维甲酸诱导MESPU35胚胎干细胞系定向分化神经细胞的最佳条件(英文)

Optimal condition of directional-differentiation of neurons from retinoic-acid induced MSESPU35 embryonic stem cell lines

背景:神经轴突再生是治疗中枢神经系统损伤必须克服的困难之一,包括移植神经干细胞、胚胎干细胞和许旺细胞等在内的细胞移植疗法已取得显著疗效,然而供体细胞不足的瓶颈限制了其发展。目的:观察维甲酸诱导MESPU35胚胎干细胞分化过程,找到其最佳分化为神经细胞的条件。设计:非随机对照实验。单位:解放军第三军医大学组织胚胎学教研室。材料:实验于2000-01/05在解放军第三军医大学基础部组织胚胎学教研室完成。发情期昆明种小鼠18只,雌12只,雄6只,2∶1同笼交配,阴栓检出日记为受孕1d。MESPU35胚胎干细胞株。方法:孕13～16d的小鼠胚胎,去头,胸腹腔脏器及四肢后,制备饲养层细胞。①采用饲养层贴壁培养增殖MESPU35胚胎干细胞,经典4-/4+法(即拟胚体自然生长4d,不加维甲酸,随后4d添加维甲酸诱导形成高比例神经拟胚体的方法)诱导其神经定向分化,不同浓度血清培养拟胚体,相差显微镜观察不同血清浓度下神经样拟胚体并计数。②免疫细胞化学技术观察各个分化时相点(5,9,14d)和不同维甲酸浓度下分化细胞形态学特征,流式细胞仪计数分化神经细胞比例。主要观察指标:①维甲酸诱导MESPU35胚胎干细胞分化后神经拟胚体形成中突起和细胞体长度估计测量。②免疫细胞化学染色分化细胞形态观察及流式细胞仪测量分化细胞比例。结果:①相差显微镜观察发现不同血清浓度对拟胚体形成后神经定向分化有一定影响,血清浓度过高和过低降低了拟胚体神经分化比例,5%血清浓度分化比例高。②免疫细胞化学观察维甲酸诱导MESPU35胚胎干细胞分化形成的NF200阳性细胞和胶质纤维酸性蛋白阳性细胞的比例随分化时相点和维甲酸浓度升高而升高,NF200阳性细胞形态由无突起变为多极细胞,胶质纤维酸性蛋白阳性细胞突起由短变长,最后联结成网状。③流式细胞仪检测分化产生的胶质纤维酸性蛋白、NF200阳性细胞比例变化类似免疫细胞化学。结论:维甲酸配合合适的血清浓度、分化时相点等条件能够诱导MESPU35胚胎干细胞高比例神经分化,其分化调节以浓度依赖模式和时间依赖模式进行。

BACKGROUND： Neural axon regeneration is one of the difficulties that must be overcome in treatment of injury of central nerve system. Significant therapeutic effects have been obtained in transplantation of neural stem cells （NSCs）, embryonic stem cells （ESCs） and Schwann cells. But the bottleneck situation of insufficiency of cell provider has limited the development on it. OBJECTIVE： To observe directional-differentiation of retinoic-acid induced ESCs so as to find optimal condition for neuronal differentiation. DESIGN： Non-randomized controlled experiment was designed. SETTING： Teaching-Research Room of Histology. and Embryology, Department of Basic Medicines, Third Military Medical University of Chinese PLA MATERIALS： The experiment was performed in Staff Room of Histology and Embryology, Third Military Medical University of Chinese PLA from January to May 2000. Eighteen Kunming mice in disoestrus were employed, of which, 12 mice were female and 6 mice male. They were placed in same cage at ratio of 2：1 for mating. The date of pregnancy was recorded. MESPU35 ESC line was prepared. METHODS： Removed head, internal organs and four limbs, feeder-layer cells were prepared from the embryo of mouse of 13-16 d pregnancy. ① Feeder-layer adherent culture was used to proliferate MESPU35 ESCs. Classic 4-/4＋ method [The embryoid body （EB） grew naturally for 4 days, without retinoic acid added. In the coming 4 days, retinoic acid was added to induce neural EB of high proportion] was applied to induce the directional differentiation of the nerve. EB was cultured with serum of different concentrations. Phase contrast microscope was used to observe nerve-like EB in serum of different concentrations and to count numbers, ②Immunocytoehemical technique was used to observe cellular morphological characters at various differentiating phase spots （5^th, 9^th, 14^th days） and with retinoic acid at various concentrations. Flow cytometer （FCM） was used to count the proportion of differentiated neurons. MAIN OUTCOME MEASURES： ①Estimated measurement of the length of process and cell body during formation of neural EB after retinoic-acid induced differentiation of MESPU35 ESCs. ②Observation of cell morphology with immunoeytochemical staining and proportion of differentiated cells assayed with FCM. RESULTS： ①It was discovered with phase contrast microscope that serum of different concentrations affect neural directional differentiation after EB formation to certain extent. Excessively high and low concentrations of serum reduced the pruportion of neural differentiation of EB. The differentiating proportion is high in serum with 5% concentration. ②It is observed with immunocytochemical technique that the proportions of NF200 positive cell and glial fibrillary acidicprotein （GFAP） positive cell in differentiation of MESPU35 ESCs induced by retinoic acid were increased with phase spots in differentiation and increased concentration of retinoic acid. NF200 positive cell is transformed as multipolar neurons from absence of process in morphology. The processes of GFAP positive cell became longer and linked among each other as reticular pattern finally. ③It was assayed with FCM that the proportion changes of GFAP positive cell and NF200 positive cell manufactured in differentiation were similar to immunocytochemical one.CONCLUSION： Retinoic acid in combination with proper concentration of serum and differentiating phase spots can induce neural-differentiation of MESPU35 ESC at high proportion and its differentiating regulation is in the patterns of concentration dependence and time dependence.