功能化多肽水凝胶细胞外基质对神经干细胞增殖分化的调控研究

Functionalized polypeptide hydrogel matrix for ex vivo modulation of proliferation and differentiation of neural stem cells

目的探讨一种整合精氨酸-甘氨酸-天冬氨酸（RGD）环肽的功能化多肽水凝胶RADA16-c（RGDfK）细胞外基质对神经干细胞（NSCs）增殖分化的调控影响，以期为涉及NSCs增殖分化调控因素的研究提供一种能高度模拟体内三维条件的体外仿生研究模型。方法收集新生1d龄SD大鼠脊髓组织，采用完全培养基培养脊髓源性NSCs，采用细胞免疫荧光染色观察NSCs形态并鉴定。实验分3组，即Ⅰ型胶原组、基本序列为16个氨基酸残基的两性多态水凝胶（RADA16）组和RADA16-c（RGDfK）组，分别选择Ⅰ型胶原、RADA16及RADA16-c（RGDfK）[由ac-（RADA）4-CONH2和ac-（RADA）4-GG-cyclo（RGDtK）按摩尔比3：1物理混合而成]这3种水凝胶，采用AR2000EX型流变仪测量水凝胶的弹性模量，建立水凝胶细胞外基质与脊髓源性NSCs三维共培养模型，通过噻唑蓝（MTT）法检测不同水凝胶细胞外基质体系中NSCs的增殖活性，及细胞免疫化学染色和激光共聚焦观测技术观察NSCs的分化表达。结果（1）RADAl6及RADA16-c（RGDfK）水凝胶弹性模量在生理条件下分别为（0．42±0．07）kPa及（0．47±0．09）kPa，2者间差异无统计学意义（P〉0．05）；而Ⅰ型胶原弹性模量为（0．87±0．12）kPa，明显强于其他2组，差异有统计学意义（P〈0．05）。（2）RADA16-cRGDfK）细胞外基质中神经球形态细胞团均匀分布，克隆球直径相当，介于200～300μm。RADA16组和RADA16-（cRGDfK）组较Ⅰ型胶原组具有更好的促细胞增殖特性，其中RADA16-（cRGDfK）组对NSCs体外增殖的支持能力最优，与其他2组比较差异具有统计学意义（P〈0．05）。（3）RADA16组和RADA16-c（RGDfK）组在血清诱导环境下可见大量神经前体细胞分化为神经胶质细胞，神经元分化比例较低，分别为17．6％±3．1％及19．0％±3．6％，与Ⅰ型胶原组神经元分化比例（10．6％±2.3％）差异均有统计学意义（P〈0．05）。结论功能化多肽水凝胶RADA16-c（RGDfK）细胞外基质具有优良的神经细胞相容性和细胞黏附性，对体外脊髓源性NSCs的增殖分化有良好的促进作用，可作为神经细胞组织工程研究的新型载体。

Objective In vitro model with three dimensional cell culture provides the appealing biomimetic platform to probe the biological characteristics of multiple stem cells, which serves as an important in vitro tools to investigate regulating factors controlling the proliferation and differentiation of neural stem cells （NSCs）. The present study aims to reconstruct an integrated poly-peptide hydrogel made extracellular matrix （ECM） enhanced with cyclo-RGD molecular [c（RGDfK）] for the exploration of NSCs bio-characteristics. Methods Spinal cords from one-d-old SD rats were collected and spinal-derived NSCs were induced in the complete medium; immunofluorescence staining was employed to observe the NSCs morphology and identify NSCs. Three hydrogel including type I collagen, self-assembly poly-peptide nanofiber hydrogel （SAPNH） of RADA16 and RADA16-c（RGDfK） were employed to serve as culturing ECM of spinal-derived NSCs to mimic the ex vivo 3-D culturing. With the rheological analysis, cyto-morphological observation was performed, NSCs proliferation was observed by MTT assay, and cell immunochemistry and confocal microscopy were employed to detect the NSCs differentiation. Results SAPNH born appropriate elastic module conducive to the cellular adhesion and proliferation of neural cells （RADA16 and RADA16-c （RGDflO= （0.42 ±0.07） kPa and （0.47 ±0.09） kPa, without significant difference （P〉0.05）; however, the elastic module in the type I collagen group was （0.87±0.12） kPa, which was significantly stronger than the two groups （P〈0.05）. Uniform distribution of neuron-shape cells was noted in the extracellular matrix of RADA16-c （RGDfK） cells, with almost the diameter of cell sphere （200-300μm）; cells in the RADA16 and RADA16-c （RGDfK） had better growth characteristics than the other two groups; RADA16-c（RGDfK） had significantly better cellular adhesion and proliferation of neural cells as compared with RADAI6 and collagen groups （P〈0.05）. A large number of neural precursor cells differentiated into neurogliocytes was noted in the RADA16 group and RADA16-c （RGDfK） group, with low proportion of neuronal differentiation （17.6%±3.1% and 19.0%±3.6%, respectively）; this proportion of neuronal differentiation was significantly higher than that in the type I collagen group （10.6%±2.3%, P〈0.05）. Conclusions The functionalized SAPNH enhanced with c（RGDIK） presents the excellent biocompatibility and promotes the adhesion and proliferation of spinal NSCs. Serving as the engineered cellular vector, functionalized SAPNH has laid a solid foundation for the studies of neural regeneration and repair with novel neuro-engineering techniques in the subsequent researches.