聚碳酸亚丙酯—壳聚糖微球复合电纺丝载药系统在脊髓损伤修复中的应用研究

发布时间：2018-06-12 02:10

本文选题：聚碳酸亚丙酯 + 硫酸软骨素酶ABC　；参考：《山东大学》2016年博士论文

【摘要】：研究背景：脊髓损伤(SCI)后的再生修复和功能重建一直是医学界难以解决的问题。SCI在大多数国家的年发病率为20-40/100万,在我国随着经济的快速发展和交通运输效率的不断提高而呈逐年上升趋势。SCI后致残率高,常常导致截瘫等严重功能障碍,给患者及其家庭和社会带来极其沉重的经济和社会负担。一直以来,人们认为中枢神经系统(CNS)没有再生能力,自从Richardson在《Nature》上发表论文,中枢神经系统的再生能力才被人们逐渐认知。近年来的实验结果表明,CNS的再生修复能力与细胞的类型、发育程度以及细胞与损伤位置的距离有关,也与损伤后髓磷脂降解产生废物,离子稳态破坏,神经递质释放改变,递质受体功能障碍以及免疫炎性反应有关。同时,脊髓损伤往往会改变脊髓复杂的解剖结构,损害脊髓正常生理功能,所以损伤后恢复相应的解剖结构是功能恢复的必要前提。组织工程材料以其良好的力学性能及可塑性等优点逐渐成为脊髓损伤后再生修复的一个可能方向。众多研究表明,生物材料作为神经干细胞的载体,既可以承载神经营养因子等细胞因子,又可以为损伤后的脊髓提供力学支撑,减少创伤区域的瘢痕形成。理想的生物材料应具有良好的组织相容性,并且较少或完全不会引起其他不良反应；具有优秀的可塑造性及力学特性,以便于加工成为各种目标形态；若想承载细胞,需有良好的界面使细胞便于粘附生长；还应有稳定的或可调节的降解速率,以便适应不同细胞、营养因子发挥作用的时间；并且降解产物应无毒副作用。常见的组织工程材料主要有：(1)天然可降解聚合物；(2)人工合成可降解聚合物；(3)人工合成非可降解聚合物；(4)复合材料。这些材料的应用形式有支架、微球、电纺丝膜片、套管等。脊髓损伤后可应用的药物除了甲基强的松龙得到普遍的认可和使用外,其他药物尚未获得广泛认可或者由于其他的限制因素无法实际应用。目前有多种药物在实验中显示出了良好的效果,包括类固醇、阿片受体拮抗剂、抗脂质过氧化反应剂、神经生长因子、环磷酸腺苷(cAMP)和硫酸软骨素酶ABC (ChABC)等。本文研究探讨了可降解高分子生物材料聚碳酸亚丙酯(PPC)电纺丝和壳聚糖微球复合材料缓释载体的制作方法,以及其在脊髓损伤修复中应用的可行性,并研究了以该缓释材料为载体局部缓释不同药物对脊髓损伤后轴突再生及功能重建的作用。制作生物可降解载药高分子材料由于载药缓释材料良好的生物相容性,可降解性与持续释放药物等特点,其在脊髓损伤中的应用研究日渐增多。多数高分子可降解材料均在体内降解为酸性物质,有可能为脊髓损伤部位带来二次损伤,所以本研究选用了PPC,其可在体内最终降解为二氧化碳和水,且拥有很好的生物相容性；壳聚糖具有优异的生物相容性及可降解性,可以溶于水,为荷载蛋白类药物并保持其活性提供可靠保障。各缓释材料制作简述如下：制备单独载db-cAMP的PPC电纺丝:称量过的PPC原料和db-cAMP粉末放入30mm×50mm的小烧杯中,加入乙腈,搅拌6小时,得到乳白色均匀溶液。溶液中db-cAMP与PPC的质量比为1：9。利用静电纺丝机进行纺丝,使用接地的锡纸作为接收装置,得到单独载db-cAMP的电纺丝膜片。制备含ChABC的壳聚糖微球：称量过的壳聚糖(CS)粉末放入30mm×50mm的小烧杯中,加入1%乙酸水溶液,搅拌6小时,得到溶液A。准确称量聚乙烯毗咯烷酮(PVP)粉末,配制1%的PVP水溶液,微热搅拌3小时,放入4℃冰箱中,得到溶液B。取ChABC,加入上述PVP水溶液中,搅拌得到溶液C。将溶液C与溶液A混合,搅拌均匀后,得到溶液D。配置1%的多聚磷酸钠水溶液,将溶液D缓慢滴加到多聚磷酸钠水溶液中,滴完搅拌3小时,得到乳状浑浊液,将此浑浊液离心分离,离心力2000g,并用水洗两次,沉淀冻干,获得含有ChABC的CS微球。制备含有ChABC的电纺丝-微球和含有ChABC与db-cAMP的电纺丝-微球膜片：将得到的含有ChABC的CS微球定量加入二氯甲烷中,进行超声分散10分钟,然后加入PPC原料和db-cAMP粉末,搅拌至固体全部溶解,得到乳白色溶液。利用静电纺丝设备进行纺丝,使用接地的平整锡箔纸作为接收装置。喷头至锡箔纸距离为15cm,纺丝电压为10kV。收集到载药纤维,得到电纺丝-微球载药纤维复合膜；前法中不加db-cAMP粉末即得到只含ChABC的电纺丝-微球膜片。建立大鼠脊髓损伤模型本实验希望通过研究载药缓释材料在脊髓损伤中的应用,了解高分子缓释材料在脊髓损伤治疗中的应用可能性,并观察损伤后轴突的再生情况,观察再生轴突是否可以穿透胶质瘢痕。有鉴于此,我们选择了便于观察轴突再生的损伤模型-大鼠脊髓半横断模型。造模过程简述如下：雌性Wister大鼠(200-230g)腹腔注射麻醉后固定于手术台上,剃毛消毒后,充分暴露T7-T9脊椎棘突,咬除T8棘突,打开椎弓板,辨认后正中动脉,用11号刀垂直切入脊髓,半横断右侧脊髓,确认止血后,根据分组不同,分别植入相应的载药缓释材料,逐层缝合。术后给予20万U/天青霉素,2ml腹腔注射,每日一次,连续三天,术后对大鼠进行腹部按摩,帮助其排尿,每日三次,直至其恢复自主排尿。单独载db-cAMP电纺丝在大鼠脊髓半切模型中应用的可行性及其效果的研究损伤模型造好之后,载药材料植入横断损伤部位,缝合伤口,给予术后护理。在第1,2,3,4周各时间点采用BBB评分观察评价大鼠后肢运动功能恢复情况,并取材。免疫荧光以及免疫组化染色观察轴突再生、瘢痕形成、空洞形成等指标,用于评价各载药材料对大鼠脊髓半切损伤后轴突再生以及功能恢复的作用。单独载db-cAMP的生物可降解材料PPC电纺丝在大鼠脊髓半切损伤治疗的应用中,PPC和db-cAMP的混合溶液,做成电纺丝膜片。然后在体外模拟体内环境检测db-cAMP的释放数据,得到其释放曲线。结果显示,我们的电纺丝材料可以把db-cAMP的释放时间延长至8天,并且释放速度稳定。在SCI后第1,2,3,4周各时间点应用BBB评分量表对大鼠运动功能进行评价,而后借助免疫荧光和免疫组化技术对损伤区域的轴突再生和瘢痕形成进行研究。BBB结果显示植入载药电纺丝膜片组(实验组)大鼠功能恢复比对照组以及空白PPC组好,免疫组化和免疫荧光结果显示实验组大鼠有更多的轴突长入胶质瘢痕,轴突再生更活跃,并且损伤部位瘢痕更薄。实验结果证明PPC载药电纺丝膜片可以在大鼠脊髓损伤的治疗中应用,为脊髓损伤的治疗提供了一个新的给药选择。联合db-cAMP和ChABC材料与单独用药的效果比较研究我们在PPC电纺丝载药材料的基础上,引入了CS微球,把蛋白类药物ChABC和静电纺丝联合起来,拓宽了蛋白类药物在脊髓损伤治疗中的应用渠道,也增加了脊髓损伤的治疗手段。再次验证了生物可降解高分子材料PPC电纺丝和壳聚糖微球载药体系在脊髓损伤局部缓释药物的可行性。同时验证了小分子化学药物db-cAMP和大分子蛋白类药物ChABC在脊髓损伤局部缓释的作用。实验证明,上述两种单药应用都各自具有一定效果,但促进轴突再生和功能恢复的作用并非十分显著。考虑到脊髓损伤修复障碍由不同因素构成,因此本实验进行药物联合应用,研究电纺丝-微球载药系统同时荷载两种药物的可行性,验证其联合应用时在脊髓损伤的再生修复方面有无协同作用,在缓释方面有无相互影响,并分别和单独给药相比较。检测单独载药和联合载药材料中药物的体外释放曲线,结果显示,与单独载药时相比较,联合载药时的药物释放曲线并无明显差别。在损伤后1,2,3,4周各时间点用BBB评分法对大鼠运动功能进行评分,并应用免疫荧光和免疫组化观察轴突再生和瘢痕形成。结果证实,各实验组中轴突再生以及运动功能恢复均优于对照组；载有ChABC的材料组,瘢痕壁不连续,瘢痕疏松；单独缓释db-cAMP组和单独ChABC组在功能恢复方面无显著性差异；联合用药组的轴突再生以及运动功能恢复情况均好于单独给药组,组间BBB评分有显著性差异(p≤0.05)本研究的主要结论：1.可降解PPC电纺丝以及壳聚糖微球拥有很好的生物组织相容性以及局部缓释药物能力,并且可以通过调节电纺丝的直径、拉伸率、断裂伸长率和壳聚糖微球直径等物理特性调整其降解速度以达到优化药物缓释的目的。2.载有双丁酰环磷酸腺苷(dibutyryl cyclic adenosine monophosphate, db-cAMP)的PPC电纺丝材料可以促进大鼠SCI后轴突再生、运动功能恢复、减少胶质瘢痕形成。3.载有硫酸软骨素酶ABC (ChABC)的壳聚糖微球复合PPC电纺丝材料可以很好地缓释ChABC,阻止空洞内瘢痕壁形成,使轴突更容易通过损伤区域,促进大鼠脊髓损伤后运动功能恢复。4.联合载ChABC 和 db-cAMP的电纺丝-微球材料与缓释单一药物材料相比进一步改善了SCI后大鼠的运动功能,使损伤区域残存神经纤维增多、胶质瘢痕与空洞接触面变得不规则,瘢痕减少。实验结果表明,PPC电纺丝和壳聚糖微球复合材料缓释载体可稳定地在脊髓损伤局部释放治疗药物,并且其降解和缓释药物的速度可调控。该复合材料可为神经系统局部缓释药物治疗提供良好的载体,并能为CNS损伤修复、抗肿瘤治疗及退行性疾病等领域的研究提供新的研究平台。本实验结果显示该复合材料缓释载体具有良好的临床应用前景。
[Abstract]:Background: regenerative repair and functional reconstruction after spinal cord injury (SCI) have been a problem that is difficult to solve in the medical field. The annual incidence of.SCI in most countries is 20-40/100 million. In China, with the rapid development of economy and the increasing of transportation efficiency, the rate of disability is high after the year of.SCI, and it often leads to paraplegia and so on. Heavy dysfunction has brought extremely heavy economic and social burdens on patients and their families and society. It has been thought that the central nervous system (CNS) has no regenerative capacity. Since Richardson has published papers on , the regenerative ability of the central nervous system has been gradually recognized. In recent years, the experimental results show that the CNS is reproduced. The ability of biorepair is related to the type of cell, the degree of development and the distance between the cell and the location of the injury. It is also related to the degradation of myelin after injury, the destruction of the ions, the change of neurotransmitter release, the dysfunction of the transmitter receptor and the inflammatory response. Spinal cord normal physiological function, so recovery of the corresponding anatomical structure after injury is a necessary prerequisite for functional recovery. Tissue engineering materials, with its good mechanical properties and plasticity, have gradually become a possible direction for regenerative repair after spinal cord injury. Many studies have shown that biological materials can be used as the carrier of neural stem cells. Cell factors such as neurotrophic factors can also provide mechanical support for the injured spinal cord and reduce scar formation in the trauma area. Ideal biomaterials should have good histocompatibility, and less or completely do not cause other adverse reactions. Target morphology; in order to carry cells, a good interface is needed to make cells easy to adhere to growth; there should also be a stable or adjustable degradation rate to adapt to different cells, the time for the function of nutrient factors to play; and the degradation products should have no side effects. (1) natural biodegradable polymers; 2) synthetic biodegradable polymers; (3) artificial synthesis of non degradable polymers; (4) composite materials. The applications of these materials include scaffolds, microspheres, electrospun diaphragms, cannula and other drugs. Other drugs that can be applied after spinal cord injury are widely recognized and used, except for methyl strong pine. Other restrictive factors are not practical. A variety of drugs have shown good results in the experiment, including steroids, opioid receptor antagonists, anti lipid peroxidation, nerve growth factors, cAMP and ABC (ChABC). This paper studies the polymerization of biodegradable polymer biomaterials. The preparation of propyl carbonate (PPC) electrospun and chitosan microspheres composite materials and the feasibility of its application in the repair of spinal cord injury, and the effect of local sustained release drugs on spinal cord regeneration and functional reconstruction after spinal cord injury by using the sustained release material as carrier. Due to the good biocompatibility, biodegradability and sustained release drug, the drug release material has been applied in the spinal cord injury. Most of the biodegradable materials are degraded into acidic substances in the body and may cause two damage to the spinal cord injury. Therefore, PPC is selected in this study. The final degradation is carbon dioxide and water, and has good biocompatibility; chitosan has excellent biocompatibility and biodegradability, can dissolve in water, provide a reliable guarantee for the load protein drugs and maintain its activity. The preparation of each release material is as follows: the preparation of the single loaded db-cAMP PPC electrospun: the weighing PPC raw material In a small beaker of 30mm x 50mm with db-cAMP powder, adding acetonitrile and stirring for 6 hours, a homogeneous solution of milk white is obtained. The mass ratio of db-cAMP to PPC in the solution is spinning with 1:9. electrospun machine and the earthing tin paper is used as the receiving device to obtain a single db-cAMP containing electrospun film. The preparation of chitosan microspheres containing ChABC is called. The excess chitosan (CS) powder was put into the small beaker of 30mm x 50mm, adding 1% acetic acid water solution, stirring for 6 hours, getting the solution A. to accurately weigh the polyvinylpyrrolidone (PVP) powder, preparing 1% PVP water solution, stirring for 3 hours, and putting the solution B. to ChABC in the 4 centigrade refrigerator, and stirring to get the solution C. to dissolve in the solution. The mixture of liquid C and solution A is mixed, and after mixing, the solution of polyphosphate sodium polyphosphate solution D. configuration 1% is obtained, and the solution D is slowly dripped into the sodium polyphosphate solution. After stirring for 3 hours, the turbidity liquid is obtained. The turbidity liquid is centrifuged and centrifuged for 2000g, and the CS microspheres containing ChABC are obtained by washing two times and precipitating the freeze-drying. The preparation contains C. HABC electrospun microspheres and electrospun microsphere diaphragm containing ChABC and db-cAMP: the CS microspheres containing ChABC were added into dichloromethane for 10 minutes, then PPC raw materials and db-cAMP powders were added to the solid solution to get a milk white solution. The leveling tin foil is used as the receiving device. The distance of the spray head to the foil paper is 15cm, the spinning voltage is 10kV., and the electrospun microsphere carrier fiber composite membrane is obtained. The electrospun microsphere film containing only ChABC is obtained without db-cAMP powder in the former method. The application of sustained-release materials in spinal cord injury is used to understand the possibility of the application of macromolecule sustained-release materials in the treatment of spinal cord injury, and to observe the regeneration of the axons after injury and to observe whether the regeneration axons can penetrate glial scar. The model process is briefly described as follows: female Wister rat (200-230g) is fixed on the operating table after intraperitoneal injection of anesthesia. After shaving is sterilized, the spinal spinous process is fully exposed, T8 spinous process is bitten, the vertebral arch plate is opened, the posterior median artery is identified, the spinal cord is cut through the spinal cord with No. 11 knife and the right side of the spinal cord is semi transected. After the hemostasis is confirmed, the corresponding groups are implanted the corresponding according to the different grouping and implantation, respectively. The drug sustained-release material was sutured by layer by layer. 200 thousand U/ days postoperatively, penicillin and 2ml were injected intraperitoneally, once a day for three days. After the operation, the rats were massaged in the abdomen to help their urination, three times a day, until their spontaneous urination was restored. The feasibility and effect of the application of db-cAMP electrospun in the rat spinal cord semi cut model were studied. After the injury model was built, the drug loaded materials were implanted into the damaged parts of the transection, suturing the wound and giving the postoperative nursing. The recovery of the motor function of the hind limbs of the rats was evaluated by BBB score at the time point of the 1,2,3,4 week, and the materials were obtained. Immunofluorescence and immunohistochemical staining were used to observe the axon regeneration, scar formation, cavity formation and so on. Effect of drug loading materials on axonal regeneration and functional recovery after spinal cord hemimaxulic injury in rats. A single db-cAMP biodegradable material PPC electrospun was used in the treatment of spinal cord hemi cut injury in rats. A mixed solution of PPC and db-cAMP was made into an electrospun film. Then the release data of db-cAMP were detected in an in vitro model in vivo. The release curves show that our electrospun materials can prolong the release time of db-cAMP to 8 days, and the release rate is stable. The motor function of rats is evaluated by BBB scale at every time point of SCI after 1,2,3,4 week, and then the axon regeneration and scar formation in the damaged area by immunofluorescence and immunohistochemical technique. The results of the study of.BBB showed that the functional recovery of the electrospun film group (experimental group) was better than the control group and the blank PPC group. The results of immunohistochemistry and immunofluorescence showed that the experimental group had more axon growth into the glial scar, the axon regeneration was more active, and the scar was thinner. The experimental results proved that PPC was loaded with medicine. The spun film can be used in the treatment of spinal cord injury in rats, providing a new choice for the treatment of spinal cord injury. Comparison of the effects of db-cAMP and ChABC and the effect of the single drug use. On the basis of the PPC electrospun drug loading materials, we introduced CS microspheres, combined the egg white drugs ChABC and electrostatic spinning. The application channels of protein drugs in the treatment of spinal cord injury also increase the treatment of spinal cord injury. The feasibility of the biodegradable polymer PPC electrospun and chitosan microsphere drug delivery system in the local release of spinal cord injury is verified again. Small molecular chemical drugs, db-cAMP and macromolecule proteins, are verified. The effect of drug ChABC on local sustained release of spinal cord injury. Experiments have shown that these two single drug applications have their own effect, but the role of promoting axonal regeneration and functional recovery is not very significant. Considering that the repair of spinal cord injury is made up of different factors, the combined application of drugs in this experiment is to study electrospun microsphere loading. The feasibility of the simultaneous loading of two drugs to verify the synergistic effect of the combined application of the drug on the regeneration and repair of spinal cord injury. There was no significant difference in the drug release curve of the combined drug loading. The motor function of rats was scored by BBB scoring at 1,2,3,4 weeks after injury, and the axon regeneration and scar formation were observed by immunofluorescence and immunohistochemistry. The results showed that the regeneration of axon and the recovery of motor function in the experimental groups were all better than those of the control group. In the material group carrying ChABC, the scar wall was discontinuous and the scar was loose; there was no significant difference in functional recovery between the single release db-cAMP group and the single ChABC group. The axon regeneration and the motor function recovery of the combined drug group were better than the individual administration group, and the BBB score between the groups was significantly different (P < 0.05). The main conclusions of this study were: 1. Biodegradable PPC electrospun and chitosan microspheres have good biocompatibility and local sustained release drug ability, and can adjust the degradation rate by adjusting the diameter, tensile rate, elongation at break and diameter of chitosan microspheres to achieve the goal of.2. containing dibutylopp. The PPC electrospun material of dibutyryl cyclic adenosine monophosphate (db-cAMP) can promote the regeneration of axon, recovery of motor function after SCI, and reduce the formation of.3. containing chondroitinase ABC (ChABC) containing ABC (ChABC) and PPC electrospun material, which can prevent the formation of cicatricial walls in the cavity. It makes the axon easier to pass through the damaged area, promoting the motor function after the spinal cord injury in rats to restore the motor function of.4. combined with ChABC and db-cAMP, and further improve the motor function of the rats after SCI compared with the sustained release single drug material, and increase the residual nerve fiber in the damaged area, and the glial scar and the cavity contact surface becomes irregular. The results show that the PPC electrospun and Chitosan Microsphere Composite sustained-release carrier can release the drugs locally in the spinal cord injury, and the rate of its degradation and sustained release drugs can be regulated. The composite can provide a good carrier for the local sustained release drug treatment of the nervous system, and can repair the CNS damage and resist the damage. The study of tumor treatment and degenerative diseases provides a new research platform. The results of this experiment show that the composite sustained-release carrier has good prospects for clinical application.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R651.2

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