多聚唾液酸修饰聚己内酯纳米纤维三维支架的脊髓损伤治疗研究

发布时间：2018-08-15 13:48

【摘要】：抑制继发性损伤和恢复神经系统受损通路是治疗脊髓损伤(Spinal cord injury,SCI)的基本策略。多聚唾液酸(Polysialic acid,PSA)是一种内源性多糖聚合物,与神经细胞黏附因子结合后,促进细胞迁移、突触形成、神经元和髓鞘再生等,在中枢神经系统的受损通路修复中起重要作用。本研究采用PSA与生物可降解材料聚己内酯(Polycaprolactone,PCL)复合,选择肾上腺皮质激素药物甲基泼尼松龙(Methylprednisolone,MP)为治疗药物,采用静电纺丝技术分别制备PCL、PCL/MP、PCL/PSA、PCL/MP/PSA四种纳米纤维三维支架。研究电纺参数(PCL浓度、溶剂配比、PSA浓度)对纤维形貌和直径的影响,研究表明随着PCL浓度的增加和高挥发性溶剂(丙酮)比例的上升,纤维串珠状结构减少、直径增加,随着PSA浓度的增加,纤维直径减少,并确定PCL浓度150 mg/mL、PSA浓度15mg/mL、溶剂(N,N-二甲基甲酰胺:丙酮=1:1,v/v)为最理想的纳米纤维三维支架参数,在此参数下PCL、PCL/MP、PCL/PSA、PCL/MP/PSA四种纳米纤维的直径分别为 564.59 ±141.43 nm、512.85 ±69.85 nm、500.34 ±150.7 nm、421.09±57.4 nm。采用红外分光光度法分析纳米纤维三维支架组成,研究发现PCL、MP、PSA制备成膜后特征峰良好未发生明显偏移,表明三种组分保持物理性混合。以pH 7.4 PBS为释放介质研究纳米纤维三维支架的体外释药行为,研究发现PSA含量增加会加快MP释放速率,PSA浓度15 mg/ml时PCL/MP/PSA纳米纤维三维支架12 h内MP累积释放量达96%。以pH 7.4 PBS为介质研究纳米纤维三维支架的体外降解特性并采用扫描电子显微镜(Scanning electron telescope,SEM)观察纤维形貌,研究发现纳米纤维三维支架24h内降解组分主要为MP和PSA,PCL/MP/PSA纤维表面由于MP和PSA降解出现大量空洞状结构;PCL组分降解缓慢,可以长期维持支架结构的稳定,4w后部分纤维发生断裂。以原代星形胶质细胞为模型,分别采用四唑盐(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide,MTT)比色法和 SEM研究纳米纤维三维支架的细胞毒性和相容性,结果显示细胞与PCL、PCL/MP、PCL/PSA、PCL/MP/PSA四种纳米纤维三维支架共孵育2 d后细胞存活率均大于80%,7 d后细胞在PCL/MP/PSA纳米纤维三维支架表面紧密黏附并深入深层孔隙结构生长,表明纳米纤维三维支架具有良好的生物安全性和细胞相容性。以雌性SD大鼠为模型动物,采用脊髓全横断法建立SCI模型并在脊髓缺损处移植纳米纤维三维支架。选择肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白介素-6(Interleukin-6,IL-6)和细胞凋亡蛋白Caspase-3为检测指标研究纳米纤维三维支架对脊髓继发性损伤的抑制作用。术后24 h,PCL、PCL/PSA组与SCI组(创伤对照组)相比TNF-α、IL-6、Caspase-3含量无显著性差异;而PCL/MP、PCL/MP/PSA组与SCI组相比TNF-α、IL-6、Caspase-3含量显著降低,表明应用MP治疗脊髓损伤可抑制早期损伤部位TNF-α、IL-6和Caspase-3蛋白表达,减轻继发性损伤。以雌性SD大鼠为模型动物,采用脊髓全横断法建立SCI模型并在脊髓缺损处移植纳米纤维三维支架。采用Basso,Beattie and Bresnahan(BBB)运动学功能评分考察脊髓损伤大鼠的运动功能。结果显示,术后7w与SCI组相比,各治疗组BBB评分均有显著提高,PCL/MP/PSA组的运动学功能恢复状况显著优于其他各治疗组,表明MP和PSA联合治疗效果最佳。术后7 w,取脊髓断端组织制备石蜡切片进行LFB染色并采用透射电子显微镜(Transmission electron telescope,TEM)观察组织超微结构,PCL/MP/PSA组髓鞘数目、直径及板层状结构完整性显著优于其他各组。采用免疫组织化学法和免疫荧光法研究神经元和NF200、GFAP蛋白表达,PCL/MP/PSA组与其他各组相比神经元数量和NF200含量显著增加,GFAP含量显著降低,表明MP和PSA联合治疗可以有效提高神经元和轴突存活率,并抑制星形胶质细胞反应性增生。进一步采用免疫荧光法研究Ibal蛋白表达,PCL、PCL/PSA组与SCI组相比Ibal含量无显著性差异;PCL/MP、PCL/MP/PSA组与SCI组相比Ibal含量显著降低,应用MP治疗脊髓损伤可抑制损伤部位小胶质细胞活化。本研究结果表明,PCL/MP/PSA纳米纤维三维支架可以发挥MP和PSA的联合治疗效果,有效抑制脊髓损伤后急性炎症反应和细胞凋亡蛋白Caspase-3表达,改善脱髓鞘化并抑制星形胶质细胞活化增殖,提高神经元和轴突生存率,最终改善脊髓损伤后大鼠的运动学功能,为组织工程支架应用于脊髓损伤的修复治疗提供新思路。
[Abstract]:Polysialic acid (PSA) is an endogenous polysaccharide polymer that, when combined with neuronal adhesion factors, promotes cell migration, synapse formation, neuronal and myelin sheath regeneration in the central nervous system. In this study, PCL, PCL/MP, PCL/PSA and PCL/MP/PSA nanofibers were prepared by electrospinning with the combination of PSA and biodegradable polycaprolactone (PCL), methylprednisolone (MP) as a therapeutic drug. Three-dimensional scaffolds. The effects of electrospinning parameters (PCL concentration, solvent ratio, PSA concentration) on fiber morphology and diameter were studied. The results showed that with the increase of PCL concentration and the ratio of high volatile solvents (acetone) increasing, the bead-like structure of the fibers decreased and the diameter of the fibers increased. With the increase of PSA concentration, the diameter of the fibers decreased. The optimum scaffolding parameters of nanofibers were determined by the concentration of 15mg/mL and solvent (N,N-dimethylformamide:acetone=1:1,v/v). The diameters of PCL,PCL/MP,PCL/PSA,PCL/MP/PSA nanofibers were 564.59+141.43 nm,512.85+69.85 nm,500.34+150.7 nm,421.09+57.4 nm, respectively. Three-dimensional scaffolds were composed of PCL, MP and PSA. The results showed that the characteristic peaks of PCL, MP and PSA did not deviate significantly after preparation, indicating that the three components remained physically mixed. The cumulative release of MP in nanofiber scaffolds was 96% within 12 hours. The degradation characteristics of nanofiber scaffolds in vitro were studied by using pH 7.4 PBS as medium. The morphology of the scaffolds was observed by scanning electron microscopy (SEM). The degradation components of nanofiber scaffolds were mainly MP and PSA, PCL/MP/PSA fibers within 24 hours. The degradation of MP and PSA resulted in a large number of hollow structures on the surface; the degradation of PCL components was slow, which could maintain the stability of the scaffold structure for a long time, and some fibers broke after 4 weeks. Tetrazolium salt (3-(4,5-dimethylthiazolyl-2) - 2,5-diphenyltetrazolium bromide (MTT) colorimetry and SEM were used to study the nanoparticles respectively. The cytotoxicity and compatibility of the three-dimensional scaffolds showed that the cell viability was more than 80% after incubation with PCL, PCL/MP, PCL/PSA, PCL/MP/PSA three-dimensional scaffolds for 2 days. After 7 days, the cells adhered tightly to the surface of the three-dimensional scaffolds of PCL/MP/PSA nanofibers and grew deep into the pore structure, indicating the three-dimensional branches of nanofibers. SCI model was established in female SD rats by transection of the spinal cord and three-dimensional scaffolds were transplanted into the spinal cord defect. Tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and apoptotic protein Caspase-3 were selected. To investigate the inhibitory effect of three-dimensional nanofiber scaffolds on secondary spinal cord injury, there was no significant difference in the contents of TNF-a, IL-6 and Caspase-3 between PCL, PCL and PSA groups and SCI group (trauma control group) 24 hours after operation, while the contents of TNF-a, IL-6 and Caspase-3 in PCL/MP, PCL/MP/PSA groups were significantly lower than those in SCI group. It can inhibit the expression of TNF-alpha, IL-6 and Caspase-3 protein at the early stage of injury and alleviate the secondary injury. SCI model was established in female SD rats by transection of the spinal cord and three-dimensional scaffolds were transplanted into the spinal cord defect. Basso, Beattie and Bresnahan (BBB) kinematic function scores were used to investigate the movement of spinal cord injury rats. The results showed that the BBB scores of each treatment group were significantly higher than those of SCI group at 7 weeks after operation. The recovery of kinematics function of PCL/MP/PSA group was significantly better than that of other treatment groups, indicating that the combination of MP and PSA had the best therapeutic effect. The number, diameter and lamellar structural integrity of myelin sheath in PCL/MP/PSA group were significantly better than those in other groups. The expression of NF200 and GFAP in neurons and neurons was studied by immunohistochemistry and immunofluorescence. The number of neurons and NF200 content in PCL/MP/PSA group were significantly increased compared with other groups. In addition, GFAP content decreased significantly, indicating that MP and PSA combined treatment can effectively improve the survival rate of neurons and axons, and inhibit the reactive proliferation of astrocytes. The results showed that PCL/MP/PSA nanofiber three-dimensional scaffold could effectively inhibit the expression of caspase-3, ameliorate demyelination and inhibit astrocyte activation after spinal cord injury. Plasmacyte activation and proliferation can improve the survival rate of neurons and axons, and ultimately improve the motor function of rats after spinal cord injury.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08

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