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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: Axonal regeneration is one of the most difficult problems that must be overcome in the treatment of central nervous system injury. Cell transplantation including transplantation of neural stem cells, embryonic stem cells and Schwann cells has achieved a significant effect. However, the bottleneck of donor cell insufficiency Its development. OBJECTIVE: To observe the differentiation of MESPU35 embryonic stem cells induced by retinoic acid and to find out the optimal conditions for its differentiation into nerve cells. Design: Non-randomized controlled experiment. Unit: Department of Histology and Embryology, Third Military Medical University. Materials: The experiment was performed at the Department of Embryology, Third Military Medical University from January 2000 to January 2005. Oestrus Kunming mice 18, 12 females, 6 males, 2: 1 with the cage mating, diuretic detection diary for pregnancy 1d. MESPU35 embryonic stem cell line. METHODS: Mouse embryos 13 ~ 16 days pregnant, go head and neck, thoracoabdominal organs and limbs were prepared feeder cells. ① The MESPU35 embryonic stem cells were adherent cultured by feeder layer culture. The classical 4- / 4 + method (that is, the natural growth of embryoid body was performed for 4 days without any retinoic acid and then induced by retinoic acid for 4 days to induce the formation of high proportion of neural embryoid bodies) The neuro-oriented differentiation, different concentrations of serum-cultured embryoid bodies, phase contrast microscope under different serum concentrations of neuroblastoid embryos were counted and counted. ② Immunocytochemistry was used to observe the morphological characteristics of differentiated cells at different time points (5, 9, 14d) and different concentrations of retinoic acid. Flow cytometry was used to count the proportion of differentiated neurons. MAIN OUTCOME MEASURES: (1) Retinoic acid induces projections and cell body length estimation in neurite-like embryoid body after differentiation of MESPU35 embryonic stem cells. ② immunocytochemical staining of differentiated cells morphology and flow cytometry to measure the proportion of differentiated cells. Results ① Phase contrast microscopy showed that different concentrations of serum had some effect on the neuro-directional differentiation after the formation of embryoid bodies. The high and low serum concentrations reduced the proportion of neural differentiation of embryoid bodies and the high proportion of 5% serum concentration. (2) Immunocytochemistry showed that the proportion of NF200 positive cells and glial fibrillary acidic protein positive cells induced by retinoic acid induced MESPU35 embryonic stem cells increased with the time of differentiation and the concentration of retinoic acid increased. The morphology of NF200 positive cells changed from non-prominence For multipolar cells, glial fibrillary acidic protein-positive protrusions by the short variable length, the final link into a network. ③ flow cytometry to detect differentiation of glial fibrillary acidic protein, NF200-positive cells similar to the changes in immunocytochemistry. CONCLUSION: Retinoic acid can induce a high proportion of neural differentiation of MESPU35 embryonic stem cells with proper serum concentration, differentiation phase point and other conditions, and its differentiation regulation is in a concentration-dependent and time-dependent manner.