丝素蛋白纳米纤维直径对嗅鞘细胞生长和迁移影响的研究

发布时间：2018-04-05 08:30

  本文选题：嗅鞘细胞　切入点：丝素蛋白　出处：《苏州大学》2011年硕士论文

【摘要】：目的探讨不同直径丝素蛋白纳米纤维(silk fibroin nanofibers,SFS)对嗅鞘细胞(olfactory ensheathing cells,OECs)生长及迁移的影响,为脊髓损伤(spinal cord injury,SCI)修复材料的选择提供实验基础和理论依据。 方法(1)SFS的制备:运用静电纺丝技术制备SFS,并通过扫描电镜(scanning electron microscope,SEM)观察。(2)OECs的纯化培养:取SD大鼠嗅球新鲜分离的OECs,采用改良差速贴壁法纯化培养。(3) OECs与SFS的复合培养:将纯化后的OECs分别接种至以左旋多聚赖氨酸(poly-l-lysine,PLL)(对照组)和SFS(400nm,1200nm)(实验组)包被盖玻片的培养皿中进行复合培养。(4)检测指标:复合培养不同时间点行倒置相差显微镜和扫描电镜观察细胞形态、生长、分布及与材料黏附情况。神经生长因子受体p75(nerve growth factor receptor p75,NGFR p75)及胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫荧光染色测定细胞表现型,噻唑兰(Thiazoyl Blue Tetrazolium Bromide,MTT)法测定支架材料上OECs的增殖情况,死活细胞染色试剂及流式细胞术检测细胞生存率,细胞的迁移通过Leica AF6000活细胞工作站拍摄得到图像,活细胞工作站动态跟踪观察OECs生长迁移并计算细胞迁移轨迹、迁移行为、迁移效率、迁移速率。 结果(1)倒置相差显微镜示SFS上培养的OECs与对照组相比,细胞沿丝素纤维迁移,细胞突起方向走向较一致,形态特征无显著差异。(2)扫描电镜观察显示SFS平均直径约为(395±3)nm、(1210±7)nm,纤维呈三维立体网状结构,分布均匀,OECs与材料结合紧密,且细胞突起方向与纤维走向较一致,形态与对照组无显著性差异。(2)免疫荧光染色:培养4d时,免疫荧光染色显示实验组NGFRp75/GFAP阳性,OECs在SFS材料上仍保留其特有的表现型。(3)MTT检测显示:培养第4d时,对照组OECs的吸光度值较1200nm直径的材料OECs的吸光度值有显著差异(p0.05),培养第7d时,对照组OECs的吸光度值较实验组吸光度值有显著差异(p0.05),而且OECs在400nmSFS材料上的吸光度值比在1200nmSFS材料上的吸光度值高,有统计学意义(p0.05)。400nmSFS相比1200nmSFS更能促进OECs的增殖。(4)死活细胞染色试剂检测显示:在培养第4、7天时,实验组细胞形态较对照组正常,成活率较高,两组死亡细胞数比较无显著性差异。(5)流式细胞术检测结果显示:在培养第4天时,实验组OECs较对照组OECs的凋亡率无显著差异(p0.05)。(6)活细胞工作站观察显示:细胞的迁移速率,迁移效率和对照组相比均无显著地统计学差异(p0.05),两种直径的SFS对OECs在纤维上的迁移速率、迁移效率没有显著影响。 结论不同直径丝素蛋白纳米纤维对嗅鞘细胞的生长与迁移均具有支持和引导作用,SFS有望成为一种新型的组织工程支架材料结合OECs移植修复脊髓损伤。
[Abstract]:Objective to investigate the effects of fibroin nanofibers (SFSs) of different diameters on the growth and migration of olfactory ensheathing cells (OECs) in olfactory ensheathing cells (OECs), and to provide experimental and theoretical basis for the selection of repair materials for spinal cord injury-induced spinal cord injury (sci).Methods preparation of SFS: SFSs were prepared by electrospinning technique, and the purification and culture of OECs from olfactory bulb of SD rats were observed by scanning electron microscopy (SEM). The cells were freshly isolated from olfactory bulb of SD rats and cultured by modified differential adherent method. The compound culture of OECs and SFS was carried out.Culture: the purified OECs was inoculated into a culture plate coated with L-poly-l-lysineine (control group) and SFS400nmnmnmnmnmnmnmnmnmnmHN (experimental group) respectively. The detection index: inverted phase contrast microscopy was performed at different time points in the compound culture.The morphology of cells was observed by microscope and scanning electron microscope.Growth, distribution and adhesion to materials.Neuronal growth factor receptor p75(nerve growth factor receptor p75 (NGFR-p75) and glial fibrillary acidic protein (GFAP) were used to detect the phenotype of the cells. The proliferation of OECs on the scaffold was measured by Thiazoyl Blue Tetrazolium BromideMTTassay.The survival rate of cells was detected by cell staining reagent and flow cytometry. The cell migration was captured by Leica AF6000 living cell workstation. The living cell workstation dynamically tracked the growth and migration of OECs and calculated the migration path and migration behavior.Migration efficiency, migration rate.Results (1) the inverted phase contrast microscope showed that the OECs cultured on SFS moved along the fibroin fibers and in the same direction as the cells in the control group.The average diameter of SFS was about 1210 卤7 nm, the fiber was three-dimensional reticular structure, the distribution of OECs was close to the material, and the direction of cell process was consistent with the direction of fiber, and the average diameter of SFS was about 1210 卤7 nm by scanning electron microscope (SEM), which showed that the fiber had a three-dimensional reticular structure, and the direction of cell process was the same as that of the fiber.There was no significant difference in morphology between the control group and the control group. Immunofluorescence staining showed that the NGFRp75/GFAP positive cells of the experimental group still retained their specific phenotype on the SFS material at the 4th day of culture.The absorbance value of OECs in control group was significantly different from that of OECs in diameter of 1200nm.The absorbance value of OECs in the control group was significantly higher than that in the experimental group, and the absorbance value of OECs on 400nmSFS was higher than that on 1200nmSFS.Compared with 1200nmSFS, P0.05N 路400nmSFS could promote the proliferation of OECs. The staining reagent showed that the cell morphology of the experimental group was normal and the survival rate was higher than that of the control group at the 7th day of culture.There was no significant difference in the number of dead cells between the two groups. The results of flow cytometry showed that the apoptosis rate of OECs in experimental group was not significantly different from that of OECs in control group on the 4th day of culture. The observation of living cell workstation showed that the migration rate of cells was higher than that of control group.There was no significant difference in migration efficiency between the two groups. There was no significant difference between the two diameters of SFS on the migration rate and efficiency of OECs on fiber.Conclusion fibroin nanofibers of different diameters can support and guide the growth and migration of olfactory ensheathing cells. SFS is expected to be a new tissue engineering scaffold combined with OECs transplantation to repair spinal cord injury.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】

相关期刊论文 前10条

1 左保齐;张锋;孙春光;白伦;;再生桑蚕丝素/柞蚕丝素蛋白静电纺无纺网结构的研究[J];高分子材料科学与工程;2007年04期

2 黄红云,王洪美,修波,王锐,卢明,陈琳,亓树彬陈林,张泽华,武海青,苟成青,程建铎,陆晓白,刘宗惠;嗅鞘细胞移植治疗脊髓损伤临床试验的初步报告[J];海军总医院学报;2002年01期

3 王锐,田增民,刘爽,王亚明,孙君昭,陈琳,赵虎林,王洪伟,卢旺盛,郭欣茹;嗅鞘细胞移植治疗脊髓截瘫110例[J];海军总医院学报;2005年03期

4 胡志俊;马迎辉;王拥军;舒冰;周重建;;新生鼠嗅鞘细胞培养的几种纯化方法的比较[J];生物医学工程与临床;2009年01期

5 陈琳,黄红云,王锐,王洪美,修波,李秉辰;嗅鞘细胞移植治疗多发性硬化1例[J];中华神经外科疾病研究杂志;2004年01期

6 范志海;沈忆新;陆政峰;成茂华;董启榕;;成年大鼠嗅球嗅鞘细胞的分离、培养、纯化与生物学特性的研究[J];苏州大学学报(医学版);2007年04期

7 黄红云,王洪美,顾征,陈琳,李荧,宋英伦,郝伟,李炳辰,张健,张峰,苟成青;嗅鞘细胞移植治疗运动神经元病/肌萎缩侧索硬化的初步报告[J];中国临床康复;2004年13期

8 郑遵成;刘超;张振兴;王道奎;郑修启;冀永久;郑建森;;嗅鞘细胞移植治疗陈旧性脊髓损伤48例(英文)[J];中国临床康复;2006年09期

9 陈琳;黄红云;刘彦铖;郗海涛;张峰;张健;王洪美;苟成青;刘瑞文;姜超;江昭;谢自行;罗春燕;;嗅鞘细胞移植治疗肌萎缩侧索硬化症中期安全性评价[J];中国临床康复;2006年25期

10 陈琳;黄红云;王洪美;王福斌;郗海涛;;嗅鞘细胞移植改善橄榄体桥脑小脑萎缩患者神经功能:1例报告[J];中国临床康复;2006年29期

，

本文编号：1713978