体外诱导猪Müller细胞定向分化为光感受器细胞的研究

摘要

背景 大鼠、小鼠Müller细胞在体外能诱导分化为表达视网膜光感受器细胞特异标记的细胞,但目前有关成年猪Müller细胞分化为视网膜光感受器细胞的研究鲜有报道.目的 研究成年猪Müller细胞于体外定向诱导后分化成视网膜第一级传导神经元光感受器细胞.方法 消化成年猪眼视网膜组织,分离Müller细胞行体外继代单层贴壁培养.使用定向分化培养基对P2、P3和P4代Müller单层贴壁细胞及先形成细胞球悬浮培养2—3d,然后再对贴壁细胞进行诱导分化,最后结合细胞形态和免疫荧光染色结果鉴定Müller细胞以及诱导分化效果.结果 免疫荧光染色结果显示,P2,P3和P4 Müller细胞均能表达Müller细胞特异蛋白分子标记,即谷氨酸合成酶(GS),P3 Müller细胞还同时表达另一种Müller细胞特异蛋白分子标记,神经胶质纤维酸性蛋白(GFAP).免疫荧光染色分析并计算视网膜光感受器细胞特异标记视紫质Rhodopsin阳性率,P2 Müller单层贴壁分化细胞为(27.99±6.53)％,P2悬浮细胞球分化为(16.54±3.40)％.P2悬浮细胞球诱导分化后形态更趋向神经元,而P2代Müller单层贴壁细胞分化后形态仍趋向于成纤维细胞.随着培养代数的增加,细胞球定向分化Rhodopsin阳性表达程度逐渐减弱,P2、P3和P4 Rhodopsin阳性率分别为(56.23±7.32)％、(35.26±8.55)％和(12.68±3.18)％,差异无统计学意义(F=2.618,P=0.099).同代细胞随着诱导时间的延长,Rhodopsin阳性表达有所减弱,差异无统计学意义(P=0.099),但分化细胞形态更为细长.结论 成年猪Müller细胞离体培养后可定向分化为表达视网膜光感受器样细胞特异标记的细胞.细胞球悬浮培养2～3d,以及延长诱导分化时间都能使得分化细胞形态更为细长.%Background Recent studies indicated that rat and mouse Müller cells can be induced and differentiated into photoreceptor-like cells in vitro, but it is not known whether this also happens to adult pig Müller cells nowadays.Objective This study was to test whether adult pig Müller cells can be differentiated to the retinal photoreceptors (the primary transmission neurons of the retina) in vitro.Methods Müller cells were isolated from the neural retina of adult pig eyes and cultured and passaged.The 3rd and 4th generation of cells were themonolayerly cultured,and the cells forced to form spheres in suspension in altra-low adherent dishes for 2-3 days first and then reseeded in normal adherent plates,and both of them were cultured in a specifically formulated medium to induce the differentiation of retinal photoreceptor.The cells was verified by immunocytochemistry.Cell morphology and immunofluorescence staining were utilized to measure the efficacy of the differentiation.Results The 2nd,3rd and 4th generation of Müller cells expressed glutamate synthetase (GS) , a specific maker of Müller cells.Inaddition, the 3rd generation of cells also expressed glial fibrillary acidic protein (GFAP) and another specific maker of Müller cells.Three visual fields under fluorescence-microscope were randomly chosen to calculate the average positive ratio of rhodopsin,a specific marker of mature photoreceptors.The photoreceptor differentiation ratios of the 2nd generation of cells for monolayer culture only and with additional sphere suspension culture were (27.99±6.53 (％ and (16.54±3.40) ％ , respectively.With passages, the number of rhodopsin positive cells gradually decreased, and the intensity of rhodopsin expression gradually weakened.The directed rhodopsin positive ratios of the 2nd,3rd and 4th generation of cells from sphere formation were (56.23±7.32)％ , (36.26 ±8.55)％ and (12.68 ±3.18)％ , respectively.Although the rhodopsin expression was weakened over passages,the differentiated cells were more slender and elongated.There was no statistic ally significant difference between different groups (F =2.618, P =0.099).Conclusions Adult pig Müller cells can be differentiated into retinal photoreceptors in vitro.The morphology of the differentiated cells appears moreslender and elongates if the sphere-induced differentiation method is used and/or the directed differentiation time is further extended.