神经干细胞联合激活态雪旺细胞与PCL支架生物相容性的实验研究

发布时间：2018-06-08 06:43

  本文选题：神经干细胞 + 激活态雪旺细胞　； 参考：《天津医科大学》2017年硕士论文

【摘要】：【目的】本课题拟通过检测激活态雪旺细胞(activated Schwann cells,ASCs)和非激活态雪旺细胞(Schwann cells,SCs)之间营养因子的表达差异,探究体外培养对ASCs的功能状态影响,及ASCs、SCs诱导神经干细胞(neural stem cells,NSCs)向神经元分化的作用;通过培养ASCs、SCs于静电纺丝聚己内酯(PCL)纤维支架上,对比ASCs、SCs与PCL纤维支架的生物相容性;通过培养NSCs或联合ASCs于静电纺丝PCL纤维支架上,观察细胞分布、增殖、分化,探究ASCs和NSCs共培养体系与静电纺丝PCL纤维支架的生物相容性。【方法】通过结扎Wistar大鼠坐骨神经7天,模拟坐骨神经挫伤模型,提取ASCs;由正常坐骨神经提取SCs,分别培养至第3代,提取总蛋白,通过Western Blot对比分析ASCs和SCs的营养因子在蛋白水平的表达差异;提取Wister孕鼠E14.5天胚胎海马区NSCs,通过免疫荧光染色进行细胞鉴定及观察NSCs的多向分化能力;通过ASCs和SCs的条件培养基半量换液诱导NSCs分化,观察神经元分化比例及轴突长度;3D培养ASCs和SCs在静电纺丝PCL纤维支架上,通过CCK-8实验检测细胞活性与增殖,结晶紫染色观察细胞分布及寻找合适细胞密度,激光共聚焦显微镜观察细胞粘附和形态;将NSCs单纯或联合ASCs培养于静电纺丝PCL纤维支架上,通过CCK-8实验检测NSCs增殖,通过激光扫描电镜观察细胞形态和分布,通过激光共聚焦显微镜观察NSCs神经元的分化和ASCs MBP的表达。【结果】1.传至第3代的ASCs较SCs高表达BDNF、NGF(P0.05),而NT-3、MBP的表达则低于SCs(P0.05);半量换液7天后,ASCM组,神经元分化比例为36.06±7.04%;SCM组分化比例为17.22±3.78%;M组分化比例为5.78±3.03%。ASCM组神经元分化比例高于其他两组(P0.05)。ASCM组,神经元轴突长度为161.33±67.44μm;SCM组神经元轴突长度为110.33±60.34μm;M组,神经元轴突长度为97.55±54.09μm。ASCM组神经元轴突长于其余两组(P0.05)。2.静电纺丝PCL纤维支架的纤维直径集中在7.93±1.41μm,纤维随机分布能形成3D培养结构;结晶紫染色观察:一定数量的雪旺细胞紧贴纤维纵向分布,且细胞种植密度为2×10^4个/cm2时细胞增殖良好;通过S-100染色,激光共聚焦显微镜观察:细胞呈典型的梭型,并且细胞紧贴纤维分布,与结晶紫染色观察结果相一致;CCK-8实验检测:ASCs在PCL支架上比SCs的增殖更快,第7天OD值高于SCs(P0.05)。3.神经干细胞在静电纺丝PCL纤维支架上培养,1-7天OD值逐渐增大,在第6-7天时,趋于平缓,在第7天时细胞增殖达到最高峰;通过βⅢ-Tubulin、GFAP、O4免疫荧光染色,利用激光共聚焦显微镜进行观察:神经干细胞分化7天后,能够分化为星形胶质细胞,神经元和少突胶质细胞;NSCs与ASCs共培养于静电纺丝PCL支架上后,分化7天进行疫荧光染色共聚焦显微镜观察:一定数量的ASCs能够表达MBP,神经干细胞能够分化为神经元,并且神经元多分布于表达MBP的ASCs周围。【结论】1.体外培养条件下传代至第3代的ASCs仍能保持一定的激活表型,且能促进NSCs向神经元方向分化;2.静电纺丝PCL支架具有3D培养结构,与ASCs具有良好的生物相容性,且纤维对细胞分布具有一定导向性作用;3.NSCs、ASCs共培养体系在静电纺丝PCL支架上能够形成3D培养,可以进行体内移植探究。
[Abstract]:[Objective] to investigate the difference in the expression of nutritional factors between activated Schwann cells (ASCs) and non active Schwann cells (Schwann cells, SCs) in the active state, and to explore the effect of in vitro culture on the functional state of ASCs, and the effect of ASCs and SCs induced neural stem cells (neural stem) to neuron differentiation. The biocompatibility of ASCs, SCs and PCL fiber scaffolds was compared by ASCs and SCs on the electrostatic spinning polyhexyl (PCL) fiber scaffold. The biocompatibility of the cell distribution, proliferation and differentiation between the NSCs or the combined ASCs on the electrostatic spinning PCL fiber scaffold was observed and the biocompatibility between the ASCs and NSCs co culture system and the electrostatic spinning PCL fiber scaffold was investigated. Methods by ligating the sciatic nerve of Wistar rats for 7 days, the sciatic nerve contusion model was simulated and ASCs was extracted. SCs was extracted from the normal sciatic nerve and cultured to third generations. The total protein was extracted respectively. The expression difference between ASCs and SCs in the protein level was analyzed by Western Blot, and NSCs in the hippocampus of E14.5 Day Embryo of Wister pregnant mice was extracted. Immunofluorescence staining was used to identify and observe the multidirectional differentiation of NSCs; NSCs differentiation was induced by semi mass transfer of ASCs and SCs conditioned medium, and the proportion of neuron differentiation and the length of axon were observed. 3D culture ASCs and SCs were used to detect cell activity and proliferation by CCK-8 test and crystal violet staining. The cells were distributed and looked for the appropriate cell density. The adhesion and morphology of the cells were observed by laser confocal microscopy. The NSCs pure or combined ASCs was cultured on the electrospun PCL fiber scaffold. The proliferation of NSCs was detected by the CCK-8 experiment. The morphology and distribution of the cells were observed by laser scanning electron microscopy. The points of NSCs neurons were observed by laser confocal microscopy. Expression of ASCs and MBP. [results] 1. to third generation of ASCs higher than SCs expressed BDNF, NGF (P0.05), and NT-3, MBP was lower than SCs (P0.05), 7 days after the half volume of liquid, ASCM group, the proportion of neuron differentiation was 36.06 + 7.04%, the proportion of differentiation was 17.22 + 3.78%, the proportion of differentiation group of 5.78 + neurons differentiation was higher than the other In the two group (P0.05) group.ASCM, the axon length of the neuron was 161.33 + 67.44 m, and the axon length of the SCM group was 110.33 + 60.34 mu m; the axon of the neuron axon length was 97.55 + 54.09 mu m.ASCM, and the axon of the neuron was longer than the other two groups (P0.05). The fiber diameter of the.2. electrostatic spinning PCL fiber stent was 7.93 + 1.41 mu m, and the random distribution of fiber could form 3D. Culture structure; crystal violet staining observation: a certain number of Schwann cells were closely attached to the fiber longitudinal distribution, and the cell proliferation was good when the cell planting density was 2 x 10^4 /cm2; the cells were observed by S-100 staining and laser confocal microscope: the cells showed a typical spindle type, and the cells were closely attached to the fiber distribution, and the results were in accordance with the crystal violet staining results; CCK-8 Experimental detection: the proliferation of ASCs on the PCL scaffold is faster than that of SCs. The seventh day OD value is higher than that of SCs (P0.05).3. neural stem cells on the electrospun PCL fiber scaffold. On the 1-7 day, the OD value gradually increases. At the 6-7 day, it tends to be gentle and the cell proliferation reaches the peak at seventh days; through the beta III -Tubulin, GFAP, O4 immunofluorescence staining, and laser copolymerization. The focal microscope was observed: 7 days after differentiation, the neural stem cells differentiated into astrocytes, neurons and oligodendrocytes. NSCs and ASCs were co cultured on the electrospun PCL scaffold and were differentiated for 7 days by immunofluorescence staining confocal microscopy: a certain number of ASCs could express MBP, and neural stem cells could differentiate into nerves. And the neurons were distributed around the ASCs of the expression of MBP. [Conclusion] 1. in vitro culture, the third generation of ASCs still can maintain a certain activation phenotype, and can promote the differentiation of NSCs to the neuron; 2. electrospun PCL scaffold has a 3D culture structure, and has good biocompatibility with ASCs, and the fiber is distributed to the cells. 3.NSCs ASCs co culture system can form 3D culture on electrospinning PCL scaffolds and can be transplanted in vivo.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2;R318.08

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