本文選題：膠原　切入點：碳納米管　出處：《中國人民解放軍醫(yī)學院》2014年碩士論文　論文類型：學位論文

【摘要】：目的 探討膠原-碳納米管復合物薄膜對胚胎大鼠皮層神經元突起發(fā)育和NSCs分化的影響，，為該材料應用于體外大腦皮層構建和體內中樞神經系統(tǒng)損傷的修復奠定基礎。 材料與方法 一、膠原-碳納米管復合物薄膜的制備：我們選用了SWCNTs和鼠尾I型膠原作為原料，利用超聲的方法進行混勻來制備膠原-碳納米管混合溶液，最后利用烘干技術制作成膠原-碳納米管復合物薄膜。 二、膠原-碳納米管復合物薄膜的物理學和生物學評價：我們將制備好的膠原-碳納米管復合物薄膜進行掃描電子顯微鏡觀察其表面形貌，通過電化學工作站測量其導電性，通過阿爾瑪藍試劑來評價材料與胚胎大鼠皮層神經元、NSCs的生物相容性。 三、膠原-碳納米管復合物薄膜對胚胎大鼠皮層神經元突起發(fā)育的影響：將胚胎大鼠皮層神經元接種在材料的表面，在體外培養(yǎng)3d后，對細胞進行固定，進行微管相關蛋白-2的免疫熒光染色，并采用IMAGE J軟件中的Neurite Tracer插件對神經元突起的數(shù)量和長度進行測量記錄，并進行統(tǒng)計分析。同時，通過SEM對體外培養(yǎng)7d的皮層神經元與材料接觸的表面形貌進行觀察。 四、膠原-碳納米管復合物薄膜對胚胎大鼠皮層NSCs分化的影響：我們將胚胎大鼠皮層NSCs接種在材料的表面，通過撤掉EGF、bFGF，來觀察體外培養(yǎng)7d后，材料對NSCs分化為不同譜系神經細胞的影響，并進行統(tǒng)計分析。此外，采用SEM對體外培養(yǎng)7d后，NSCs與材料接觸的表面形貌進行觀察。 結果 一、膠原-碳納米管復合物薄膜的制備：1、分離所得的鼠尾I型膠原溶液清澈透亮，黏度適中，膠原濃度約為3mg/ml；2、制備的膠原-碳納米管混合溶液肉眼觀察無明顯聚集物存在，TEM觀察：1.5mg/ml和2mg/ml的膠原-碳納米管溶液中的聚集物相對較多；3、制備的膠原-碳納米管復合物薄膜肉眼觀察均勻程度高，無明顯雜質存在。 二、膠原-碳納米管復合物薄膜的物理學和生物學評價： 1、SEM觀察，不同濃度的膠原-碳納米管復合物薄膜的均勻程度不同，同透射電鏡結果一致，1.5mg/ml和2mg/ml濃度組的膠原-碳納米管復合物薄膜出現(xiàn)的聚集物較多，表面形貌相對不均勻； 2、電化學工作站導電性測量結果發(fā)現(xiàn)：從0mg/ml到2mg/ml的不同濃度的膠原-碳納米管復合物薄膜的電阻率（*m）分別為：9.19x104，14.1，5.77x10-1，1.89x10-3，1.54x10-4； 3、阿爾瑪藍生物相容性檢測結果發(fā)現(xiàn)： 體外培養(yǎng)7d后，從0mg/ml到2mg/ml的不同濃度的膠原-碳納米管復合物薄膜上，胚胎大鼠皮層神經元的平均氧化還原率（%）分別為：48.36±5.22，55.32±5.15，55.37±2.68，44.39±2.82，34.66±2.97，差異有統(tǒng)計學意義（P0.01），且0mg/ml組與1.5mg/ml組，0.5mg/ml組與1mg/ml組無統(tǒng)計學差異，余各組氧化還原率不同（P0.01）。 體外培養(yǎng)7d后，從0mg/ml到2mg/ml的不同濃度的膠原-碳納米管復合物薄膜上，胚胎大鼠皮層NSCs的平均氧化還原率（%）分別為：20.89±0.45，25.03±2.78，19.78±1.21，19.35±1.20，19.42±0.60，差異有統(tǒng)計學意義（P0.01），且0.5mg/ml組與0mg/ml、1mg/ml、1.5mg/ml、2mg/ml組氧化還原率不同（P0.01）。 三、膠原-碳納米管復合物薄膜對胚胎大鼠皮層神經元突起發(fā)育的影響 1、體外培養(yǎng)3d后，從0mg/ml到2mg/ml的不同濃度組的膠原-碳納米管復合物薄膜材料上的神經元的突起的數(shù)量（個）分別為：1.17±0.64，1.06±0.25，1.49±0.78，1.04±0.19，1.06±0.35，差異有統(tǒng)計學意義（P0.01），且1mg/ml組與其他各組神經元突起的數(shù)量不同（P0.01）； 2、體外培養(yǎng)3d后，從0mg/ml到2mg/ml的不同濃度組的膠原-碳納米管復合物薄膜材料上的神經突起的平均長度（μm）分別為：56.13±26.88，68.23±48.73，84.48±32.53，73.08±23.74，77.15±27.67，差異有統(tǒng)計學意義（P0.05），且0mg/ml組與1mg/ml、2mg/ml組突起的平均長度不同（P0.05）。 四、膠原-碳納米管復合物薄膜對胚胎大鼠皮層NSCs分化的影響 1、體外培養(yǎng)7d后，在0mg/ml到2mg/ml的不同材料表面上，胚胎大鼠皮層NSCs自發(fā)分化為MAP-2抗體陽性的神經元的比例（%）分別為：25.69±0.92，28.54±1.05，28.15±0.96，24.27±1.66，24.02±1.21，差異有統(tǒng)計學意義（P0.01），且0mg/ml組與0.5mg/ml、1mg/ml組，0.5mg/ml組與0mg/ml、1.5mg/ml、2mg/ml組，1mg/ml組與0mg/ml、1.5mg/ml、2mg/ml組分化為神經元的比例不同（P0.05）； 2、體外培養(yǎng)7d后，在0mg/ml到2mg/ml的不同材料表面上，胚胎大鼠皮層NSCs自發(fā)分化為GFAP抗體陽性的星型膠質細胞的比例（%）分別為：44.02±0.61，27.59±0.11，32.19±1.23，30.25±2.23，23.89±1.33，差異有統(tǒng)計學意義（P0.01），且1.5mg/ml組與0.5mg/ml、1mg/ml組分化為星型膠質細胞的比例相同（P0.05），余各組兩兩比較均不相同（P0.05）。 3、體外培養(yǎng)7d后，在0mg/ml到2mg/ml的不同材料表面上，胚胎大鼠皮層NSCs自發(fā)分化為OLIGODENDROCYTE抗體陽性的少突膠質細胞的比例（%）分別為：25.98±11.41，39.78±6.74，35.91±4.64，32.90±7.14，27.07±3.21，各組間比較無統(tǒng)計學差異。 結論 1、膠原-碳納米管復合物薄膜的生物相容性與材料的濃度及細胞的種類有關，0.5~1.5mg/ml組的生物相容性較好，而2mg/ml組的生物相容性較差。 2、不同濃度的膠原-碳納米管復合物薄膜對神經元突起的發(fā)育的影響不同，1mg/ml的膠原-碳納米管復合物薄膜可以促進胚胎大鼠皮層神經元突起的發(fā)育。 3、不同濃度的膠原-碳納米管復合物薄膜對NSCs分化的影響不同，0.5mg/ml和1mg/ml的膠原-碳納米管復合物薄膜可以促進胚胎大鼠NSCs向神經元分化；0.5mg/ml、1mg/ml、1.5mg/ml和2mg/ml的膠原-碳納米管復合物薄膜均能抑制胎大鼠皮層NSCs向星型膠質細胞分化，且2mg/ml組最明顯；不同材料對胚胎大鼠皮層NSCs向少突膠質細胞分化比例無差別。
[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.

【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741.05

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