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三维打印胶原蛋白-壳聚糖复合支架中大鼠神经干细胞的生长和分化情况 被引量:1

Growth and differentiation of rat neural stem cells on coilagen-chitosan composite scaffold fabricated by three-dimensional printing
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摘要 目的观察三维打印胶原蛋白-壳聚糖复合生物支架中大鼠神经干细胞(NSCs)生长和分化情况。方法取妊娠1d~16d的SD大鼠胚胎大脑皮质,分离培养和鉴定大鼠NSCs。采用低温三维打印联合冷冻干燥技术制备胶原蛋白一壳聚糖复合生物支架,将NSCs移植入三维支架共培养,并将常规悬浮培养NSCs为对照组(3组,每组5个复孔),三维打印胶原蛋白一壳聚糖复合支架共培养的NSCs为观察组(3组,每组5个复孔),使用倒置相差显微镜、扫描电镜观察细胞在支架上的生长状况。分别采用细胞计数试剂盒和免疫荧光法检测NSCs的吸光度值和分化情况。结果NSCs培养第3代神经球中免疫荧光检测提示Nestin蛋白表达阳性,诱导7d后NSCs的免疫荧光检测提示神经元特异性核蛋白(NeuN)和神经胶质纤维酸性蛋白(GFAP)表达阳性。三维打印胶原蛋白-壳聚糖复合支架为三维多孔立体结构,四边形孔径为(510±63)μm。显微镜及扫描电镜观察结果显示NSCs可在支架中很好地生长、分化。观察组共培养1、4、7、11、14d吸光度值均高于对照组[(0.34±0.04)比(0.21±0.03)、(0.70±0.08)比(0.35±0.04)、(0.92±0.11)比(0.67±0.08)、(0.75±0.09)比(0.42±0.05)、(0.53±0.07)比(0.27±0.03)],差异均有统计学意义(均P〈0.05)。三维打印胶原蛋白-壳聚糖复合支架周围和内部存在NeuN和GFAP阳性细胞。结论三维打印技术可制备微观结构可控的胶原蛋白-壳聚糖复合支架,且具有良好的生物相容性,适宜NSCs生长和分化. Objective To observe growth and differentiation of rat neural stem cells(NSCs) on collagen-chitosan (CG-CS) composite scaffold fabricated by three-dimensional(3D) printing. Methods Cerebral cortex was taken from 14-16 d SD rat embryo and NSCs were isolated, cultured and identified. CG-CS composite biological scaffolds were fabricated by 3 D printing and freeze-drying technique. Three control groups had routine suspension culture NSCs; three observation groups had NSCs cocultured with CG-CS scaffolds. Cell growth and differentiation on scaffolds were observed by inverted phase contrast microscope, scanning electron microscope, Cell Counting Kit-8 ( CCK-8 ) assay and immunofluorescence assay. Results In the 3rd generation of NSCs, expressions of Nestin protein, neuron specific nuclear protein(NeuN) and glial fibrillary acidic protein(GFAP) were positive in immunofluorescenee tests. CG-CS composite scaffolds were successfully made by porous spatial structures with quadrilateral aperture of (510 ± 63 )~Lm. NSCs grew and differentiated well on CG-CS scaffolds under microscope and scanning electron microscope; absorbance values in observation group after 1,4, 7, 11, 14 d of culture were significantly higher than those in control groupl (0. 34 ± 0. 04) vs (0. 21 ± 0.03), (0.70±0.08) vs (0.35±0.04), (0.92 ±0.11) vs (0.67±0.08), (0.75 ±0.09) vs (0.42 ± 0.05), (0.53±0.07) vs (0.27 ±0.03)] (P〈0.05). NeuN and GFAP positive NSCs could be observed outside and inside of biological scaffolds. Conclusion 3D printing technology can fabricate microstructurecontrollable CG-CS composite biological scaffolds, which have good biocompatibility and benefit growth and differentiation of NSCs.
出处 《中国医药》 2017年第3期442-446,共5页 China Medicine
基金 国家自然科学基金(81271392、81301050) 中国博士后科学基金(2013M542583)
关键词 神经十细胞 生物支架 胶原蛋白 壳聚糖 三维打印 Neural stem cells Biological scaffold Collagen Chitosan Three-dimensional printing
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