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目的:我们的目标是从源于胚胎体(EBs)的小鼠胚胎干细胞(mESCs)中分离运动神经元样细胞前体(MNLCPs),以用于发展针对运动神经元疾病的药物筛选试验和移植疗法。天然Shh蛋白(或Shh通路激动剂)和维甲酸诱导的胚胎体中,MNLCPs和未分化细胞的含量是不确定的。如果不把未分化的细胞从细胞培养中充分去除,其可能会干涉药物筛选试验或在移植后增生。我们开发了一种以密度梯度离心法为基础的富集MNLCPs的方法。方法:我们用Wichterle等人2008年改进的方法,将mESCs(HBG3:eGFP:HB9)扩大和分化。通过化学和酶学的无研磨处理,含有绿色荧光蛋白的MNLCPs和未分化细胞的胚胎体被小心轻轻地离解成单细胞。利用OptiprepTM8%~20%逐步梯度离心技术将MNLCPs回收。拥有绿色荧光蛋白的MNLCPs的含量由流式细胞仪检测。结果:我们的结果表明,在胚胎体形成前,mESCs在明胶包被的培养板上生长,其分化为MNLCPs的能力减少。比较mESCs在明胶,明胶与PMEFs,及PMEFs包被的培养板上的生长发现,mESCs在PMEFs包被的培养板上产生含绿色荧光蛋白的MNLCPs的得率为(54.1±11.0)%(x±s;n=12),在明胶包被的培养板上的得率为(2.8±1.1)%(x±s;n=9)。用密度梯度离心法获得的含绿色荧光蛋白的MNLCPs的平均含量为(87.7±5.5)%(x±s;n=3)。结论:我们的数据表明,不使用细胞分选器,无研磨解离和密度梯度离心法也能用于富集具有高存活率的MNLCPs。有必要对MNLCPs在体外、体内和表型上进行进一步的生理学意义(如神经轴突的生长及形成神经肌肉接头的能力)上的鉴定。
Objective: Our goal is to isolate motor neuron-like cell precursors (MNLCPs) from mouse embryonic stem cells (mESCs) derived from embryonic bodies (EBs) for the development of drug screening assays and grafts for motor neuron diseases therapy. The content of MNLCPs and undifferentiated cells in the natural Shh protein (or Shh pathway agonist) and retinoid-induced embryonic bodies is indeterminate. If undifferentiated cells are not adequately removed from the cell culture, they may interfere with drug screening assays or proliferate after transplantation. We developed a method to enrich MNLCPs based on density gradient centrifugation. METHODS: We expanded and differentiated mESCs (HBG3: eGFP: HB9) using a modified approach by Wichterle et al. Embryos of MNLCPs containing green fluorescent protein and undifferentiated cells are carefully and gently dissociated into single cells by chemically and enzymatically grinding-free. MNLCPs were recovered using Optiprep ™ 8% to 20% step-gradient centrifugation. The content of MNLCPs with green fluorescent protein was detected by flow cytometry. Results: Our results show that mESCs are grown on gelatin-coated plates prior to embryoid body formation and their ability to differentiate into MNLCPs is reduced. Comparison of the growth of mESCs on gelatin, gelatin and PMEFs, and PMEFs coated plates showed that mESCs produced green fluorescent protein-containing MNLCPs on PMEFs coated plates with (54.1 ± 11.0)% (x ± s; n = 12). The yield on gelatin-coated plates was (2.8 ± 1.1)% (x ± s; n = 9). The average content of green fluorescent protein-containing MNLCPs obtained by density gradient centrifugation was (87.7 ± 5.5)% (x ± s; n = 3). Conclusions: Our data show that without using a cell sorter, both grind-free and density gradient centrifugation can also be used to enrich MNLCPs with high viability. It is necessary to identify MNLCPs for further physiological significance in vitro, in vivo and phenotype, such as the growth of axons and their ability to form neuromuscular junctions.