壳聚糖作为中枢神经系统支架材料的可行性研究

Feasibility study of chitin as a central nervous system scaffold material

目的探索壳聚糖作为中枢神经系统组织工程支架材料的可行性。方法选择孕14～16dWistar大鼠剖宫取胎鼠脑进行分离、培养,经巢蛋白、胶原纤维酸性蛋白、特异性烯醇化酶和半乳糖脑苷酯免疫细胞化学染色鉴定获得神经干细胞,种植于壳聚糖膜和明胶膜表面培养,然后行巢蛋白免疫细胞化学染色,观察神经干细胞在壳聚糖膜和明胶膜表面的贴附、生长和分化状态,以及壳聚糖、明胶的降解速度。结果从胎鼠大脑分离获得的神经干细胞,巢蛋白染色呈阳性反应,可分化形成神经元和胶质细胞,分化细胞分别呈胶原纤维酸性蛋白、特异性烯醇化酶和半乳糖脑苷酯染色阳性反应。神经干细胞接种后12h即贴附于壳聚糖膜表面,24h壳聚糖膜和明胶膜均贴附牢固;48h明胶膜表面神经球周围首先出现带突起的分化细胞,壳聚糖膜迟至生长第3天才出现。神经干细胞接种生长后第3天,壳聚糖膜部分降解,明胶膜完全降解;第4天膜表面长满分化细胞;第5、6天壳聚糖膜大部分降解,分化细胞部分死亡。结论壳聚糖与神经干细胞生物相容,具有促细胞贴附和分化作用,由于机械性能差,不宜作为中枢神经组织工程的结构性生物支架材料,但可以单独或者联合促贴附物明胶作为促神经干细胞贴附和分化的支架表面修饰剂。

Objective To explore the feasibility of chitosan as a tissue engineering scaffold material for central nervous system. Methods Neural stem cells from the brain of 14-16 day embryonic Wistar rats were isolated and cultured. These cells were idenlified by immunohistochemical staining with nestin, glial fibrillary acidic protein （GFAP）, specific enolase （SE） and galactocerebroside （GalC） and seeded on the surfaces of Chitosan films and gelatin films at a density of 10^5cells/ml. The cultured neural stem cells were stained by nestin immunohistochemical staining, then their attachment, growth and differentiation state and the degradation speed of chitosan and gelatin were observed. Results Neural stem cells isolated from fatal rat brain were nestin positive and could differentiated into neurons and glial cells. Which were GFAP, SE and GalC immune positive cells. Neural stem cells started attaching on the surface of chitosan films and in 12 hours and attached firmly on chitosan and gelatin films in 24 hours after seeding and the differentiated cells with processes were found on gelatin and chitosan films in 48 hours or 3 day respectively. On the 3 rd day after growth of seeding neural stem cells, the chitosan film partly degraded, but the gelatin film dissolved completely. The differentiated cells were grown fully on the film at 4 th day. On the 5 th day most part of chitosan was degraded and the differentiated cells died partly. Conclusion Chitosan is biocompatible with neural stem cells in vitro. It can promote the attachment and differentiation of cells on films, but due to its poor mechanical properties, chitosan is unappropriate in application of tissue engineering structural scaffold biomaterial for central nervous system. But it can be used alone or in combination with gelatin as a surface modification material to accelerate neural stem cell attachment and differentiation.