视网膜干细胞体外诱导分化为神经节细胞的初步研究  

Differentiation of retinal stem cells into ganglion cells in vitro

作　　者：李依孺[1] 叶茂[1] 吴楠[1] 王一[1] 

机构地区：[1]第三军医大学西南医院全军眼科中心,重庆400038

出　　处：《第三军医大学学报》2014年第10期1026-1030,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金青年科学基金(30901645)~~

摘　　要：目的探讨诱导视网膜干细胞（retinal stem cell，RSC）分化为视网膜神经节细胞（retinal ganglion cell，RGC）的体外培养方法，提供视神经损伤后细胞移植治疗所需的种子细胞。方法悬浮培养胎龄14dLE大鼠来源的RSC，传2代流式细胞仪和免疫荧光鉴定后，用5％血清、BDNF、DAPT、DAPT联合BDNF4组不同方式诱导14d。流式细胞仪行成熟RGC特异性标志物thyl．1鉴定，并对DAPT联合BDNF组不同诱导时间点行RGC相关标志物math5、bm3b、thyl．1和神经干细胞标志物nestin、ki67表达的鉴定。结果传2代的RSCsnestin、ehx10、ki67表达强阳性，部分细胞math5表达阳性；5％血清、BDNF、DAPT、DAPT联合BDNF组诱导RSCs14d后thy1．1阳性率分别为（11．38±1．58）％、（19．10±2．29）％、（23．90±1．09）％、（31．31±2．98）％，两两比较差异均有统计学意义（P〈0．05）。DAPT联合BDNF组诱导3～5d后部分细胞nestin、ki67、bm3b、math5均阳性表达。结论使用DAPT联合BDNF诱导，可以提高RSCs在体外分化成RGCs的比率，并且诱导3～5d后，RGCs相关标志物出现，细胞仍具有一定增殖能力。Objective retinal ganglion cells （RGCs） To investigate in vitro in order how to induce to provide the cell transplantation. Methods RSCs were isolated from the the retinal stem cells （RSCs） differentiation into seed cells for the repair of optic nerve injury after retina of LE rats at embryonic days 14, and then identified by flow cytometry and immunofluorescence staining after sub-culturing for 2 culture generations. Then the obtained cells were cultured in 4 different ways, 5% FBS （ control ）, BDNF （ 10 ng/mL ）, DAPT （25 μmol/L）, and DAPT ＋ BDNF for 14 d. Flow cytometry was used to identify specific marker CD90. 1 （thy1. 1） of mature RGCs and markers math5, and brn3b, and neural stem cells＇ markers nestin, ki67 of DAPT ＋ BDNF group at different time points. Results The obtained RSCs at passage 2 were strongly positive to nestin, chxl0, and ki67, and some of them even to math5. The positive rate of thyl. 1 was （ 11.38 ± 1.58 ） %, （ 19.10 ±2.29） %, （23.90 ± 1.09） % and （31.31 ± 2.98 ） % respectively, in the RSCs cultured in the mediums containing 5%FBS, BDNF, DAPT, and DAPT ＋ BDNF, with significant difference between any 2 groups （P 〈 0. 05）. DAPT ＋ BDNF induction for 3 to 5 d resulted in positive expression of nestin, ki67, brn3b and math5 in some cells. Conclusion DAPT ＋ BDNF enhances the differentiation of RSCs into RGCs in vitro, and the cells express RGCs relative markers and remain reproductive capacity after 3 to 5 days＇ induction.

关 键 词：视网膜干细胞 视网膜神经节细胞 细胞培养 

分 类 号：R322.91[医药卫生—人体解剖和组织胚胎学] R329-33[医药卫生—基础医学]