骨髓间充质干细胞分化为光感受器样细胞的体外诱导和微环境研究

Induce and differenctiation of marrow mesenchymal stem cell into photoreceptor-like cell in vitro and microenvironment

背景近年来间充质干细胞（MSCs）在眼科方面已成功诱导分化为角膜细胞、视网膜神经节细胞（RGCs）、视网膜神经样细胞等，但诱导成为光感受器细胞及其微环境的研究尚少。目的研究骨髓MSCs（BMSCs）在体外定向分化为光感受器样细胞的可能性及其所需的微环境。方法分别将第2代BMSCs细胞株和视网膜色素上皮（RPE）细胞株进行常规培养和传代，取第4代人BMSCs细胞株及第3代RPE细胞株用于实验。将BMSCs分为诱导组和对照组，诱导组的BMSCs与含有20μg／L碱性成纤维细胞生长因子（bFGF）、20μg／L表皮生长因子（EGF）及20μg／L脑源性神经营养因子（BDNF）的骨髓干细胞培养基（MSCM）及RPE细胞的条件培养液共培养进行BMSCs的诱导分化，而对照组的BMSCs仅用MSCM培养基进行培养，倒置显微镜下观察分化细胞的形态学变化。采用免疫细胞化学法检测RPE细胞诱导BMSCs3、5、7d时细胞中视紫红质蛋白的阳性表达率，以鉴定分化细胞的表型。采用逆转录PCR（RT—PCR）法检测RPE细胞诱导BMSCs5d、7d时细胞中视紫红质和恢复蛋白mRNA的表达情况。结果培养的BMSCs形态均一，呈纺锤形或梭形，RPE细胞形态均一，呈多边形或六边形，细胞内充满色素颗粒，生长良好。诱导后的部分BMSCs呈神经样细胞形态，细胞变圆，突起增长，突起末端可见一级、二级分支，相互间连接呈网状。免疫细胞化学法检测表明，诱导组细胞诱导后第3、5、7天细胞中可见视紫红质的表达，表达率分别为（5．83±0．29）％、（20．36±0．32）％和（29．80±2．30）％，明显高于对照组的（0．71±0．35）％、（2．56±0．24）％和（2．32±0．42）％，差异均有统计学意义（t3d=41．510，t5d=107．290，t7d=30．036，P〈0．01）。RT—PCR法检测显示，诱导后5d、7d诱导组细胞中视紫红质和恢复蛋白mRNA表达的灰度值均明显高于对照组，差异均有统计学意义（视紫红质mRNA：t5d=103．506，t7d=122．584，P〈0．01；恢复蛋白mRNA：t5d=106．674，t7d=189．992，P〈0．01）。结论采用含bFGF。

Background Marrow mesenchymal stem cells （MSCs） has been successful induced to differentiate into corneal cells,retinal ganglion cells （RGCs） and retinal neuron-like cells in recent years, but there are few studies about MSCs induced into photoreceptor cells and their microenvironment. Objective The aim of this study was to explore the induce and differentiation of bone marrow mesenehymal stem cells （BMSCs） into photoreeeptor-like cells in vitro and mieroenvironment. Methods The second generation of human BMSCs strain and RPE cells strain were cultured and passaged, respectively, and the fourth generation of BMSCs and the third generation of RPE cells were used in the experiment. BMSCs were eoeuhured using the mesenchymal stem cells medium （MSCM） contained 20 μg/L basic fibroblast growth factor （bFGF） ,20 μg/L epithelial growth factor （EGF） and 20μg/L brain derived neurotrophie factor （BDNF） with RPE cells to induce the differentiation of BMSCs in the induced group, and BMSCs were cultured in MSCM only in the control group. The morphology of induced and differentiated cells were observed under the inverted microscope. Inmmunocytoehemistry was used in induced for 3-, 5-,7-day cells to detect the expression rate of rhodopsin protein for the identification of phenotype of the differentiated cells. RT-PCR was used in induced for 5-,7-day cells to detect the expressions of rhodopsin mRNA and recoverin mRNA. Results Cultured BMSCs grew well with the spindle shape,and passaged RPE cells showed the uniform size and polygon shape with the abundant pigment in the cells. Some induced cells appeared to be the neuron-like cells with round shape and long prominence and the secondary reticular branches. The expression rates of rhodopsinin the cells were （5.83±0.29）%, （20. 36±0. 32）% and （29.80±2. 30）% in the third,fifth and seventh day after induce, which were significantly higher than （0.71 ±0. 35 ） % , （ 2. 56±0.24 ） % and （ 2. 32±0. 42 ） % of control cells （ t3 d = 41. 510, t5 d = 107. 290, t7 d = 30. 036, P〈0. 01 ）. The grey scales of rhodopsin mRNA and recoverin mRNA were significantly elevated in the induced and differentiated cells compared with control cells in the fifth and seventh day （ rhodopsin mRNA ：t5 d = 103. 506, t7 d = 122. 584, P〈0. 01 ;reeoverin mRNA ：t5 d = 106. 674, t7 d = 189. 992 ,P〈0. 01 ）. Conclusions BMSCs can be successfully indueed to differentiate into photoreeeptor cells after cocultured by conditioned medium with RPE cells.