羊水干细胞定向及稳定分化为神经元样细胞体系的建立

The system for directional and stable differentiation of amniotic fluid stem cells into neuron-like cells

背景:利用干细胞分化代替受损神经元的设想,已经逐渐受到科研工作者的关注。胚胎干细胞和诱导性多能干细胞虽然具有良好的神经元分化潜能,但由于接种活体动物具有致瘤性,限制了其进一步的深入研究。目的:建立稳定的羊水干细胞分选、培养、成神经诱导分化体系,探讨其作为神经元再生种子细胞的可行性。方法:经B超引导下,穿刺获取孕19-22周期间的羊水样本10 mL,磁珠分选其中c-Kit阳性的羊水干细胞。免疫荧光染色鉴定羊水干细胞标记物Oct-4、Sox2;RT-PCR检测羊水干细胞多次传代后干性标记物c-Kit、Oct-4、Sox2、Nestin的表达;羊水干细胞悬滴培养4 d观察拟胚体的形成情况;采用"两阶段"分步诱导羊水干细胞向神经元方向分化,免疫荧光染色观察Neuro D、Tuj1的表达。结果与结论:①羊水中可分选出大约1%的c-Kit阳性羊水干细胞;②(75.0±4.6)%的羊水干细胞表达Oct-4,(86.0±2.8)%的羊水干细胞表达Sox2;③RT-PCR方法检测干性标记物c-Kit、Oct-4、Sox2、Nestin的表达量并不随细胞传代数的增加而改变;④经悬滴培养可形成拟胚体并表达Oct-4;⑤免疫荧光证实未经诱导的羊水干细胞表达神经元标记物Tuj1,但缺乏典型的神经元形态特征;RT-PCR证实不同羊水干细胞标本都能检测到Tuj1的表达,同一样本多次传代也能稳定检测到Tuj1的表达;⑥羊水干细胞经碱性成纤维细胞生长因子、脑源性神经营养因子、神经营养因子3的两阶段分步诱导后表达神经干细胞标记物Neuro D和神经元标记物Tuj1,具有神经元样细胞的形态特征。

BACKGROUND:Neuronal regeneration using stem cell differentiation has gained a lot of attentions from researchers.Although embryonic stem cells and induced pluripotent stem cells have good potential for neuronal differentiation,a high risk of tumor development in vivo limits the further study.OBJECTIVE:To establish a stable system for sorting,culture and neuronal differentiation of amniotic fluid stem cells,and to explore the feasibility as seed cells for neuronal regeneration.METHODS:Amniotic fluid sample(10 mL)was obtained at 19-22 weeks of pregnancy under B-ultrasound guidance,and amniotic fluid stem cells were isolated by c-Kit magnetic beads.The markers Oct-4 and Sox2 of amniotic fluid stem cells were identified by immunofluorescence.The expression levels of c-Kit,Oct-4,Sox2 and Nestin in amniotic fluid stem cells after multiple passages were detected by RT-PCR.Then,the cells were cultured by hanging drop for 4 days to observe the embryoid bodies-like structures.Amniotic fluid stem cells were induced to differentiate into neurons using two-stage method.The expression levels of Neuro D and Tuj1 were observed by immunofluorescence.RESULTS AND CONCLUSION:(1)About 1%of amniotic fluid stem cells were positive for c-Kit.(2)(75.0±4.6)%of amniotic fluid stem cells expressed Oct-4 and(86.0±2.8)%of the cells expressed Sox2.(3)The expression levels of c-Kit,Oct-4,Sox2 and Nestin detected by RT-PCR did not change with passage times.(4)Embryoid bodies-like structures formed after hanging drop culture.(5)Immunofluorescence results showed that amniotic fluid stem cells expressed neuronal marker Tuj1,but without the typical morphological features.RT-PCR detected the expression of Tuj1 in different amniotic fluid stem cell specimens as well as in the same sample after several passages.(6)Amniotic fluid stem cells could have the characteristics of neuron-like cells after induction with basic fibroblast growth factor,brain-derived neurotrophic factor,and neurotrophin factor 3 in two stages,and could express neural stem cell marker Neuro D and neuronal marker Tuj1.