發(fā)布時間：2018-08-15 13:48

【摘要】：抑制繼發(fā)性損傷和恢復(fù)神經(jīng)系統(tǒng)受損通路是治療脊髓損傷(Spinal cord injury,SCI)的基本策略。多聚唾液酸(Polysialic acid,PSA)是一種內(nèi)源性多糖聚合物,與神經(jīng)細(xì)胞黏附因子結(jié)合后,促進(jìn)細(xì)胞遷移、突觸形成、神經(jīng)元和髓鞘再生等,在中樞神經(jīng)系統(tǒng)的受損通路修復(fù)中起重要作用。本研究采用PSA與生物可降解材料聚己內(nèi)酯(Polycaprolactone,PCL)復(fù)合,選擇腎上腺皮質(zhì)激素藥物甲基潑尼松龍(Methylprednisolone,MP)為治療藥物,采用靜電紡絲技術(shù)分別制備PCL、PCL/MP、PCL/PSA、PCL/MP/PSA四種納米纖維三維支架。研究電紡參數(shù)(PCL濃度、溶劑配比、PSA濃度)對纖維形貌和直徑的影響,研究表明隨著PCL濃度的增加和高揮發(fā)性溶劑(丙酮)比例的上升,纖維串珠狀結(jié)構(gòu)減少、直徑增加,隨著PSA濃度的增加,纖維直徑減少,并確定PCL濃度150 mg/mL、PSA濃度15mg/mL、溶劑(N,N-二甲基甲酰胺:丙酮=1:1,v/v)為最理想的納米纖維三維支架參數(shù),在此參數(shù)下PCL、PCL/MP、PCL/PSA、PCL/MP/PSA四種納米纖維的直徑分別為 564.59 ±141.43 nm、512.85 ±69.85 nm、500.34 ±150.7 nm、421.09±57.4 nm。采用紅外分光光度法分析納米纖維三維支架組成,研究發(fā)現(xiàn)PCL、MP、PSA制備成膜后特征峰良好未發(fā)生明顯偏移,表明三種組分保持物理性混合。以pH 7.4 PBS為釋放介質(zhì)研究納米纖維三維支架的體外釋藥行為,研究發(fā)現(xiàn)PSA含量增加會加快MP釋放速率,PSA濃度15 mg/ml時PCL/MP/PSA納米纖維三維支架12 h內(nèi)MP累積釋放量達(dá)96%。以pH 7.4 PBS為介質(zhì)研究納米纖維三維支架的體外降解特性并采用掃描電子顯微鏡(Scanning electron telescope,SEM)觀察纖維形貌,研究發(fā)現(xiàn)納米纖維三維支架24h內(nèi)降解組分主要為MP和PSA,PCL/MP/PSA纖維表面由于MP和PSA降解出現(xiàn)大量空洞狀結(jié)構(gòu);PCL組分降解緩慢,可以長期維持支架結(jié)構(gòu)的穩(wěn)定,4w后部分纖維發(fā)生斷裂。以原代星形膠質(zhì)細(xì)胞為模型,分別采用四唑鹽(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide,MTT)比色法和 SEM研究納米纖維三維支架的細(xì)胞毒性和相容性,結(jié)果顯示細(xì)胞與PCL、PCL/MP、PCL/PSA、PCL/MP/PSA四種納米纖維三維支架共孵育2 d后細(xì)胞存活率均大于80%,7 d后細(xì)胞在PCL/MP/PSA納米纖維三維支架表面緊密黏附并深入深層孔隙結(jié)構(gòu)生長,表明納米纖維三維支架具有良好的生物安全性和細(xì)胞相容性。以雌性SD大鼠為模型動物,采用脊髓全橫斷法建立SCI模型并在脊髓缺損處移植納米纖維三維支架。選擇腫瘤壞死因子-α(Tumor necrosis factor-α,TNF-α)、白介素-6(Interleukin-6,IL-6)和細(xì)胞凋亡蛋白Caspase-3為檢測指標(biāo)研究納米纖維三維支架對脊髓繼發(fā)性損傷的抑制作用。術(shù)后24 h,PCL、PCL/PSA組與SCI組(創(chuàng)傷對照組)相比TNF-α、IL-6、Caspase-3含量無顯著性差異;而PCL/MP、PCL/MP/PSA組與SCI組相比TNF-α、IL-6、Caspase-3含量顯著降低,表明應(yīng)用MP治療脊髓損傷可抑制早期損傷部位TNF-α、IL-6和Caspase-3蛋白表達(dá),減輕繼發(fā)性損傷。以雌性SD大鼠為模型動物,采用脊髓全橫斷法建立SCI模型并在脊髓缺損處移植納米纖維三維支架。采用Basso,Beattie and Bresnahan(BBB)運動學(xué)功能評分考察脊髓損傷大鼠的運動功能。結(jié)果顯示,術(shù)后7w與SCI組相比,各治療組BBB評分均有顯著提高,PCL/MP/PSA組的運動學(xué)功能恢復(fù)狀況顯著優(yōu)于其他各治療組,表明MP和PSA聯(lián)合治療效果最佳。術(shù)后7 w,取脊髓斷端組織制備石蠟切片進(jìn)行LFB染色并采用透射電子顯微鏡(Transmission electron telescope,TEM)觀察組織超微結(jié)構(gòu),PCL/MP/PSA組髓鞘數(shù)目、直徑及板層狀結(jié)構(gòu)完整性顯著優(yōu)于其他各組。采用免疫組織化學(xué)法和免疫熒光法研究神經(jīng)元和NF200、GFAP蛋白表達(dá),PCL/MP/PSA組與其他各組相比神經(jīng)元數(shù)量和NF200含量顯著增加,GFAP含量顯著降低,表明MP和PSA聯(lián)合治療可以有效提高神經(jīng)元和軸突存活率,并抑制星形膠質(zhì)細(xì)胞反應(yīng)性增生。進(jìn)一步采用免疫熒光法研究Ibal蛋白表達(dá),PCL、PCL/PSA組與SCI組相比Ibal含量無顯著性差異;PCL/MP、PCL/MP/PSA組與SCI組相比Ibal含量顯著降低,應(yīng)用MP治療脊髓損傷可抑制損傷部位小膠質(zhì)細(xì)胞活化。本研究結(jié)果表明,PCL/MP/PSA納米纖維三維支架可以發(fā)揮MP和PSA的聯(lián)合治療效果,有效抑制脊髓損傷后急性炎癥反應(yīng)和細(xì)胞凋亡蛋白Caspase-3表達(dá),改善脫髓鞘化并抑制星形膠質(zhì)細(xì)胞活化增殖,提高神經(jīng)元和軸突生存率,最終改善脊髓損傷后大鼠的運動學(xué)功能,為組織工程支架應(yīng)用于脊髓損傷的修復(fù)治療提供新思路。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08

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