含IKVAV两亲性肽自组装凝胶二维诱导神经干细胞的分化  

Two-dimensional differentiation of neural stem cells induced by self-assembled hydrogel from IKVAV-containing peptide amphiphile

作　　者：宋玉林[1] 郑启新[2] 吴凯[1] 殷明[1] 

机构地区：[1]南昌大学第二附属医院骨科,江西省南昌市330006 [2]华中科技大学同济医学院协和医院骨科,湖北省武汉市430000

出　　处：《中国组织工程研究与临床康复》2009年第34期6667-6670,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:脊髓基质支架是构建脊髓组织工程的关键因素,目前,各种无机或有机高分子材料均不是构建脊髓组织工程理想支架材料,含有活性结构的多肽在体液或培养液促发下可形成与脊髓黏弹性一致多孔支架,有望成为优良脊髓组织工程支架材料。目的:将鼠神经干细胞接种于含IKVAV两亲性肽自组装凝胶表面,观察其对细胞分化影响。设计、时间及地点:细胞水平对照观察,实验于2006-10/11在华中科技大学同济医学院协和医院骨科实验室完成。材料:多肽序列C16H31O-A3G4D2IKVAV(MW=1438.31,纯度=96.06%)为自行设计合成。乳鼠由华中科技大学同济医学院实验动物中心提供。方法:机械分离法从乳鼠大脑皮质获取神经干细胞;10g/L两亲性肽溶液在DMEM/F12触发下自组装为三维多孔凝胶;1×108L-1神经干细胞悬液分别接种于凝胶表面(实验组)及多聚赖氨酸包被的盖玻片表面(对照组)培养2周,免疫细胞化学染色鉴定。主要观察指标:①分离细胞免疫组织化学染色观察巢蛋白、神经元特异性烯醇化酶及胶质纤维酸性蛋白表达。②凝胶纳米纤维形态。③凝胶表面免疫细胞化学染色观察巢蛋白、神经丝及胶质纤维酸性蛋白表达。结果:分离的细胞为巢蛋白阳性的神经干细胞,可分化为神经元特异性烯醇化酶阳性神经元及胶质纤维酸性蛋白表达阳性胶质细胞;透射电镜示凝胶由纳米纤维构成,纤维直径有3～5nm,长度100～1500nm;培养7d后,实验组观察到细胞长出突起,可发现神经丝阳性神经元样细胞,标记为红色荧光,胶质纤维酸性蛋白阳性胶质细胞样细胞,标记为绿色荧光;对照组仅形成未分化细胞球,巢蛋白阳性,标记为绿色荧光。结论:含IKVAV肽自组装三维凝胶与神经干细胞有良好的细胞相容性,可诱导神经干细胞分化为神经元及胶质细胞。BACKGROUND： Spinal cord matrix scaffold is a key factor in spinal cord tissue engineering. Currently, neither inorganic nor organic polymers are ideal scaffold materials for spinal cord tissue engineering. The polypeptide with active structure in humor or culture solution could form porous scaffold with coincident visco-elasticity with spinal cord, which may become excellent scaffold material for spinal cord tissue engineering. OBJECTIVE： Mouse neural stem cells （NSCs） were implanted in the surface of self-assembled hydrogel from IKVAV-centaining peptide amphiphile （IKVAV-PA） to observe its influence on cell differentiation. DESIGN, TIME AND SETTING： Contrast observation at cellular level. The experiment was performed at the Laboratory of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from October to November 2006. MATERIALS： Polypeptide sequence C16H310-A3G4D21KVAV （Mw =1 438.31, purity=96.06%） was self-made. Neonatal mice were provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. METHODS： NSCs harvested from the cerebral cortex of neonatal mice were dissociated mechanically and cultivated in serum-free media; three-dimensional self-assembly hydrogel from lwt% amphiphilic peptide was formed with addition of DMEM/F12. NSCs at density of 1 xl08/L were respectively incubated in the surface of hydrogel （experimental group） and the coverslips covered with polylysine （control group） for two weeks, and were examined immunocytochemically. MAIN OUTCOME MEASURES： Isolated cells were immunohistochemically examined for nestin, neuron specific enolase （NSE） and glial fibrillary acidic protein （GFAP）; hydrogel nanofiber morphology was observed. Expression of nestin, NSE and GFAP of the hydrogel surface was detected. RESULTS： The nestin-positive cells obtained were able to be differentiated into NSE-positive neurons and GFAP-positive glial cells; transmission electron micr

关 键 词：神经干细胞 分化 自组装 凝胶 

分 类 号：R318[医药卫生—生物医学工程]