季铵盐壳聚糖三维支架复合GNDF载间充质干细胞向神经样细胞分化

背景：壳聚糖类水凝胶因其良好的生物相容性、可降解性及对药物的缓释作用，作为支架材料近年来在组织损伤修复领域逐渐成为研究热点。 目的：探索大鼠骨髓间充质干细胞在季铵盐壳聚糖温敏凝胶支架上生长、向神经样细胞定向分化的可行性，为治疗神经系统损伤寻找理想的组织工程材料。 方法：季铵盐壳聚糖与β-甘油磷酸钠复合制成温敏凝胶，扫描电镜观察凝胶的三维结构，MTT法评价凝胶浸提液对骨髓间充质干细胞活力的影响；将牛血清白蛋白加载于凝胶支架，紫外光谱吸收法分析凝胶支架对牛血清白蛋白的缓释效果。接种大鼠骨髓间充质干细胞于凝胶支架，扫描电镜观察在支架缓释胶质细胞源性神经营养因子作用下，骨髓间充质干细胞的生长、分化情况，免疫荧光技术检测神经元烯醇化酶的表达。 结果与结论：季铵盐化壳聚糖与甘油磷酸钠复合所得凝胶支架，其多孔性特点明显，有温敏特性，对蛋白的缓释效果良好，承载大鼠骨髓间充质干细胞后，对其增殖无明显不利影响。在凝胶支架缓释的胶质细胞源性神经营养因子作用下，骨髓间充质干细胞呈现神经样细胞形态，表达神经元特异性标记物神经元烯醇化酶。说明季铵盐壳聚糖温敏凝胶对胶质细胞源性神经营养因子的缓释效果良好，其凝胶支架具有多孔径、良好生物相容性特点，可承载大鼠骨髓间充质干细胞体外生长和向神经元定向分化。

Neuron-like differentiation of mesenchymal stem cells induced by quaternary chitosan thermosensitive hydrogel scaffolds combined with glial cell line-derived neurotrophic factor

BACKGROUND：In recent years, chitosan-based thermosensitive hydrogel, as scaffold materials, have received more and more attentions in the field of tissue repair because of good biocompatibility, biodegradability and drug-sustained release.
OBJECTIVE：To explore the directed differentiation and growth of rat bone marrow mesenchymal stem cells on the quaternary chitosan thermosensitive hydrogel scaffold and to look for more ideal tissue engineering materials for the treatment of nervous system damage.
METHODS：The thermosensitive hygrogel scaffold was prepared using hydroxypropyltrimethyl ammonium chloride chitosan （HACC） andβ-glycerophosphate （β-GP）. The spatial structure of scaffold was observed by scanning electronic microscope. Effect of leaching liquor from the HACC/β-GP scaffold on the viability of bone marrow mesenchymal stem cells was detected by （4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） assay. The albumin from bovine serum was combined with the scaffold, and the slow-release effect of the scaffold was detected by ultraviolet absorption spectrometry. Bone marrow mesenchymal stem cells were incubated onto the compound scaffold at 3 passages. The adhesion, growth and differentiation of bone marrow mesenchymal stem cells on the compound scaffold were observed by the scanning electron microscope. Neuron-specific enolase was detected by immunofluorescence.
RESULTS AND CONCLUSION：The porosity and thermal sensitivity of HACC/β-GP scaffold and slow-release effect of glial cellline-derived neurotrophic factor were apparent. The results of MTT showed that the compound scaffold cannot take apparent negative effects to the proliferation of bone marrow mesenchymal stem cells. After inoculation, bone marrow mesenchymal stem cells permeated the porous structure of the scaffold and adhered to the scaffold. Under the role of glial cellline-derived neurotrophic factor, bone marrow mesenchymal stem cells showed neuron-like cellmorphology and cells co-cultured with the compound scaffold expressed the marker of neurons, neuron-specific enolase. Under the role of slow-release glial cellline-derived neurotrophic factor, bone marrow mesenchymal stem cells can grow wel in vitro and differentiate into neuron-like cells on the HACC/β-GP scaffold.