very safe. In summary, the target delivery system modified by biopeptides was designed and prepared to help the active target delivery strategy to solve the drug failure. It is proved that the delivery system can show good efficacy and ideal therapeutic index through various ways. High efficiency and low toxicity is a long-term goal of drug research. This study can provide experimental experience for the development of spinal cord injury and central nervous system related diseases. Theoretical basis.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R651.2

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本文編號：2088854