光损伤鼠视网膜片培养上清液诱导 MSCs 分化为视网膜样细胞的研究

目的：应用大鼠视网膜片光损伤后的培养上清液，在体外诱导大鼠骨髓间充质干细胞（ mesenchymal stem cells ， MSCs）成为视网膜样细胞的可能性。 方法：贴壁筛选法分离、培养大鼠MSCs ，流式细胞仪对其细胞纯度鉴定。取材大鼠视网膜神经上皮层作为视网膜片，常规石蜡切片HE染色鉴定各层组织完整性。电镜观察大鼠视网膜片光损伤程度。制备3种诱导分化大鼠MSCs的条件培养液。3种条件培养液均培养诱导至第3代大鼠MSCs 7～8d，用RT-PCR检测视紫红质（Rhodopsin）、神经元特异性烯醇化酶（neuron-specific enolase，NSE）、胶质纤维酸性蛋白（ glial fibrillary acidic protein ，GFAP）等视网膜细胞标志物在诱导后细胞中的表达情况。 结果：HE染色显示大鼠视网膜片取材结构完整，电镜显示大鼠视网膜片光损伤后结构损伤严重。 RT-PCR鉴定：条件培养液诱导大鼠MSCs 7～8d，条件培养液Ⅰ组Rhodopsin （0．3915±0．00644）、NSE （0．2019±0．00682）、GFAP （0.1972±0．00211），条件培养液Ⅱ组Rhodopsin（0．0983±0．00319）、NSE （0．1048±0．00323）、GFAP （0．1040±0.00254），条件培养液Ⅲ组Rhodopsin（0．0044±0．00126）、NSE（0．0498±0．00149）、GFAP（0．0467±0．00333），组间差异有统计学意义。 结论：光损伤大鼠视网膜片培养上清液可诱导大鼠MSCs分化为视网膜样细胞，为干细胞治疗视网膜变性疾病提供新思路。

In vitro differentiation of MSCs into retina-like cells by the supernatant fluid of light-injured neurosensory retina

AIM： To explore the possibility of inducing rat mesenchymal stem cells （ MSCs） into retina-like cells by the supernatant fluid of light-injured neurosensory retina in vitro.METHODS： MSCs were isolated and attached to the wall of culture dishes by their specific adherent ability. Then the cells were characterized by flow cytometry.The neurosensory retina was isolated from retina of SD rat and it was tested by hematoxylin-eosin （ HE ） staining.The pathological changes of light-injured neurosensory retina was observed under transmission electron microscope. Three kinds of supernatant fluid of light -injured neurosensory retina of SD rats were prepared.The third passage of MSCs were cultured with these mixed medium for 7-8d, we used RT-PCR to see whether they could express rhodopsin, neuron-specific enolase （NSE）, and glial fibrillary acidic protein （ GFAP ） , and positive cells were counted and analyzed. RESULTS： HE staining showed the retinal sheets included full-thickness neural retina.Neurosensory retina＆amp;nbsp;developed ultrastructural destructions by light injury.RT-PCR showed that the medium of mixed I expressed higher positive rate of rhodopsin （0.3915±0.00644）, NSE （0.2019± 0.00682）, GFAP （0.1972 ±0.00211） than the medium of mixed Ⅱ rhodopsin （0.0983 ±0.00319）, NSE （0.1048 ± 0.00323）, GFAP （0.1040±0.00254） and medium of mixedⅢrhodopsin（0.0044±0.00126）, NSE （0.0498±0.00149）, GFAP （0.0467±0.00333）.The difference of intergroup has statistical significance. 〈br〉 CONCLUTION：The supernatant fluid of light-injured neurosensory retina of SD rats can induce MSCs to differentiate into retina-like cells and provide new insights of stem cell therapy for retinopathy.