胶原—碳纳米管复合物薄膜对胚胎大鼠皮层神经元突起发育和神经干细胞分化的影响

发布时间：2018-03-16 09:11

本文选题：胶原　切入点：碳纳米管　出处：《中国人民解放军医学院》2014年硕士论文　论文类型：学位论文

【摘要】：目的探讨胶原-碳纳米管复合物薄膜对胚胎大鼠皮层神经元突起发育和NSCs分化的影响，，为该材料应用于体外大脑皮层构建和体内中枢神经系统损伤的修复奠定基础。材料与方法一、胶原-碳纳米管复合物薄膜的制备：我们选用了SWCNTs和鼠尾I型胶原作为原料，利用超声的方法进行混匀来制备胶原-碳纳米管混合溶液，最后利用烘干技术制作成胶原-碳纳米管复合物薄膜。二、胶原-碳纳米管复合物薄膜的物理学和生物学评价：我们将制备好的胶原-碳纳米管复合物薄膜进行扫描电子显微镜观察其表面形貌，通过电化学工作站测量其导电性，通过阿尔玛蓝试剂来评价材料与胚胎大鼠皮层神经元、NSCs的生物相容性。三、胶原-碳纳米管复合物薄膜对胚胎大鼠皮层神经元突起发育的影响：将胚胎大鼠皮层神经元接种在材料的表面，在体外培养3d后，对细胞进行固定，进行微管相关蛋白-2的免疫荧光染色，并采用IMAGE J软件中的Neurite Tracer插件对神经元突起的数量和长度进行测量记录，并进行统计分析。同时，通过SEM对体外培养7d的皮层神经元与材料接触的表面形貌进行观察。四、胶原-碳纳米管复合物薄膜对胚胎大鼠皮层NSCs分化的影响：我们将胚胎大鼠皮层NSCs接种在材料的表面，通过撤掉EGF、bFGF，来观察体外培养7d后，材料对NSCs分化为不同谱系神经细胞的影响，并进行统计分析。此外，采用SEM对体外培养7d后，NSCs与材料接触的表面形貌进行观察。结果一、胶原-碳纳米管复合物薄膜的制备：1、分离所得的鼠尾I型胶原溶液清澈透亮，黏度适中，胶原浓度约为3mg/ml；2、制备的胶原-碳纳米管混合溶液肉眼观察无明显聚集物存在，TEM观察：1.5mg/ml和2mg/ml的胶原-碳纳米管溶液中的聚集物相对较多；3、制备的胶原-碳纳米管复合物薄膜肉眼观察均匀程度高，无明显杂质存在。二、胶原-碳纳米管复合物薄膜的物理学和生物学评价： 1、SEM观察，不同浓度的胶原-碳纳米管复合物薄膜的均匀程度不同，同透射电镜结果一致，1.5mg/ml和2mg/ml浓度组的胶原-碳纳米管复合物薄膜出现的聚集物较多，表面形貌相对不均匀； 2、电化学工作站导电性测量结果发现：从0mg/ml到2mg/ml的不同浓度的胶原-碳纳米管复合物薄膜的电阻率（*m）分别为：9.19x104，14.1，5.77x10-1，1.89x10-3，1.54x10-4； 3、阿尔玛蓝生物相容性检测结果发现：体外培养7d后，从0mg/ml到2mg/ml的不同浓度的胶原-碳纳米管复合物薄膜上，胚胎大鼠皮层神经元的平均氧化还原率（%）分别为：48.36±5.22，55.32±5.15，55.37±2.68，44.39±2.82，34.66±2.97，差异有统计学意义（P0.01），且0mg/ml组与1.5mg/ml组，0.5mg/ml组与1mg/ml组无统计学差异，余各组氧化还原率不同（P0.01）。体外培养7d后，从0mg/ml到2mg/ml的不同浓度的胶原-碳纳米管复合物薄膜上，胚胎大鼠皮层NSCs的平均氧化还原率（%）分别为：20.89±0.45，25.03±2.78，19.78±1.21，19.35±1.20，19.42±0.60，差异有统计学意义（P0.01），且0.5mg/ml组与0mg/ml、1mg/ml、1.5mg/ml、2mg/ml组氧化还原率不同（P0.01）。三、胶原-碳纳米管复合物薄膜对胚胎大鼠皮层神经元突起发育的影响 1、体外培养3d后，从0mg/ml到2mg/ml的不同浓度组的胶原-碳纳米管复合物薄膜材料上的神经元的突起的数量（个）分别为：1.17±0.64，1.06±0.25，1.49±0.78，1.04±0.19，1.06±0.35，差异有统计学意义（P0.01），且1mg/ml组与其他各组神经元突起的数量不同（P0.01）； 2、体外培养3d后，从0mg/ml到2mg/ml的不同浓度组的胶原-碳纳米管复合物薄膜材料上的神经突起的平均长度（μm）分别为：56.13±26.88，68.23±48.73，84.48±32.53，73.08±23.74，77.15±27.67，差异有统计学意义（P0.05），且0mg/ml组与1mg/ml、2mg/ml组突起的平均长度不同（P0.05）。四、胶原-碳纳米管复合物薄膜对胚胎大鼠皮层NSCs分化的影响 1、体外培养7d后，在0mg/ml到2mg/ml的不同材料表面上，胚胎大鼠皮层NSCs自发分化为MAP-2抗体阳性的神经元的比例（%）分别为：25.69±0.92，28.54±1.05，28.15±0.96，24.27±1.66，24.02±1.21，差异有统计学意义（P0.01），且0mg/ml组与0.5mg/ml、1mg/ml组，0.5mg/ml组与0mg/ml、1.5mg/ml、2mg/ml组，1mg/ml组与0mg/ml、1.5mg/ml、2mg/ml组分化为神经元的比例不同（P0.05）； 2、体外培养7d后，在0mg/ml到2mg/ml的不同材料表面上，胚胎大鼠皮层NSCs自发分化为GFAP抗体阳性的星型胶质细胞的比例（%）分别为：44.02±0.61，27.59±0.11，32.19±1.23，30.25±2.23，23.89±1.33，差异有统计学意义（P0.01），且1.5mg/ml组与0.5mg/ml、1mg/ml组分化为星型胶质细胞的比例相同（P0.05），余各组两两比较均不相同（P0.05）。 3、体外培养7d后，在0mg/ml到2mg/ml的不同材料表面上，胚胎大鼠皮层NSCs自发分化为OLIGODENDROCYTE抗体阳性的少突胶质细胞的比例（%）分别为：25.98±11.41，39.78±6.74，35.91±4.64，32.90±7.14，27.07±3.21，各组间比较无统计学差异。结论 1、胶原-碳纳米管复合物薄膜的生物相容性与材料的浓度及细胞的种类有关，0.5~1.5mg/ml组的生物相容性较好，而2mg/ml组的生物相容性较差。 2、不同浓度的胶原-碳纳米管复合物薄膜对神经元突起的发育的影响不同，1mg/ml的胶原-碳纳米管复合物薄膜可以促进胚胎大鼠皮层神经元突起的发育。 3、不同浓度的胶原-碳纳米管复合物薄膜对NSCs分化的影响不同，0.5mg/ml和1mg/ml的胶原-碳纳米管复合物薄膜可以促进胚胎大鼠NSCs向神经元分化；0.5mg/ml、1mg/ml、1.5mg/ml和2mg/ml的胶原-碳纳米管复合物薄膜均能抑制胎大鼠皮层NSCs向星型胶质细胞分化，且2mg/ml组最明显；不同材料对胚胎大鼠皮层NSCs向少突胶质细胞分化比例无差别。
[Abstract]:objective
To investigate the effects of collagen and carbon nanotube composite film on embryonic rat cortical neuron neurite development and NSCs differentiation, lay the foundation for the application of the material in the building and repair of the cerebral cortex in vitro in the central nervous system damage.
Materials and methods
A, collagen - carbon nanotube composite film preparation: we chose SWCNTs and collagen type I as raw material, blending and preparation of collagen - carbon nanotubes mixed solution by ultrasonic method, finally using drying technology into production of collagen / carbon nanotube composite film.
Two, physics and biological evaluation of collagen - carbon nanotube composite film: we prepared collagen - carbon nanotube composite film by scanning electron microscopy to observe the surface morphology, measuring the conductivity by electrochemical workstation, by Alma blue reagent evaluation materials and cortex of rat embryonic neurons, the compatibility of NSCs biological.
Three, effects of collagen and carbon nanotube composite film on cortical neuronal neurite development of embryonic rat: inoculation of embryonic rat cortical neurons on the surface of the material, in 3D after cultured in vitro, the cells were fixed, immunofluorescence staining of microtubule associated protein -2, and measured the record with IMAGE in J software the Neurite Tracer plug-in on the number and length of neurites, and statistical analysis. At the same time, through the observation of surface morphology of cortical neurons and contact material SEM on 7d cultured in vitro.
Four, effects of collagen and carbon nanotube composite film on the differentiation of embryonic rat cortical NSCs: we will embryonic rat cortex NSCs inoculated on the surface of the material removed by EGF, bFGF, and to observe the 7d in vitro, material on the differentiation of NSCs into different lineages of nerve cells, and statistical analysis. In addition, the SEM of 7D cultured in vitro after contact with the material surface morphology of NSCs were observed.
Result
A, collagen - carbon nanotube composite film preparation: 1, isolated from rat tail collagen type I was clear, moderate viscosity, collagen concentration is about 3mg/ml; 2, the preparation of collagen - carbon nanotubes mixed solution observed without obvious aggregation, TEM and 2mg/ml were observed: 1.5mg/ml collagen the aggregates of carbon nanotubes in solution is relatively large; 3, the preparation of collagen - carbon nanotube composite film observed high degree of uniformity, no obvious impurity.
Two, physics and biological evaluation of collagen - carbon nanotube composite film:
1, SEM observation, the degree of uniformity of different concentrations of collagen - carbon nanotube composite films are different, consistent with the TEM results, 1.5mg/ml and 2mg/ml concentrations of collagen - carbon nanotube composite film of aggregates are relatively uneven surface topography;
2, the measurement results showed that electrochemical workstation conductivity resistivity from 0mg/ml to 2mg/ml of different concentrations of collagen - carbon nanotube composite film (*m) were: 9.19x104,14.1,5.77x10-1,1.89x10-3,1.54x10-4;
3, Alma blue biocompatibility test results found:
After cultured in vitro for 7d from 0mg/ml to 2mg/ml of different concentrations of collagen - carbon nanotube composite film, the average oxidation of embryonic rat cortical neurons and the reduction rate (%) respectively: 48.36 + 5.22,55.32 + 5.15,55.37 + 2.68,44.39 + 2.82,34.66 + 2.97, the difference was statistically significant (P0.01), and 0mg/ml group and 1.5mg/ml group, 0.5mg/ml group and 1mg/ml group showed no significant difference in other groups, the redox rate of different (P0.01).
After cultured in vitro for 7d from 0mg/ml to 2mg/ml of different concentrations of collagen - carbon nanotube composite film, the average NSCs of embryonic rat cortical oxidation reduction rate (%) respectively: 20.89 + 0.45,25.03 + 2.78,19.78 + 1.21,19.35 + 1.20,19.42 + 0.60, the difference was statistically significant (P0.01), and group 0.5mg/ml and 0mg/ml. 1mg/ml, 1.5mg/ml, 2mg/ml group of oxidation reduction rate of different (P0.01).
Three, effects of collagen and carbon nanotube composite film on cortical neurons of rat embryonic development.
1, after cultured in vitro for 3D from 0mg/ml to 2mg/ml in different concentration groups of collagen - carbon nanotube composite films on neuronal processes (a) respectively: 1.17 + 0.64,1.06 + 0.25,1.49 + 0.78,1.04 + 0.19,1.06 + 0.35, the difference was statistically significant (P0.01), and the number of 1mg/ml group and other groups different neuronal processes (P0.01);
2, 3D were cultured in vitro, the average neurite length from 0mg/ml to 2mg/ml in different concentration groups of collagen - carbon nanotube composite film material on the (m) respectively: 56.13 + 26.88,68.23 + 48.73,84.48 + 32.53,73.08 + 23.74,77.15 + 27.67, the difference was statistically significant (P0.05), and group 0mg/ml and 1mg/ml. The average length of 2mg/ml group raised different (P0.05).
Four, effects of collagen and carbon nanotube composite film on the differentiation of embryonic rat cortical NSCs
1, after cultured in vitro for 7d in 0mg/ml to 2mg/ml on the surface of different materials, the proportion of the NSCs cortex of embryonic rats spontaneously differentiate into MAP-2 positive neurons of the (%) respectively: 25.69 + 0.92,28.54 + 1.05,28.15 + 0.96,24.27 + 1.66,24.02 + 1.21, the difference was statistically significant (P0.01), and group 0mg/ml and 0.5mg/ml. 1mg/ml group, 0.5mg/ml group and 0mg/ml, 1.5mg/ml, 2mg/ml group, 1mg/ml group and 0mg/ml, 1.5mg/ml, 2mg/ml were divided into different proportion of neurons (P0.05);
2, after cultured in vitro for 7d in 0mg/ml to 2mg/ml on the surface of different materials, the proportion of the NSCs cortex of embryonic rats spontaneously differentiate into astrocytes of GFAP antibody positive (%) were: 44.02 + 0.61,27.59 + 0.11,32.19 + 1.23,30.25 + 2.23,23.89 + 1.33, the difference was statistically significant (P0.01), and 1.5mg/ml and group 0.5mg/ml, group 1mg/ml differentiated into astrocytes (P0.05), the same proportion of more than 22 groups were different (P0.05).
3, after cultured in vitro for 7d in 0mg/ml to 2mg/ml on the surface of different materials, the proportion of the NSCs cortex of embryonic rats spontaneously differentiate into oligodendrocyte OLIGODENDROCYTE of antibody positive (%) were: 25.98 + 11.41,39.78 + 6.74,35.91 + 4.64,32.90 + 7.14,27.07 + 3.21, no significant difference between the two groups.
conclusion
1, the type and concentration and cell compatibility of collagen materials - carbon nanotube composite film biological, 0.5~1.5mg/ml group of good biocompatibility, while the 2mg/ml group had poor compatibility.
2, the influence of the development of different concentrations of collagen - carbon nanotube composite film on the neurites of different 1mg/ml collagen / carbon nanotube composite film can promote neurite growth of embryonic rat cortical neurons.
3. The effects of different concentrations of collagen - carbon nanotube composite film on differentiation of NSCs 0.5mg/ml and 1mg/ml, the collagen - carbon nanotube composite film can promote neuronal differentiation of rat embryonic NSCs; 0.5mg/ml, 1mg/ml, 1.5mg/ml and 2mg/ml collagen / carbon nanotube composite films could inhibit the fetal rat cerebral cortex to NSCs astrocyte differentiation, and 2mg/ml group was the most obvious; different materials on embryonic rat cortex NSCs to oligodendrocyte differentiation ratio of no difference.

【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741.05

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