蜂毒明肽介导脊髓损伤靶向治疗的研究

发布时间：2018-07-01 19:12

本文选题：蜂毒明肽 + 姜黄素　；参考：《西南大学》2015年硕士论文

【摘要】：脊髓损伤(spinal cord injury, SCI)是一种由机械创伤、炎症及其他因素造成的脊髓病理损伤的疾病,是脊柱损伤中最严重的一种并发症。脊髓损伤往往导致损伤节段以下肢体严重的功能障碍,因此患者不得不接受长期甚至终身的治疗和护理。这不仅给患者本人带来身体和心理的严重伤害,还给患者家庭及整个社会造成了巨大的经济负担。尽管在脊髓损伤治疗过程中,我们已经在药学和手术治疗方面获得了一些经验,但是目前临床上对脊损损伤的治疗与康复仍然没有切实有效的治疗方案和药物,这对患者及其亲属乃至主治医生而言都是一个巨大的困扰。目前临床公认的用于治疗脊髓损伤的药物以甲基强的松龙(methylprednisolone, MP)为主。然而大量的临床经验表明甲基强的松龙对脊髓损伤的治疗效果有限,而其副作用却非常明显。此外,现在临床上使用的大剂量甲基强的松龙冲击疗法也受到了质疑。因为大剂量甲基强的松龙在治疗脊髓损伤的同时也会引起严重的感染甚至可以导致死亡。因此在脊髓损伤治疗方面我们亟需探索出更为有效的药物和治疗手段来满足临床需要。本研究的目的在于,构建以蜂毒明肽(apamin)为导向分子的主动靶向性递药系统治疗脊髓损伤,为中枢神经系统相关疾病的治疗提供理论依据和实验基础。本研究的内容主要包括：蜂毒明肽修饰的聚合物胶束的制备及表征和靶向聚合物胶束的体内评价两个方面。首先以中枢神经系统高亲和性的蜂毒明肽作为靶向分子,以两亲性高分子材料聚乙二醇-二硬脂酰乙醇胺(PEG-DSPE)作为药物载体材料,以具有抗炎、抗氧化药效的脂溶性化合物姜黄素(CUR)作为模型药物,选用以上三种临床上正在使用或者正在进行临床实验的成分来构建靶向胶束给药系统apamin-PM-CUR。然后,建立小鼠脊髓损伤模型,并从行为学、电生理学及组织学三个方面对治疗后造模小鼠恢复情况进行评价。最后对所构建的靶向胶束给药系统apamin-PM-CUR的安全性进行评估。具体实验方案及结果如下。蜂毒明肽修饰的聚合物胶束的制备及表征。首先利用蜂毒明肽的活性氨基基团和NHS活化PEG-DSPE之间的酰胺反应合成得到生物靶向材料apamin-PEG-DSPE,用核磁共振氢谱仪和生物质谱仪对产物进行结构鉴定和分子量测定。实验结果表明通过该法可以成功合成聚合物胶束靶向材料apamin-PEG-DSPE。然后以脂溶性的姜黄素作为模型药物,采用薄膜水化法制备apamin-PM-CUR口PM-CUR胶束。所制备胶束并测得胶束粒径可达50nm,大小均一,分散良好,包封率为79.11%。通过X-射线光电子能谱(X-PS)分析和X-射线粉末衍射(X-RD)分析实验可知,胶束表面确实有修饰的多肽存在,而且姜黄素在聚合物胶束中也完全包载。胶束在4℃放置3个月后,并未发现有姜黄素泄露,自身的粒径和分散性也无明显变化。体外释放度实验表明apamin-PM-CUR和PM-CUR在释放度上无明显区别,由此可见蜂毒明肽的修饰并不影响胶束的释药行为。蜂毒明肽胶束的体内评价。首先,采用Allen's垂直重物打击法建立小鼠脊髓损伤模型,通过BMS评分、MEP检测以及组织学观察结果证实造模成功。然后,在正式实验中将造模小鼠分为apamin-PM-CUR组、PM-CUR、甲基强的松龙组和生理盐水组连续给药7天,从行为学(BMS评分)、电生理学(MEP)、组织学(H E、HRP染色)3个水平对各组造模小鼠恢复情况进行评估。实验证明,PM-CUR组与生理盐水组情况类似小鼠恢复情况并不好。该组小鼠存活率低,仅存的少数小鼠直到给药后24周下肢仍然不能自由活动,MEP-N1潜伏期也大于10ms,说明该组小鼠神经通路受阻。通过组织切片可知该组小鼠损伤组织部位炎症严重,脊髓的结构完整性和功能完全性并未得到恢复。相反的apamin-PM-CUR组小鼠恢复良好,恢复效果甚至优于阳性对照MP组小鼠的状况。该组小鼠在给药后24周,小鼠存活率约为80%,而且下肢能够较为协调地运动,神经通路恢复良好,受损脊髓部位神经元再生较多,组织空洞减少,神经功能恢复较好。在安全性方面,靶向胶束中的apamin含量大约是apamin-PEG-DSPELD50的1/700,EEG实验可以证明在低于1 00mg/kg的给药剂量下,注射apamin-PEG-DSPE的小鼠脑电波和生理盐水组小鼠脑电图相似,振幅稳定、波动平稳。充分说明经PEG-DSPE定点修饰的apamin具有很好的安全性。综上所述,本文设计并制备了由生物多肽修饰的靶向递药系统,借助主动靶向递送策略,解决了药物不能足量进入脊髓损伤部位的问题,并通过多种途径,证明了该递药系统在体内能够表现出良好的药效和较理想的治疗指数。高效低毒是药物研究的长远目标,本研究工作可为脊髓损伤以及中枢神经系统相关疾病药物的研发提供实验经验和理论基础。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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