胚胎神经干细胞和听囊细胞体外分化的实验研究

发布时间：2018-08-23 14:21

【摘要】：目的：研究胚胎神经干细胞和听囊细胞体外分化为毛细胞的潜能；探讨胚胎神经干细胞在内耳再生研究领域的价值。 方法：分离孕9.5-10.5天C5786/L小鼠胚胎的后脑区域的神经管组织和听囊组织,采用无血清培养,进行体外培养、扩增,并通过免疫荧光化学染色(巢蛋白)进行鉴定。取传至第4代神经干细胞和听囊细胞,接种于盖玻片上。添加血清培养基,培养15天后行免疫荧光检测,与RT-PCR检测。观察神经干细胞和听囊细胞的体外分化特点。 结果：采用无血清培养法成功地培养出胚胎神经干细胞球和听囊细胞球,采用神经球分析法收集细胞球进行纯化与传代,免疫荧光检测示：来源于神经管和听囊的细胞球巢蛋白(nestin)表达阳性,证实其都具有神经干细胞的特点。添加含血清培养基培养15天后,免疫荧光检测证实所获得的神经干细胞和听囊细胞均可以在体外分化为表达毛细胞标记物MyosinV Ⅶa的细胞。 结论：胚胎神经干细胞和听囊细胞能够在体外培养的条件下分化为毛细胞样细胞：胚胎神经干细胞可以用于研究内耳毛细胞分化过程中的重要基因表达与调控因子、揭示聋病发生的机制及耳蜗的细胞移植治疗的可行性。
[Abstract]:Aim: to study the potential of embryonic neural stem cells and auditory follicle cells to differentiate into hair cells in vitro and to explore the value of embryonic neural stem cells in the field of inner ear regeneration. Methods: the neural tube tissues and auditory bursa tissues in the posterior brain of 9.5-10.5 days C5786 / L mouse embryos were isolated, cultured in vitro and amplified by serum-free culture, and identified by immunofluorescence staining (nestin). Neural stem cells and auditory sac cells were transferred to the fourth passage and inoculated on the cover glass. After 15 days of culture, serum was added to the medium and immunofluorescence assay was performed with RT-PCR. To observe the differentiation characteristics of neural stem cells and auditory sac cells in vitro. Results: embryonic neural stem cell ball and auditory sac cell ball were successfully cultured by serum-free culture. The expression of nestin (nestin) was positive in neurons from nerve tube and auditory sac, which confirmed that they had the characteristics of neural stem cells. After cultured in serum-containing medium for 15 days, the obtained neural stem cells and auditory sac cells were confirmed to be able to differentiate into cells expressing hair cell marker MyosinV 鈪，

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