体外诱导大鼠视网膜Müller细胞向神经节细胞定向分化的研究

Study on the differentiation of retinal ganglion cells from rat Müller cells in vitro

目的 研究鼠视网膜Müller细胞经体外条件培养基诱导后去分化为神经干细胞及进一步定向分化成神经节样细胞的特性.方法 实验研究.体外培养出生后7-10 d的Sprague Dawley大鼠视网膜Müller细胞,应用逆转录聚合酶链反应（RT-PCR）法及免疫荧光染色法鉴定Müller细胞纯度.取培养的第3或4代Müller细胞,用含有N2、碱性成纤维细胞生长因子和表皮生长因子的Delbeccon＇s modified Eagle＇s medium（DMEM）-F12干细胞条件培养基培养3～5 d后,再用含5%胎牛血清、脑源性神经营养因子和视黄酸的DMEM培养基诱导分化7～10 d,采用免疫荧光染色法分别鉴定去分化及再诱导分化后的细胞.结果 RT-PCR及免疫荧光染色结果显示,分离培养的视网膜Müller细胞纯度可达（95.17±2.68）%以上.干细胞条件培养基培养3～5 d后,大部分Müller细胞汇集形成细胞球,经免疫荧光染色法鉴定,显示细胞球内（95.26 ±1.35）%以上的细胞Nestin表达阳性,（90.33±4.12）%以上细胞BrdU表达阳性.将这些细胞球进一步诱导分化后,可见细胞球内细胞可向外扩展、铺开,分化出形态各异的细胞,其中约（21.14±1.49）%细胞表达神经节细胞特异性标记物Thy1.1阳性.结论 成年啮齿动物视网膜Müller细胞经体外条件培养基诱导后,可以产生具有增殖能力和分化潜能的神经干细胞,并可进一步定向分化为神经节样细胞,这为干细胞研究和视神经再生治疗等提供了新的方法和手段.

Objective To certify the ability of retinal Müller cells for producing neural stem cells in vitro and to find a method that can aquire more retinal ganglion cells from these stem cells. Methods Müller cells were isolated from rat retina, and proliferating cells were expanded in serum-containing medium. The third or fourth passage of cells were identified by RT-PCR and Immunocytochemistry analysis. For dedifferentiation, the cultured cells were transferred to the sphere-culture medium composed of DMEM/F-12 supplemented with N2,bFGF and EGF. After 3-5 days, the culture media were substituted with BDNF,RA and 5% FBS and culture was continued for 7-10 days. At last, cells in this two stages were identified by immunocytochemical analysis. Results Approximately （95. 17 ±2. 68）% of cells in the culture were Miiller cells as revealed by expressing glutamate-spartate transporters （GLAST） and glutamine synthetase （ GS） immunoreactivities. RT-PCR analysis also revealed that the culture was enriched for Müller cells and not contaminated with other retinal cells. After 3-5 days cultured in the the sphere-culture medium, the Mttller cells became round and differentiate to neurospheres. （95. 26 ± 1. 35）% of cells in the neurosphere were positively reacted for Nestin,and （90. 33 ±4. 12）% for BrdU. Neurospheres cultured for 7-10 days with 5% FBS,BDNF and RA can redifferentiate to various new cells. And the expression of Thyl. 1 which is a marker of retinal ganglion cells was observed in （21. 14 ± 1. 49）% of these cells. Conclusions Adult rodent Müller cells can generate clonal neurospheres,which consist of proliferating and multipotent cells,and redifferentiate to ganglion cells. This study may provide a novel tool in the study on stem cells and contribute to therapies for neural regeneration in retina.