【作者】 张振兴；

【导师】 郑启新；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 目的：获得均一性的神经干细胞方法：取新生1天内乳鼠的大脑皮层,机械分离出神经干细胞,无血清培养液培养,倒置相差显微镜观察干细胞的生长情况,Nestin免疫荧光法对神经干细胞进行鉴定结果：分离培养出的细胞聚集成神经球,呈悬浮状,可在体外大量增殖和长期存活。Nestin染色呈阳性结论：体外成功地分离及培养了大鼠NSCs目的：设计与合成功能化自组装多肽水凝胶方法：采用固相法合成多肽RADA16与RADA16-IKVAV,将两者混合制备功能化自组装多肽水凝胶IKVAVmx。原子力显微镜观察其形态学特征结果：IKVAVmx可自组装形成水凝胶,原子力显微镜证实其由纳米纤维交织而成结论：成功地合成了IKVAVmx自组装多肽水凝胶目的：检测功能化自组装多肽水凝胶体外与神经干细胞的生物相容性方法：采用无血清培养法从新生大鼠皮层分离培养NSCs。将NSCs分别接种到功能化自组装多肽水凝胶IKVAVmx(实验组)与自组装多肽水凝胶RADA16(对照组)表面。用CCK-8法检测细胞在两种水凝胶中的增殖能力。倒置显微镜观察细胞在两种水凝胶中的形态。激光扫描共聚焦显微镜观察细胞迁移情况。应用Nestin、MAP2、GFAP和CC-1免疫荧光染色,检测神经干细胞分化情况。Western blot检测两种水凝胶中NSCs的MAP2蛋白表达情况。结果：实验组中NSCs的增殖及迁移能力均明显高于对照组(P<0.05)；实验组中MAP2阳性细胞百分率较对照组显著提高(P<0.05),GFAP阳性细胞百分率较对照组显著降低(P<0.05)。实验组中MAP2的蛋白表达含量明显高于对照组(P<0.05)。结论：功能化自组装多肽水凝胶具有良好的细胞生物相容性,从而为神经组织工程提供了一个有前景的生物材料。更多还原

【Abstract】 Objective:To culture rat neural stem cells (NSCs) the in vitro. Methods:NSCs harvested from the cerebral cortex of neonatal one-day rats were triturated and cultivated in serum-free medium. The morphology of growth of cells was demonstrated by inverted phase contrast microscopy. Immunofluorescence staining with Nestin was used to identify NSCs. Result:The cells isolated and cultured grew in a manner of neurospheres and suspended in the medium. The cells were capable of proliferating and maintaining long-term survival in vitro. Conclusion:NSCs derived from cerebral cortex of neonatal rats were successfully isolated and cultured in vitro.Objective:To design and synthesize a new self-assembling peptide hydrogel scaffold.Methods:Peptide RADA16 and RADA16-IKVAV were synthesized by solid phase method. The functionalized self-assembling peptide named IKVAVmx was then made by mixing the above two peptide solutions. The morphological features of IKVAVmx were studied by atom force microscope (AFM). Result:IKVAVmx could self-assemble to form a hydrogel. AFM showed that self-assembly hydrogel was made up of interconnected nanofibers.Conclusion:IKVAVmx peptide was synthesized and could self-assemble into hydrogel.Objective:To observe the of functionalized self-assembling peptide hydrogel with neural stem cells in vitro. Methods:NSCs were isolated from the cerebral cortex of neonatal rats and cultured in serum-free medium. NSCs were seeded on the surface of both the functionalized self-assembling peptide hydrogel (experimental group) and RADA16 self-assembling peptide hydrogel (control group). The proliferation behavior of the cells was evaluated by CCK-8 assay. The cell migration was detected by laser scan confocal microscope. Immunofluorescence staining with Nestin, MAP2, GFAP, and CC-1 was used to assess the differentiation of NSCs. The protein expression of MAP2 was determined by Western blot analysis. Result:The power of cell proliferation and migration in experimental group was significantly higher than that of control group (P<0.05). Compared with control group, the percentage of MAP2 positive cells increased significantly (P<0.05) and the percentage of GFAP positive cells decreased significantly (P<0.05) in experimental group. Western blot analysis indicated that MAP2 activity in IKVAVmx scaffold was significantly higher than that in pure RADA16 scaffold (P<0.05). Conclusion:The functionalized self-assembling peptide hydrogel has good biocompatibility with NSCs, suggesting that it may be a promising biomaterial for neural tissue engineering.更多还原