骨髓间充质干细胞向光感受器样细胞和视网膜神经样细胞的分化

Differentiation of bone marrow mesenchymal stem cells to retinal nerve-like cells and photoreceptor-like cell

目的探讨与新生鼠视网膜神经细胞共培养诱导骨髓间充质干细胞(bone marrow stromal cells,BMSC)的分化情况。方法体外贴壁培养法分离培养Lewis大鼠BMSC,记录BMSC的增殖情况并进行鉴定;制备细胞爬片并在双层培养板内与新生鼠视网膜神经细胞共培养,观察BMSC的神经存活和分化情况,并行免疫荧光染色鉴定。结果早期的BMSC分裂较缓慢,第2天进入快速增殖期,细胞生长活跃,大约持续至第5天,细胞分裂进入平台期,增殖缓慢或轻微下降。流式细胞仪显示,90.27%的3代BMSC处于G0-G1期,其余9.73%的细胞处于G2期、S期和M期;93.28%的4代BMSC处于G0-G1期,其余6.72%的细胞处于G2期、S期和M期。流式细胞仪检测示3代和4代BMSC抗原CD90+/CD45-分别为92%、90%,体外获得了较高纯度的BMSC。MTT法检测共培养后3d、5d、7d的细胞存活率分别为(65.48±9.95)%、(73.56±8.68)%、(76.84±8.65)%,与对照组[(66.14±10.45)%、(70.11±9.60)%、(73.21±6.98)%]比较,差异均无统计学意义(均为P>0.05)。免疫荧光染色显示,共培养组诱导后部分BMSC视蛋白、巢蛋白于3d后、微管蛋白于5d后出现阳性反应,对照组均为阴性。结论 BMSC具有向光感受器样细胞和视网膜神经样细胞分化的潜能。

Objective To explore the differentiation of bone marrow mesenchymal stem cells （ BMSC ） induced by cocultivation with retinal nerve cell from newborn mice.Methods BMSC from newborn Lewis rats were separated and adherently cultured in vitro.Proliferation of BMSC was recorded and indentified,cell climb of BMSC tablet was made,and retinal nerve cell from newborn mice in double culture plate were cocultivated,and BMSC neural survival and differentiation were observed and indentified by immunofluorescence assay.Results The proliferation of BMSC in early stage was slow,rapidly from two days to five days,then slowly.The third and forth passaged BMSC antigen CD90 ＋ /CD45 - were 92% and 90%,respectively,high purity of BMSC were obtained in vitro.The cell survival rate of cocultivation detected by MTT at 3 days,5 days,7 days were （ 65.48±9.95 ） %,（ 73.56±8.68 ） % and （ 76.84±8.65 ） %,respectively,compared with control group,which were （ 66.14±10.45） %,（ 70.11±9.60 ） % and （ 73.21±6.98 ） %,there was no statistical difference （ all P 0.05） .Immunohistochemical assay indicated that the experimental group started to appear positive reactions of apparent protein,nest protein and microtubule protein amid a part of BMSC after induction from 3 days and 5 days,respectively, while control group was all negative.Conclusion BMSC has the potential of differentiating to photoreceptor-like cells and retinal nerve-like cell.