水凝胶三维培养对神经干细胞增殖特性及分化潜能的研究实验  

An experimental study on the proliferation characteristics and differentiation potential of neural stem cells in three-dimensional hydrogel culture

作　　者：于蒙洋 钟慧琳 刘畅[1] 钟少文 邹清雁 沈波[1] 郑雨轩 唐程[1] 王斌[1] Yu Mengyang;Zhong Huilin;Liu Chang;Zhong Shaowen;Zou Qingyan;Shen Bo;Zheng Yuxuan;Tang Cheng;Wang Bin(Department of Orthopedics,Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China;Third Division,Department of Orthopedics,Second Affiliated Hospital of Luohe Medical College,Luohe,Henan 462300,China;Guangzhou Celldicine Biotech Co.,Ltd.,Guangzhou 510633,China)

机构地区：[1]广州医科大学附属第二医院骨外科,广州510260 [2]漯河市高等医学专科学校第二附属医院骨三科,河南漯河462300 [3]广州熙帝生物科技有限公司,广州510633

出　　处：《中华生物医学工程杂志》2021年第6期581-590,共10页Chinese Journal of Biomedical Engineering

基　　金：广州市科技计划项目(201904010002);广东省自然科学基金项目(2020A1515010211)。

摘　　要：目的观察对比神经干细胞(NSC)在Matrigel水凝胶培养和悬浮培养的神经干细胞的生长形态、增殖、分化潜能、细胞产量及存活率等情况。方法分离提取孕12~13 d胎鼠大脑组织中的神经干细胞,用悬浮培养、三维Matrigel水凝胶支架培养2种培养方案传代神经干细胞,分别进行形态学观察分析,免疫组织化学荧光术鉴定巢蛋白(Nestin)和Sox-2的表达,及流式细胞术鉴定免疫表型;用10%的胎牛血清诱导分化培养基诱导神经干细胞分化后,行免疫荧光染色鉴定神经干细胞的终末分化细胞:神经元、少突胶质细胞、星形胶质细胞,对应特异性标记物分别为NeuN、CNPase、GFAP。结果免疫荧光鉴定P1代NSC,Nestin和Sox-2双阳性表达;P2代流式细胞鉴定Nestin∶90%,证明从胎鼠大脑能够获取纯度较高的神经干细胞;台盼蓝染色计数表明Matrigel基质胶三维培养方案细胞产量明显升高,是悬浮培养方案的5~10倍,收取的神经干细胞的存活率较悬浮培养稍增高,但无统计学差异意义(P=0.0812)。本试验两种方法培养的NSC均具多向分化性能,均能分化成神经元、星形胶质细胞及少突胶质细胞,分化后各终末细胞所占比例基本一致(P值分别为:0.1302;0.1108;0.1605)。结论相比于悬浮培养,三维Matrigel水凝胶支架方案培养出的NSC,具有活性高,增殖能力强,消化后死亡率低,细胞产量大,能达到悬浮培养的5倍以上,能稳定传代及便于细胞形态学观察等优点,并在连续传至多代后仍能保持NSC的基本特性和多向分化潜能。Objective To investigate the morphology,proliferation,differentiation potential,growth yield and cell viability of neural stem cells(NSC)in Matrigel hydrogel culture vs suspension culture.Methods NSCs were isolated and extracted from the brain tissues of fetal rats at 12th to 13th days of pregnancy,and were then grown in suspension culture and 3D Matrigel hydrogel scaffolds,respectively,for cell passage.The morphology of NSCs was studied.Fluorescence immunohistochemistry was used to identify the expression of Nestin and Sox-2.Flow cytometry was used to identify the immunophe-notype.After induced differentiation in culture media containing 10%fetal calf serum,immunoflu-orescence staining was performed to identify the terminally differentiated cells from NSCs includ-ing neurons,oligodendrocytes,astrocytes,as well as their corresponding specific markers NeuN,CNPase,and GFAP.Results Immunofluorescence showed dual positivity of Nestin and Sox-2 ex-pression in the first-passage NSCs,and flow cytometry showed positive Nestin expression in 90%of second-passage NSCs,indicating the high purity of NSCs obtained from the brain of fetal mice.Cell counts with Trypan blue staining showed that 3D culture with Matrigel matrix gel led to signif-icantly higher yield of cells,which was 5-10 times the yield by suspension culture.The viability of the NSCs recovered in Matrigel matrix gel was slightly higher than that in suspension culture,alt-hough the difference was not statistically significant(P=0.0812).The NSCs cultured with either method in this experiment showed multipotency,and were able to differentiate into neurons,astro-cytes and oligodendrocytes with comparable proportions of terminally differentiated cells(P=0.1302,0.1108,and 0.1605,respectively).Conclusion Compared with suspension culture,NSCs cultured by 3D Matrigel hydrogel scaffold show high viability,vigorous proliferation,low mortality after digestion,and a larger cell yield more than 5 times as in suspension culture.The cultured NSCs are advantageous in stable passa

关 键 词：神经干细胞 悬浮培养 三维培养 Matrigel水凝胶支架 

分 类 号：R651.2[医药卫生—外科学]