神经干细胞三维培养及构建类神经组织的研究

发布时间：2019-06-02 23:28

【摘要】： 目前,神经干细胞(Neural stem cells, NSCs)的体外培养主要采用悬浮培养和贴壁培养体系,然而这两种培养体系都存在无法模拟体内NSCs生长微环境的缺点。而三维的细胞培养体系能够为细胞提供良好的生长环境,促进细胞分泌细胞外基质,从而形成与体内组织相近的结构。因此,本研究采用三维培养体系来培养NSCs,并构建出类神经组织结构。实验以大鼠海马脑组织来源的NSCs为种子细胞,选用天然生物材料-胶原水凝胶为支架材料。首先对胶原内呈三维生长的细胞与传统单层培养细胞及悬浮培养细胞的生长和代谢情况进行比较。进而采用先期使用增殖培养基、后期使用分化培养基的策略,使NSCs在胶原水凝胶内先增殖然后向神经细胞分化,形成相互连接的类神经网络。采用cck-8试剂盒(Cell counting kit-8)对所构建的三维网络内细胞的增殖情况进行检测；使用激光扫描共聚焦显微镜观察所构建的三维类网络结构内经死活染色和免疫荧光染色后的细胞生长和分化情况。最后,对三维灌注式培养NSCs进行了初步的研究。实验结果显示,胶原内三维培养的NSCs增殖倍数最大,4 d内可达70倍,而普通悬浮培养和单层培养的扩增倍数分别为2.5倍和35倍。葡萄糖和乳酸代谢检测结果表明,胶原内细胞的葡萄糖比消耗速率最小,比消耗速率曲线最平缓,且乳酸的比生成速率也最小。细胞-胶原构建物体外培养42 d后,构建物内的细胞活率维持在82%左右,细胞在构建物内分布较均匀,而且能够分化成神经元、星型胶质细胞和少突胶质细胞。此外,层粘连蛋白(Laminin, LN)预处理胶原蛋白海绵支架内NSCs的接种率(55.8±1.21%)较单纯用培养基处理的支架内接种率(43.1±1.18%)有显著提高。最后,灌注式生物反应器培养条件下,NSCs在支架中呈团簇生长、分布均匀、且阳性表达Nestin；灌注式三维培养利于细胞营养物质的供应,能够使pH值维持在细胞正常的耐受范围。由以上实验结果我们得出以下结论：胶原内的三维培养条件更加利于NSCs的增殖和代谢,而且本研究所获得的三维组织构建物在形态、细胞构成等方面均与神经组织相类似；经LN预处理的胶原蛋白海绵支架更利于NSCs的粘附,可提高细胞在支架上的接种率；灌注式生物反应器培养条件有助于NSCs在支架中均匀分布和增殖,保持NSCs的特性。
[Abstract]:At present, the culture of neural stem cell (Neural stem cells, NSCs) in vitro mainly adopts suspension culture and adherent culture system. However, these two culture systems can not simulate the growth microenvironment of NSCs in vivo. The three-dimensional cell culture system can provide a good growth environment for cells and promote the secretion of extracellular matrix, thus forming a structure similar to that of in vivo tissues. Therefore, in this study, three-dimensional culture system was used to culture NSCs, and construct neural tissue structure. In this experiment, NSCs from rat hippocampal brain tissue was used as seed cell, and collagen hydrogel, a natural biomaterial, was used as scaffold material. Firstly, the growth and metabolism of three-dimensional growth cells in collagen were compared with those of traditional monolayer cells and suspension culture cells. Then the strategy of using proliferation medium in advance and differentiation medium in the later stage was used to make NSCs proliferate first in collagen hydrogel and then differentiate into nerve cells to form a connected neural network. The proliferation of cells in the constructed three-dimensional network was detected by cck-8 kit (Cell counting kit-8). Laser scanning confocal microscope was used to observe the growth and differentiation of cells in the constructed three-dimensional network structure after dead and alive staining and immunofluorescence staining. Finally, the three-dimensional perfusion culture of NSCs was studied. The results showed that the proliferation multiple of NSCs in collagen three-dimensional culture was the highest, which could reach 70 times in 4 days, while the amplification multiple of common suspension culture and monolayer culture was 2.5 times and 35 times, respectively. The results of glucose and lactic acid metabolism showed that the specific consumption rate of glucose in collagen cells was the smallest, the specific consumption rate curve was the slowest, and the specific production rate of lactic acid was the smallest. After 42 days of culture in vitro, the cell survival rate in the construct was about 82%, and the cells were evenly distributed in the construct, and could differentiate into neurons, star glial cells and oligodendrocytes. In addition, the vaccination rate of NSCs in collagen sponge scaffold pretreated with laminin (Laminin, LN) (55.8 卤1.21%) was significantly higher than that treated with medium alone (43.1 卤1.18%). Finally, under the condition of perfusion bioreactor, NSCs grew in clusters and distributed evenly in the scaffold, and the three-dimensional culture with positive expression of Nestin; was beneficial to the supply of cell nutrients, and the pH value could be maintained in the normal tolerance range of cells. From the above experimental results, we draw the following conclusions: the three-dimensional culture conditions in collagen are more conducive to the proliferation and metabolism of NSCs, and the three-dimensional tissue structures obtained in this study are similar to nerve tissue in morphology and cell composition. The collagen sponge scaffold pretreated with LN is more beneficial to the adhesion of NSCs and can improve the inoculum rate of cells on the scaffold, and the culture conditions of perfusion bioreactor are helpful to the uniform distribution and proliferation of NSCs in the scaffold and to maintain the characteristics of NSCs.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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