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

发布时间：2017-12-27 22:07

本文关键词：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　出处：《中国修复重建外科杂志》2014年05期 　论文类型：期刊论文

【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(P0.05)。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(P0.05);但均低于空白对照组的100%(P0.05)。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。
[Abstract]:Objective to investigate the differentiation and differentiation of C17.2 neural stem cells (neural stem cells, NSCs) into neurons after olfactory ensheathing cells (OECs) serum-free supernatant in vitro. Methods a new 3 D Kunming mouse olfactory bulb was used to isolate and culture OECs, and the serum-free supernatant was prepared. C17.2 NSCs was cultured in H-DMEM/F12 medium containing 15%FBS, and spread to the third generation. When the cells grew to 80% fusion, they were added to OECs supernatant without serum (experimental group) and H-DMEM/F12 medium (control group) to induce culture. C17.2NSCs was used as blank control group. The growth of cells after induction were observed under inverted microscope. In 5 d after the induction, cells were collected for microtubule associated protein 2 (microtubuleassociated protein 2, MAP-2) and beta tubulin III (beta -tubulin- III) immunofluorescence staining, Western blot assay (Nestin), nestin expression of beta -tubulin- III and MAP-2 protein detection. Lactate dehydrogenase (lactate dehydrogenase, LDH) the leakage rate of cell viability was detected by MTT in parallel. Results in the experimental group, cell bodies began to contract 24 h after induction, and the differentiated cells increased significantly after 3 D. Synapse growth was observed in the control group. After 24 h induction, the morphology of cells did not change significantly. After 3 D, the cell body shrinked, the cytoplasm was condensed, and the cells lysed and broken. After induction of 5 d, immunofluorescence staining showed that the expression of beta -tubulin- III and MAP-2 in the experimental group were positive, and there was no expression in the control group and the blank control group. Western blot detection showed that the expression of Nestin protein of NSCs markers in experimental group was significantly reduced, and the expression of neuronal markers beta -tubulin- III and MAP-2 increased, compared with control group and blank control group, the difference was statistically significant (P0.05). The leakage rate of LDH in the experimental group was 130.60% + 6.86%, which was significantly lower than that in the control group (178.20% + 5.44%), and the cell viability was 62.20% + 3.82%, which was significantly higher than that in the control group (18% + 3.83%). The difference was statistically significant (P0.05), but it was lower than that in the control group 100% (P0.05). Conclusion the OECs serum-free supernatant containing OECs secreted protein can not only induce the differentiation of NSCs into neurons, but also maintain the cell viability after differentiation.
【作者单位】：解放军第163医院(湖南师范大学第二附属医院)神经外科;湖南师范大学医学院;
【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)~~
【分类号】：R329
【正文快照】： 神经干细胞(neural stem cells,NSCs)是一类能自我更新、增殖及向3种神经类型细胞分化的成体干细胞,在中枢神经系统损伤后的神经修复和再生中具有重要作用。研究表明,NSCs的分化方向受局部微环境调控,包括周围相联系的各种细胞、细胞外基质和体内各种细胞因子等[1-6],其中主要

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