睫状缘色素细胞与骨髓间充质干细胞体外共培养构建视网膜干细胞的初步研究

Preliminary study of retinal stem cells fabrication by direct co-culture of ciliary margin pigment cells and bone marrow mesenchymal stem cells

目的探索睫状缘色素细胞(pigmented cells from the ciliary margin,PCM)与骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM)体外共培养构建视网膜干细胞的可行性。方法按照组织贴壁培养方法分别分离培养原代大鼠BM和PCM,再将二者进行直接共培养(1∶2),观察细胞生长情况;MTT方法进行增殖能力检测;在促神经分化诱导液中诱导21 d后行免疫荧光染色,观察视网膜干细胞相关分子标记物包括视杆细胞Rho1D4、双极神经元CHX10和Müller胶质细胞10E4的表达。结果原代分离培养的两种细胞生长状态良好;共培养后细胞总体的增殖活性虽然显著低于单纯BM,但比单纯PCM有了一定提升;诱导分化后单纯PCM视网膜干细胞相关分子标记物表达阳性率显著高于BM,而共培养后的细胞表达阳性率显著高于单纯PCM和BM。结论采用BM与PCM共培养能够获得大量表达视网膜干细胞相关分子标记物的细胞群,有望成为视网膜干细胞来源,用于视神经损伤修复。

Objective To explore the availability of fabrication retinal stem cells（RSC） in vitro by co-culture of pigmented cells from the ciliary margin（PCM） and bone marrow mesenchymal stem cells（BM）.Methods Primary PCM and BM were isolated according to previous reports.Co-culture was performed by directly combine BM and PCM at the volume ratio of 1：2.The growth was observed by inverted microscope,while the proliferation was monitored by MTT assay.After neural induction for 21 days,RGC related markers of rod photoreceptor（RholD4）：bipolar neurons（CHX10） and M（u｜¨）ller glia（10E4） were detected by immunofluorescent staining.Results The isolated cells grew in good condition.The proliferation rate of cocultured cells was lower than BM,but higher than PCM.After induction,RGC related markers expression in PCM were significantly higher than BM,while the co-cultured cells were much higher than PCM and BM.Conclusion Direct co-culture of BM and PCM can acquire abundant RSC,and may be a promising cell source for damaged optical neuron repair.