【作者】 黄涛；

【导师】 赵洪洋；

【作者基本信息】 华中科技大学 ， 外科学（神经外科）， 2019， 博士

【摘要】 第一部分以柠檬酸作为交联剂构建壳聚糖/柠檬酸水凝胶目的:脊髓损伤是一项困扰世界多年的医学难题,至今仍无令人满意的治疗方式。神经支架可以改善脊髓损伤部位微环境,同时可以为新生轴突提供物理指引,是一种非常具有潜力的治疗方式,引起学者们的强烈关注。壳聚糖是一种天然蛋白多糖,具有组织相容性好,可降解等优势,是一种性能优异的生物材料,但它的理化性能限制了它的应用。有鉴于此,我们引入柠檬酸作为交联剂,希望以此增强壳聚糖水凝胶的强度,进而可以用于构建神经修复支架。方法:3 wt%壳聚糖溶液分别与0.3 wt%,0.33 wt%和0.38 wt%柠檬酸溶液发生反应,采用冷冻干燥法反复冻融,最终形成不同比例的壳聚糖/柠檬酸水凝胶。将不同比例的壳聚糖/柠檬酸溶液加入特制模具中形成神经损伤修复支架供动物学实验使用。使用傅立叶变换红外光谱分析法检测样本了解壳聚糖/柠檬酸的化学结构和分子相互作用。使用扫描电子显微镜分析法检测样品结构和表征。结果:3 wt%壳聚糖溶液分别与0.3 wt%,0.33 wt%和0.38 wt%柠檬酸溶液通过冷冻干燥法可形成水凝胶,凝胶结构完整,呈淡黄色半透明状,有足够的弹性维持外形。通过傅立叶变换红外光谱分析发现,壳聚糖中的氨基与柠檬酸中的羧基发生了酰化反应。通过反应,向壳聚糖分子中引入了羧基这一亲水性基团,进而促使壳聚糖中的氨基转化为酰胺键。由电子扫描电镜可以看到各种比例柠檬酸/壳聚糖水凝胶都具有良好的疏松多孔结构,随着柠檬酸含量的增加,壳聚糖-柠檬酸分子颗粒更小,排列更加平滑紧密,孔隙也更加规律。结论:壳聚糖/柠檬酸水凝胶及神经支架合成成功。壳聚糖中的氨基与柠檬酸中的羧基发生了酰化反应,形成了疏松多孔结构的水凝胶物质。第二部分不同配比壳聚糖/柠檬酸水凝胶的生物相容性和生物学活性研究目的:将新生大鼠神经元原代细胞及小鼠成神经细胞瘤细胞系(Neuro-2a细胞)与壳聚糖/柠檬酸水凝胶进行共培养,研究壳聚糖/柠檬酸水凝胶对神经细胞活性、黏附和轴突生长的作用。方法:将新生大鼠神经元原代细胞或Neuro-2a细胞,分别和不同配比的壳聚糖/柠檬酸水凝胶进行共培养,设立空白对照组、单纯壳聚糖水凝胶对照组,通过细胞核DAPI染色,在显微镜下计数细胞以检测水凝胶的毒性。同时应用CCK-8实验,检测Neuro-2a细胞在不同水凝胶中培养24小时、48小时及72小时后的增殖变化情况。此外,将Neuro-2a细胞在不同水凝胶中培养72小时后,通过RT-qPCR实验对其进行Bcl2、Bax、SOD、caspase-3的表达量检测,了解水凝胶在细胞凋亡过程中的作用情况。最后,使用鬼笔环肽染色,结合荧光显微镜观察Neuro-2a细胞轴突比例和长度,了解不同水凝胶的生物活性。结果:将新生大鼠神经元原代细胞或Neuro-2a细胞分别与壳聚糖/柠檬酸水凝胶共培养,以与单纯壳聚糖水凝胶共培养作为对照,结果显示壳聚糖/柠檬酸水凝胶培养条件下细胞计数更高,生物相容性也更好。通过CCK-8实验发现,壳聚糖/柠檬酸水凝胶各组与单纯壳聚糖组相比,具有明显促进细胞增殖的作用。此外,壳聚糖/柠檬酸水凝胶不会诱导细胞凋亡,未对神经细胞产生损伤作用。并且,壳聚糖/柠檬酸水凝胶可促进Neuro-2a细胞神经突起的显著生长。其中,3 wt%壳聚糖溶液与0.33 wt%柠檬酸溶液构成的水凝胶的作用最为显著。结论:壳聚糖/柠檬酸水凝胶具有良好的生物相容性,对神经细胞无明显毒性,同时可以促进神经细胞轴突生长。结合生物相容性和生物活性角度考虑,3 wt%壳聚糖溶液与0.33 wt%柠檬酸溶液构成的水凝胶具有最大的应用潜力。第三部分不同配比壳聚糖/柠檬酸神经支架修复大鼠脊髓横断损伤的在体研究目的:建立大鼠胸段脊髓横断模型,将不同配比壳聚糖//柠檬酸水凝胶神经修复支架植入脊髓损伤部位,观察不同配比壳聚糖//柠檬酸水凝胶神经修复支架对脊髓损伤的修复效果。方法:麻醉大鼠后,行椎板切除术暴露脊髓,于胸9-胸10节段完全横断,形成约2毫米长脊髓完全缺损,建立大鼠脊髓完全横断模型。设立假手术组,对照组,支架组。其中支架组进一步根据神经修复支架种类分为CH8/CA1组,CH9/CA1组和CH10/CA1组(各9只)。术后每周称量大鼠体重,并采用Basso-Beattie-Bresnahan(BBB)运动功能评分系统对大鼠运动功能进行测定评分。于术后2周,4周,12周三个时间点分批分组处死大鼠,并进行组织检测,包括HE染色,Masson染色,LBF染色及免疫荧光染色(NF-H,GFAP)。最终统计各项染色情况。结果:大鼠脊髓完全横断模型建立成功,各组大鼠术后第一日双侧后肢完全瘫痪,排尿功能丧失。术后各组大鼠BBB评分均有所升高,但支架组大鼠运动功能的恢复情况较对照组显著(p<0.0001)。体重监测显示支架组和对照组的大鼠体重无显著差异,表明壳聚糖/柠檬酸神经支架无明显刺激性。通过对脊髓标本观察发现,术后12周脊髓残端与神经植入物融合良好,形成光滑边界,植入物可有效连接脊髓残端。HE染色和LBF染色均显示新生细胞、组织沿预留通道长入神经支架,并且可以观察到部分神经元长入通道,髓鞘排列规律、整齐。Masson染色显示位于神经通道中的胶原沉积呈现有序排列方式。免疫荧光显示与对照组相比,支架组NF-H的表达显著升高,而GFAP的表达则较低。结论:壳聚糖/水凝胶神经修复支架对脊髓损伤修复效果良好,能促进脊髓全横断大鼠的后肢运动功能恢复,同时减少脊髓损伤周围部位细胞损伤。此外,壳聚糖/水凝胶神经修复支架还可以抑制胶质瘢痕形成,促进神经元再生和轴突生长,并诱导新生轴突髓鞘化。其中,3 wt%壳聚糖溶液与0.33 wt%柠檬酸或0.38 wt%柠檬酸比例的两种神经修复支架修复效果最好,值得进一步研究、改进以应用于脊髓损伤的临床治疗。更多还原

【Abstract】 Part 1Preparation of chitosan-based hydrogels cross-linked with citric acidObjective:Spinal cord injury is a devastating disaster in the world,there is no satisfactory curative therapy until now.Usage of scaffold not only could improve the microenvironment in the lesion site,but also could guide regenerative axonal across the lesion,so it is a greattherapy with immense potential for the spinal cord injury,which attract tremendous attention from researchers.In this study,we use citric acid as a cross-linker to improve the mechanics of the chitosan-based hydrogels.We hope the chitosan-based hydrogel cross-linked with citric acid could be used to prepare the neural scaffoldsMethod:The chitosan-based hydrogels cross-linked with different concentrations of citric acid solution were prepared via directional temperature field freezing followed by lyophilization.The samples were tested with Fourier transform spectroscopy.Scanning electron microscope was also applied to investigate the structure and morphology of the obtained samplesResults:Three kinds of chitosan-citric acid hydrogels were fabricated.The structure of formed hydrogels was intact and tight,the color was translucent light yellow,and the elastic moduli was enough to sustain the architecture.The Fourier transform spectroscopy showed the amino group in chitosan was interacted with the carboxyl group in citric acid to form the acyl group.The scanning electron microscopy showed that all kinds of citric acid/chitosan hydrogels had porous structure.And with the increased content of citric acid,the radius of chitosan-citric acid molecule became smaller,the arrangement was smoother and tighter,the porosity was more regular,and the structure was more porousConclusions:The chitosan-based hydrogels and scaffolds cross-linked with citric acid were synthesized successfullyPart 2Biocompatibility and bioactivity of chitosan-based hydrogels cross-linked with different ratio of citric acidObjective:To culture new-born rat primary neurons and mouse neuroblastoma cell line(Neuro-2a cell line)cells in different ratio of chitosan/citric acid hydrogels in vitro and to evaluate the biocompatibility and bioactivity of these hydrogelsMethods:To culture new-born rat primary neurons and neuro-2a cells in different ratio of chitosan/citric acid hydrogels,separately.In addition,pure chitosan hydrogel group and blank control group were set.According to nuclear DAPI immunofluorescence staining,stained cells were observed and counted under laser confocal microscopy to evaluate the cell toxicity of hydrogels.Furthermore,proliferation of neuro-2a cells in various conditions was assessed by CCK-8 assay at 24 hours,48 hours and 72 hours.Finally,neuro-2a cells was immunofluorescence staining with FITC-Phalloidin,neurite length of neuro-2a cell was captured under laser confocal microscopy and measured using ImageJ software in order to estimate bioactivity of hydrogelsResults:The viability of both new-born rat primary neurons and neuro-2a cells was higher on the three kinds of chitosan/citric acid hydrogels than on the pure chitosan hydrogel Furthermore,the proliferative rate of neuro-2a cells was better on these chitosan/citric acid hydrogels than on the pure chitosan hydrogel,too.Finally,average neurite length of neuro-2a cells was longer on these chitosan/citric acid hydrogels than on the pure chitosan hydrogel,which meant that the bioactivity of promoting neurite outgrowth of chitosan/citric acid hydrogels was significantConclusions:Chitosan/citric acid hydrogels had good biocompatibility,no obvious cytotoxicity,and could promote axonal elongationPart 3Effect of chitosan-based scaffolds cross-linked with different ratio of citric acid on rat spinal cord injury repairObjective:To establish transective thoracic spinal cord injury of rat,and implant different ratio of chitosan/citric acid scaffolds into the lesion site.In addition,to evaluate the therapeutic effect of various scaffolds on the rat spinal cord injury modelMethods:The rats were anesthetized with 10%chloral hydrate and were operated with laminectomy to expose the spinal cord.Then the 9th to the 10th of thoracic spinal cord was transectively cut off and about 2 millimeters spinal cord defect was left.The rat presented with spastic convulsions of hind limbs,followed by flaccid paralysis of hind limbs,the transective thoracic spinal cord injury model of rat was established.According to the manipulation,the rats were divided into three groups,including the sham group,the control group and the scaffold group.In the scaffold group,the rats were further grouped by the types of scaffolds,named the CH8/CA1 group,the CH9/CA1 group and the CH10/CA1 group(nine per group).Rat weight was measured every week after operation,and the locomoter function of SCI rat was evaluated weekly by the Basso-Beattie-Bresnahan(BBB)locomotor rating scale.By the end of the second,the fourth and the twelve post-operative week,three rats from each group were killed and the lesion site was harvested.The samples were detected by hematoxylin-eosin(HE)staining,Masson staing,Luxol fast blue(LFB)staining and immunofluescence staining(NF-H,GFAP).Finally,the picture of sample was observed and measured by the ImageJ softwareResults:The transective thoracic spinal cord injury model of rat was established successfully.On the first day after the operation,the bilateral hind limbs of the rat,except the SHAM group,were totally paralyzed,and the urinary function was lost too.The BBB score of the rats of the control group increased slightly,in contrast,the locomotor performance of the rats of the scaffold group were improved dramatically(p<0.0001)Weight monitoring of rats showed that no difference existing between the scaffold group and the control group,the fact proved that there was no apparent cytotoxicity of the scaffolds in vivo.Observing the spinal cord samples,it could be detected that the spinaltrunk and the scaffold fused well and the smooth boundary was formed between them.The scaffold could connect the defect between bilateral injured spinal cord.HE staining showed newborn cells and tissue were infiltrated into preserved channels in the scaffolds,and several regenerative neural axons could be found in the channels.Masson staining demonstrated that deposition of collagen was ranked orderly in the channels.LBF staining showed that newborn cells and tissue were grown into the channels,and myelin sheath of the axon were arranged regularly and neatly.Finally,the expression of NF-H was increased more heavily in the scaffold group than in the control group,in contrast,the expression of GFAP was lower in the scaffold groupConclusions:The chitosan/citric acid scaffold had great repair effect in the SCI.The biomaterials could promote recovery of locomotion function of the transective SCI rats,and decrease death of cells in the vicinity of the lesion site.In addition,the chitosan/citric acid scaffold could inhibit formation of glial scar on the defect of spinal cord,promote regeneration of neural cells and outgrowth of neurite,and induce remyelination of regenerative axon.Among them,Scaffolds which were fabricated by 3 wt%chitosan solution and 0.33 wt%citric acid solution or 0.38 wt%citric acid solution had better repair efficiency for the SCI.These two scaffolds had great potential for the future clinal appliance,and were worth for further investigation and improvement.更多还原