小鼠原始生殖细胞定向分化为神经样细胞的研究_临床医学论文.docVIP

小鼠原始生殖细胞定向分化为神经样细胞的研究_临床医学论文 小鼠原始生殖细胞定向分化为神经样细胞的研究_临床医学论文 【摘要】 目的：探讨微囊对原始生殖细胞(PGCs)定向分化为神经样细胞的诱导效果。方法：用原代PGCs经悬浮培养获取类胚体(EBs);将孕13 d胎鼠的大脑半球取出，将其中的神经干细胞(NSCs)作为包在微囊里的成分。待EBs贴壁后将含有NSCs的微囊放入其中，同时另外孔板中放入等量的EBs让其自然分化。将微囊诱导分化出的细胞进行NESTIN、NSE和GFAP免疫染色鉴定。结果：用微囊诱导定向分化为神经样细胞的比例显著高于自然分化的比例。结论：悬浮培养可使PGCs形成EBs，将EBs贴壁培养可自发分化形成内、中和外三个胚层的细胞，即上皮样细胞、成纤维样细胞和神经样细胞。通过微囊的诱导方法可以使EGs定向分化为神经样细胞。 【关键词】 原始生殖细胞;细胞分化;Western 免疫印迹;神经干细胞;小鼠 　　[ABSTRACT] Objective: To explore the inducing effect of microencapsulation on differentiation of PGCs(primordial germ cells) into neuronal cells. Methods: Took cerebra from the 13days old mouse embryos,encapsulated neuronal stem cells(NSCs) collected from the cerebra into microencapsulation. EBs(embryoid bodies) were formed from the PGCs collected from13 days mouse embryos by suspension culture. The microencapsulation were put in the culture plates after the EBs were plated on gelatincoated culture plates. Furthermore, the same amount of EBs were plated on gelatincoated culture plates without microencapsulation for spontaneous differentiation. The neuronal cells were detected with NESTIN, NSE and GFAP immunohistochemical staining. Results: The rate of differentiation by microencapsulation was distinctly higher than the rate of spontaneous differentiation. Conclusion: The nutritional components secreted from the microencapsulation enter the culture medium and can induce the differentiation of EBs into neuronal cells effectively. 　　[KEY WORDS] Primordial germ cell; Cell differentiation; Western blot; Neuronal stem cells; Mouse 　　原始生殖细胞(primordial germ cells, PGCs)是一种胚胎源性干细胞，具有体外分化为各种组织的能力[1]，我们已探讨了其体外自发分化的能力，发现其分化为神经样细胞的百分比最大。为了进一步提高神经样细胞的分化率，本文将重点探讨PGCs向神经样细胞定向分化的新方法，即微囊诱导法。 　　1 材料与方法 　　1.1 材料 　　ICR小鼠。 　　1.2 方法 　　1.2.1 PGCs的分离、提取和体外培养 实验使用自配13 d胎鼠，取两侧生殖嵴，用PBS洗3次后添加0.25%胰蛋白酶消化，吹打，然后过60目细胞筛，将PGCs按2.5×106/mL的密度接种于35 mm的培养瓶中进行体外培养。 　　1.2.2 悬浮培养 (1)类胚体(embryoid bodies， EBs)的形成：取分离提取的PGCs，置于培养瓶中进行悬浮培养，培养期间每隔1～2 h摇晃1次培养瓶防止PGCs贴壁。换液方法：将含细胞的培养液放入离心管里1 000 r/min离心10