成年大鼠脑膜原代组织干细胞特性细胞的分离及神经诱导分化研究

目的 分离并检测成年大鼠脑膜组织中具有干细胞特性的细胞亚群,探讨其诱导成神经细胞的能力.方法 自成年大鼠活体分离、剪取脑膜经胰酶消化制成细胞悬液,接种于培养皿,用无血清的特殊培养基培养,动态观察脑膜细胞克隆球的形成及分化过程,并用巢蛋白（Nestin）、表面抗原CD133抗体进行免疫荧光染色,对阳性细胞进行表观遗传学鉴定.分离的脑膜细胞经曲古抑菌素A（TSA）诱导培养基分别诱导分化后检测脑膜细胞向神经细胞分化成熟程度,用免疫印迹法（westen blotting）检测诱导分化后脑膜细胞内成熟神经细胞相关标志性蛋白--高分子量神经丝蛋白（NF-200）、神经元蛋白（BM88）的表达情况.结果 成年大鼠脑膜组织体外培养时不同类型细胞的贴壁时间有差异,具有干细胞特性细胞呈球形,黏着在首先贴壁的扁平基底细胞上分裂形成克隆球,克隆球细胞Nestin、CD133免疫荧光染色阳性.诱导分化后NF-200、BM88有明显表达,表明经诱导分化后脑膜细胞可分化为神经细胞.结论 活体分离成年大鼠脑膜组织部分细胞体外培养具有干细胞特性,并可向神经细胞方向分化.

Research on isolation and nerve-induced differentiation of stem-like cells form the meninges of adult rat

Objective To isolate and detect stem-like cell subsets in the meninges of adult rat, in order to explore the induced neuronal cells. Methods The meninges tissue getting from the adult rats were digested into a singal cell suspension by trypsin, then cultured in the special serum-free condition. The cloned sphere and differentiation were observed dynamically by the phase contrast microscope. Immunofluorescent staining was conducted with Nestin and CD133 anti-body. The positive cells were identified on epigenetic. Western blot assay was employed to access the meningeal cells mature nerve cells related proteins （NF- 200 and BM88） expression level after the separated meningeal cells were induced by trichostatin A （TSA）. Results The stem-like cells from the meningeal tissue of adult rats grown in the cloned sphere way in vitro which was positive expressed Nestin and CD 133 by immunofluorescence staining. Westen blotting results showed that nerve cells related markers （NF-200 and BM88 ） ex pressed in high level after induction of neuronal differentiation. Meningeal cells could differentiate into nerve cells after nerve induction. Conclusion Some cells cultured in vitro and isolated from meninges of living adult rats has the quality of stem cells and can differentiate into nerve cells.