视黄酸联合视网膜细胞培养上清液对胚胎干细胞体外分化的诱导作用

目的：探讨经过初步诱导的拟胚体在视黄酸和视网膜细胞培养上清液作用下的分化特征。方法：将胚胎干细胞(embryonic stem cells,ESC)自液氮中复苏、培养，传1代后进行拟胚体(embry-onic bodies,EB)培养。3天半的EB离心重悬后不作消化，使用视黄酸、视黄酸加鼠视网膜胶质细胞和神经细胞培养上清液、视黄酸加人视网膜色素上皮培养上清液、视黄酸加人胎儿视网膜胶质细胞培养上清液(分别记为A、B、C、D组)等进行诱导。观察诱导过程中形态学改变，培养3周时使用免疫细胞化学检测Nestin、GFAP、cytokeratin、MAP-2、rhodopsin等在诱导后细胞中的表达情况。结果：(1)形态学改变：4种条件下的早期改变基本相同，均可见多种形态的细胞；3周后：拟胚体结构基本已散开，A组：见多种形态的细胞，细胞边界欠清，饱满度下降；B组：细胞以透明度较高的圆形细胞为主；C组：拟胚体及拟胚体周围的细胞中出现了含有明显色素的细胞；D组诱导：细胞胞体较大，形态结构较为单一；(2)免疫细胞化学：4种条件下均表现为大部分细胞MAP—2阳性，未见Nestin阳性细胞；此外，A组中，未见GFAP、Cytokeratin、Rhodopsin阳性细胞；B组中，可见GFAP、Cytokeratin、Rhodopsin阳性细胞；C组仅见Cytokeratin阳性细胞；D组仅见GFAP阳性细胞。结论：在KSC的次级诱导中，视黄酸可以诱导大部分细胞成为神经细胞，多种视网膜细胞的上清液在诱导中具有一定的作用，ESC能被诱导形成与上清液来源细胞相关的细胞。眼科学报2003；19：122-125

In Vitro Differentiation of Embryonic Stem Cells Mediated by the Supernatant of Retinal Cells and Retinoid Acid

Purpose:To analyse the differentiation features of embryonic stem cells which were induced by the supernatant of retinal cells and retinoid acid (RA). Methods:Embryonic bodies (EB) were gained when embryonic stem cells (ESC) were propagated 1 generation after anabiosis. 3.5d EB without digestion were induced at four conditions sorted as condition A, B, C and D. A contains only retinoid acid (RA) in medium; B contains RA and the supernatant from retinal neurons and glia of mouse; C contains RA and the supernatant from retinal pigment epithelium of human; D contains RA and the supernatant of fetal retinal glia. The changes of embryonic stem cells were observed and the immune cytochemistry was adopted to detect the nestin, glial fibrillary acid protein (GFAP), cytokeratin, microtubule-associated protein 2 (MAP-2) and rhodopsin in induced cells. Results: (1) The changes of differentiation features of EB: The characteristics for early stage were almost same at the four conditions. The cells with various shapes could be seen around EB. EB were dispersed 3 weeks later. Additionally, the cells which were no more abounded and not clear boundary appeared at condition A, the round cells with high transparency at condition B, EB and the cells around EB with apparent pigment at condition C, the large cells with simplex shape and glia-like appearance at condition D. (2) Immune cytochemistry showed that the most induced cells were MAP-2 (+) cells, no nestin ( + ) cells appeared at the four conditions. There were no GFAP, cytokeratin or rhodopsin positive cells in condition A ; However, GFAP ( + ), cytokeratin ( + )and rhodopsin( + ) cells could be seen at condition B, cytokeratin positive cells at condition C, GFAP positive cells at condition D. Conclusion: RA can induce embryonic stem cells into neurons in second induction. Several kinds of the supernatant from retinal cells have an effect on induction of ESC. ESC can be differentiated into cells associated with the origin of the supernatant. Eye Science 2003;19:122-125.