摘要
目的构建通过京尼平（GP）交联有脑源性神经营养因子（BDNF）并携带有人源性脐带间充质干细胞（hUC—MSCs）的复合壳聚糖支架，检测该复合支架对hUC-MSCs的影响，以及hUC-MSCs在复合支架上分化，为创伤性脑外伤（TBI）后利用hUC-MSCs移植进行相关神经修复提供实验基础。方法通过低温冻干技术制备颗粒状壳聚糖多孔支架，并通过GP在支架上交联BDNF，从而构建出复合壳聚糖支架（简称CGB支架）。检测CGB支架的孔径、孔隙率等理化特征；通过酶联免疫吸附试验法（ELISA）检测CGB支架上30d经GP交联的BDNF的释放规律；通过Transwell实验系统研究支架释放的BDNF对神经干细胞（NSCs）分化的影响。结果细胞计数试剂盒（CCK-8）检测与CGB复合支架共培养的1、3、7、10d的脐带间充质干细胞（UC—MSCs）的活性分别为（87．3±6．1）％、（85．9±5．7）％、（86．1±7．5）％、（85．4±3．8）％，与阴性对照组（100．0％）比较差异无统计学意义（P〉0．05），与阳性对照组[（52．4±6．3）％、（38．0±2．1）％、（24．6±11．0）％、（11．9±3．5）％]比较差异有统计学意义（P〈0．05）。证实hUC—MSCs与CGB支架共培养后第1天的细胞黏附率明显高于其他时间段（P〈0．05）；支架通过GP交联BDNF后BDNF呈可控的稳定释放，可以持续30d以上，然而无GP交联剂的支架（CB支架）仅在前3d释放BDNF；同时，研究证实CGB支架上所释放的BDNF体外能促进NSCs的向神经元的分化。结论CGB与覆盖在其上的hUC—MSCs具有良好的生物相容性，这为TBI后通过CGB复合支架移植hUC-MSCs提供了可能。
Objective This study tested the cytotoxicity of a brain - derived neurotrophic factor （BDNF） blended chitosan scaffold with human umbilical cord mesenchymal stem cells （hUC - MSCs）, and the in vitro effect of BDNF blended chitosan scaffolds on neural stem cell differentiation with the aim of con- tributing alternative methods in tissue engineering for the treatment of traumatic brain injury （TBI）. Meth- otis The chitosan scaffold based on immobilization of BDNF by genipin （GP） as a crosslinking agent re- ferred to hereafter as a CGB scaffold was prepared by freezingdrying technique, hUC - MSCs were co - cul- tured with the CGB scaffold. Fluorescent nuclear staining （ Hoechst 33342） was employed to determine the attachment of the hUC - MSCs to CGB scaffolds on the 1 st, 3rd, 7th and 10th day of co - culture. The via- bility of hUC - MSCs adhered to the CGB scaffold was determined by digesting with O. 25% trypsin and e- valuating with the cell counting kit - 8 （ CCK - 8）. Prior to this, the diameter and porosity of CGB scaf- folds were measured. The amount of BDNF released from CGB over a 30 - days period was determined by enzyme -linked immunosorbent assay （ELISA）. Finally, we investigated whether the released BDNF can induce NSC to differentiate into neurons. Results The CGB scaffolds released BDNF in a uniform profile, whereas the CB scaffolds only released BDNF during the first 3 days. BDNF released from CGB scaffold promoted neuronal differentiation of neural stem cells（NSCs） and led to significant differences in differenti- ation rate and average neuron perimeter compared with the control group. Conclusion The results of this study demonstrate that CGB scaffolds are biocompatible with hUC -MSCs and that granular CGB scaffolds covered with hUC - MSCs are expected to generate new advances for future treatment of traumatic brain injury.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第6期1593-1596,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金（812713681）
江苏省自然科学基金（SBK201220575）
关键词
脐带间充质干细胞
壳聚糖
脑源性神经营养因子
京尼平
神经干细胞
Human umbilical cord mesenchynaal stem cells
Chitosan scaffold
Brain - de- rived neurotrophic factor
Genipin
Neural stem cell
