嗅鞘细胞无血清上清液诱导C17.2神经干细胞定向分化及分化后细胞活力检测  

DIFFERENTIATION OF C17.2 NEURAL STEM CELLS INTO NEURAL CELLS INDUCED BY SERUM-FREE CONDITIONED MEDIUM OF OLFACTORY ENSHEATHING CELLS AND CELL VIABILITY DETECTION OF DIFFERENTIATED CELLS

作　　者：王磊[1,2] 段答[1] 赵振宇[1] 滕晓华[1] 刘波[1] 葛丽特[1,2] 卢明[1,2] 

机构地区：[1]解放军第163医院(湖南师范大学第二附属医院)神经外科,长沙410003 [2]湖南师范大学医学院

出　　处：《中国修复重建外科杂志》2014年第5期633-638,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81371358);湖南省研究生科研创新项目(GX2012B230)~~

摘　　要：目的探讨体外采用嗅鞘细胞(olfactory ensheathing cells,OECs)无血清上清液诱导小鼠C17.2神经干细胞(neural stem cells,NSCs)向神经元定向分化及分化后的细胞活力。方法采用新生3 d昆明小鼠嗅球,分离培养OECs,并制备无血清上清液。C17.2 NSCs置于含15%FBS的H-DMEM/F12培养基培养,传至第3代,待细胞生长至80%融合时,分别加入OECs无血清上清液(实验组)、H-DMEM/F12培养基(对照组)诱导培养;以未诱导的C17.2NSCs作为空白对照组。倒置显微镜下观察诱导后细胞生长情况,于诱导5 d后收集细胞行微管相关蛋白2(microtubuleassociated protein 2,MAP-2)及β-微管蛋白Ⅲ(β-tubulin-Ⅲ)免疫荧光染色鉴定,Western blot检测细胞巢蛋白(Nestin)、β-tubulin-Ⅲ、MAP-2蛋白表达情况,检测乳酸脱氢酶(lactate dehydrogenase,LDH)漏出率并行MTT法检测细胞活力。结果实验组:诱导后24 h细胞胞体开始收缩,3 d后分化的细胞明显增多,突触增长;对照组:诱导后24 h细胞形态无明显改变,3 d后细胞胞体皱缩,细胞核质浓缩,并发生细胞裂解、破碎。诱导后5 d,免疫荧光染色示实验组分化后细胞β-tubulin-Ⅲ和MAP-2表达均呈阳性,对照组及空白对照组均无表达。Western blot检测显示实验组中NSCs标志物Nestin蛋白表达明显减少,神经元标志物β-tubulin-Ⅲ和MAP-2表达增加,与对照组及空白对照组比较差异均有统计学意义(P<0.05)。实验组LDH漏出率为130.60%±6.86%,显著低于对照组的178.20%±5.44%;细胞活力为62.20%±3.82%,显著高于对照组的18.00%±3.83%;比较差异有统计学意义(P<0.05);但均低于空白对照组的100%(P<0.05)。结论含有OECs分泌蛋白的OECs无血清上清液不仅能诱导NSCs向神经元分化,还能维持分化后细胞活力。Objective To study the possibility of the C17.2 neural stem cells （NSCs） differentiating into neural cells induced by serum-free condition medium of olfactory ensheathing cells （OECs） and to detect the cell viability of the differentiated cells. Methods OECs were isloated and cultured from the olfactory bulbs of 3-day-old postnatal mouse to prepare serum-free condition medium of OECs. After C17.2 NSCs were cultured with H-DMEM/F12 medium containing 15% FBS and the cell fusion reached 80%, the 3rd passage cells were induced by serum-free condition medium of OECs in the experimental group, by H-DMEM/F12 in the control group, and non-induced C17.2 NSCs served as the blank control group. The growth condition of cells was observed with inverted microscope. After 5 days, the immunofluorescence staining [microtubule-associated protein 2 （MAP-2） and β-tubulin-III] and Western blot （Nestin, β-tubulin-III, and MAP-2） were carried out to identify the neural cells derived from NSCs. The cell viabilities were measured by MTT assay and the quantity of lactate dehydrogenase （LDH） release in the medium. Results In the experimental group, the C17.2 NSCs bodies began to contract at 24 hours after induction, and the differentiated cells increased obviously with long synapse at 3 days after induction; in the control group, the cell morphology showed no obvious change at 24 hours, cell body shrinkage, condensation of nuclear chromatin, and lysis were observed at 3 days. The immunofluorescence staining showed that β-tubulin-III and MAP-2 of C17.2 NSCs were positive at 5 days after induction, and Western blot suggested that the expression of Nestin protein declined significantly and the expressions of β-tubulin-III and MAP-2 protein were increased in the experimental group, showing significant differences when compared with those in the control group and blank control group （P 〈 0.05）. The LDH release and the cell viability were 130.60% ± 6.86% and 62.20% ± 3.82% in the experimental group, and were 178.2

关 键 词：神经干细胞 嗅鞘细胞 诱导分化 细胞活力 小鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]