视网膜干细胞体外诱导分化为神经节细胞的初步研究

发布时间：2018-11-12 13:50

【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14 d LE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BDNF 4组不同方式诱导14 d。流式细胞仪行成熟RGC特异性标志物thy1.1鉴定,并对DAPT联合BDNF组不同诱导时间点行RGC相关标志物math5、brn3b、thy1.1和神经干细胞标志物nestin、ki67表达的鉴定。结果传2代的RSCs nestin、chx10、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RSCs 14 d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(P0.05)。DAPT联合BDNF组诱导3~5 d后部分细胞nestin、ki67、brn3b、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3~5 d后,RGCs相关标志物出现,细胞仍具有一定增殖能力。
[Abstract]:Objective to investigate the method of inducing retinal stem cells (retinal stem cell,RSC) to differentiate into retinal ganglion cells (retinal ganglion cell,RGC) in vitro and to provide seed cells for the treatment of optic nerve injury. Methods RSC, derived from 14 day old LE rats were identified by flow cytometry and immunofluorescence, and then induced by 5% serum and BDNF,DAPT,DAPT combined with BDNF 4 groups for 14 days. The specific marker of mature RGC was identified by flow cytometry (thy1.1), and the expression of RGC related marker math5,brn3b,thy1.1 and neural stem cell marker nestin,ki67 was identified at different time points in DAPT combined with BDNF group. Results the expression of RSCs nestin,chx10,ki67 was strongly positive in the second generation, and the positive expression of math5 was found in some of the cells. The positive rates of thy1.1 were (11.38 卤1.58)%, (19.10 卤2.29)%, (23.90 卤1.09)%, (31.31 卤2.98)%, respectively, in BDNF,DAPT,DAPT combined with BDNF group for 14 days. There were significant differences between the two groups (P0.05). DAPT combined with BDNF group after 3 days of induction of nestin,ki67,brn3b,math5 positive expression of some cells. Conclusion DAPT combined with BDNF can increase the rate of differentiation of RSCs into RGCs in vitro, and 5 days after induction, RGCs related markers appear and the cells still have the ability to proliferate.
【作者单位】：第三军医大学西南医院全军眼科中心;
【基金】：国家自然科学基金青年科学基金(30901645)~~
【分类号】：R774

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