视黄酸联合视网膜细胞共培养对胚胎干细胞体外分化的诱导作用

目的 探讨胚胎干细胞(ESC)在视黄酸联合视网膜细胞共培养诱导条件下的分化特征。方法 将ESC自液氮中复苏、培养，传1代后进行拟胚体培养。将部分3．5d拟胚体离心重悬后加入含有视黄酸的24孔板中进行诱导，另一部分加入已经培养有视网膜混合细胞的培养瓶中，培养液中同时也加入视黄酸。视网膜混合细胞中仅加入视黄酸未加拟胚体作为对照。倒置相差显微镜下观察细胞在整个诱导过程中形态学的改变并使用免疫细胞化学检测诱导细胞中巢蛋白、胶质纤维酸性蛋白、广谱细胞角蛋白、微管相关蛋白-2、视紫质蛋白表达情况。结果 (1)形态学改变仅由视黄酸诱导，诱导细胞呈多种形态；在共培养条件下，绝大多数拟胚体分化出来的细胞形态非常单一，呈透明圆形。部分原贴壁的视网膜细胞出现了明显的网状结构。(2)免疫细胞化学显示两种诱导条件均可见大部分诱导细胞MAP-2阳性，并可见Nestin阳性细胞。共培养诱导尚可见GFAP阳性、Cytokeratin阳性和Rhodopsin阳性的细胞。结论 视黄酸可以诱导大部分细胞成为神经细胞，在视黄酸和与视网膜细胞共培养诱导条件下，可以获得更为纯化的形态一致的神经样细胞，部分诱导细胞表达视网膜细胞的特性。

In vitro differentiation of embryonic stem cells induced by retinoid acid and co-culture of retinal cells

Objective To analyze the differentiation features of embryonic stem cells (ESC) induced by retinoid acid (RA) and co-culture of retinal cells. Methods ESCs were propagated 1 generation after anabiosis to gain embryonic bodies (EB).3. 5-day EB was added to 24 holes panel containing RA and the bottles cultured retinal cells and RA,and RA was added to the bottles containing retinal cells without ES as control. The changes of ESC and retinal cells were observed under the convert discrepancy microscope. Immunocytochemistry was adopted to detect the nestin, GFAP, cytokeratin, MAP-2 and rhodopsin during the inducing period. Results Various shapes of induced-cells appeared in medium containing RA. When ESCs were induced by RA and cocultured with mixed retinal cells,the differentiated-cells from EB appeared to be relatively simple with round and transparent appearance. Some sticking-wall retinal cells showed a net-shaped structure. Most induced-cells from ESC showed a positive reaction for MAP-2, and little cells were nestin positive in both medium. GFAP, cytokeratin and rhodopsin positive cells could be seen under the ESC and retinal cells co-culture condition. Conclusion RA can induce ESC into neurons in second induction. More purified cells could be gained by stimulation of RA and co-culture ESC with retinal cells. Some of induced-cells express the features of retinal cells.