新型三维多肽自组装凝胶诱导神经干细胞实验研究

Neural Stem Cells Induced by Neotype Three-dimensional Polypeptide-based Self-assembled Hydrogel

两亲性多肽与含神经干细胞(Neural stem cells,NSCs)的DMEM/F12混合,自组装为三维多孔细胞凝胶复合体,研究细胞在凝胶表面及内部的分化情况。取乳鼠大脑皮质,机械分离无血清培养法获取神经干细胞。1wt%多肽加入等体积密度为1×105/mlNSCs的DMEM/F12液,形成三维细胞凝胶复合体,细胞分布于凝胶内部(exper-imentgroup,EG);1wt%多肽与等体积DMEM/F12液混合,自组装为二维凝胶薄膜,1×105/mlNSCs接种于薄膜表面(control group,CG);无血清培养2周,免疫细胞化学法鉴定。透射电子显微镜(TEM)示凝胶由纳米纤维构成,纤维直径有3～5nm,长度为100～1500nm。获取细胞为Nestin阳性神经干细胞,纯度可达50%以上。激光扫描共聚焦显微镜(LSCM)观察:细胞在凝胶内部接种2d,存活达90%以上;1周后分化为NF及GFAP阳性细胞,分化率分别为50%±4.2%及20%±2.8%;细胞在凝胶表面1周后亦分化为NF阳性细胞及GFAP阳性细胞,分化率分别为40%±3.4%及31%±2.3%。肽基三维凝胶为细胞生存提供三维支撑,诱导细胞主要向神经元方向分化。表明肽自组装凝胶可作为新型神经组织工程支架材料。

The amphiphilic polypeptide （PA） was self-assembled into three-dimensional （3-D） porous complex of hydrogel and cells with the addition of NSCs-containing DMEM/F12. Cell differentiation in the surface and that within hydrogel were described. Cells harvested from the cerebral cortex of neonatal mice were triturated and cultivated in serum-free media. 1wt% PA was added into same volume of DMEM/F12 with cell concentration of 1 ×105/ml and self-supported into 3-D hydrogel-cell composition; cells suspended within hydrogel being maintained （Experiment group,EG）. 1wt% PA was self-assembled into two-dimensional （2-D） hydrogel films triggered by addition of DMEM/F122,and then 1 ×105/ml NSCs was seeded in the surface of films （Control group,CG）. Cells in EG and CG were incubated in serum-free media for two weeks and stained with immunocytochemistry methods. TEM showed that the hydrogel derived from PA was composed of network nanofibers with their diameter ranging from 3 to 5 nm and length ranging from 100 nm to 1.5 μm. Above 50% of cells obtained were Nestin positive cells. LSCM observations demonstrated that above 90% of cells survived two days after incubation within hydrogel,and were differentiated into NF and GFAP positive cells one week after incubation,their differentiation rates were 50%±4.2% and 20%±2.8% respectively; however,cells in CG were also differentiated into NF and GFAP positive cells,their differentiation rates were only 40%±3.4% and 31%±2.3% separately. Peptide-based hydrogel was able to provide 3-D environments for cell survival and induce primarily the differentiation of NSCs into neurons. Our data indicated that peptide-directed self-assembly of hydrogels was useful and it served as the neotype nerve tissue engineering scaffolds.