大鼠视网膜干细胞的增殖和多向分化

背景：国内对视网膜干细胞的体外分离培养及鉴定仍处于初步探索阶段。目的：体外分离、培养及鉴定新生大鼠视网膜干细胞,探讨其多向分化潜能。方法：用神经干细胞无血清培养方法分离和培养新生24h的SD大鼠睫状体细胞,第6代视网膜干细胞经胎牛血清诱导分化,应用免疫细胞化学方法检测视网膜干细胞的分化特性。结果与结论：体外培养的细胞球具有连续克隆能力,Nestin抗原阳性,BrdU标记结果显示悬浮细胞团主要由分裂增殖的细胞组成,并表达胚胎发育早期视网膜内原始细胞的特异性抗原Chx-10;诱导分化后的细胞表达星形胶质细胞特异性标志物GFAP、神经元特异性标志物NSE、感光细胞标志物Opsin、双极细胞特异性抗原PKC和节细胞特异性抗原β-tubulin,实验初步证实培养的视网膜干细胞具有神经干细胞特性,能自我更新和增殖分化成为感光细胞类型的细胞。

Proliferation and multi-differentiation of retinal stem cells in rats

BACKGROUND： Domestic studies on in vitro isolation and identification of retinal stem cells are still in the preliminary exploration stage. OBJECTIVE： To isolate, culture and identify the retinal stem cells from newborn rats in vitro and to investigate the cell multi-differentiation capacity. METHODS： The cells derived from the ciliary marginal zone （CMZ） of newborn 24-hour SD rats were isolated and cultured in vitro. The proliferating capacity and differentiating properties of the cultured cells were studied by using immunocytochemistry methods. RESULTS AND CONCLUSION： The isolated cells derived from CMZ cultured in serum-free medinum could give rise to neural spheres in the presence of basic fibroblast growth factor and epidermal growth factor. These cells could proliferate successively and generate secondary neural spheres, thus displaying potential to self-renew. These neural spheres could be expanded for up to 8 passages and could express constantly the neuroectodermal marker Nestin and Chx-10, a retinal stem cell marker, showing the undifferentiated stem cells properties in vitro. Analysis of the differentiation potential of the cultured cells in vitro showed that the cultured cells derived from CMZ were multipotential. Upon withdrawal of basic fibroblast growth factor and epidermal growth factor, and by addition of serum, the stem cells expressed cell type specific markers corresponding to neurons and glia, such as NSE, GFAP, Opsin, PKC, and β-tubulin.