骨髓间充质干细胞在大鼠视网膜光感受器细胞损伤中的应用研究

摘要

目的 探讨骨髓间充质干细胞(BMSCs)在大鼠视网膜光感受器细胞损伤中的作用机制.方法 选取24只SD大鼠,分为正常组(n=6)、光损伤组(n=6,视网膜下注射无菌生理盐水4μl)、磷酸盐(PBS)视网膜下注射组(n=6,视网膜下注射PBS 4μl,其中溶质为BMSCs)和BMSCs视网膜下移植组(n=6,视网膜下移植2xl05个BMSCs),除正常组外,其余各组光照射24 h,光照后10 d进行手术.术后14 d将眼球摘除,培养大鼠BMSCs原代及传代,制作冰冻切片,行HE染色,计算视网膜外核层层数.采取免疫荧光法,检测大鼠BMSCs表面标志物表达情况.采取TUNEL法检测细胞凋亡情况.结果 与正常组比较其视网膜外核层层数明显减少,细胞凋亡百分比有所升高;与光损伤组、PBS组相比,BMSCs组视网膜外核层层数明显增加,细胞凋亡百分比有所降低;其中BMSCs细胞在视网膜下腔不表达MAP2、 CDllb、RhOdopsin、Calretintin,部分表达Nestin阳性;同时4,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)阳性的BMSCs细胞视网膜下腔表达bFGF,不表达BDNF和CNIF.结论 大鼠实验结果表明,BMSCs视网膜下移植,可减少视网膜光感受器细胞凋亡,作用原理是可能通过视网膜下腔分泌的bFGF发挥作用.%Objective To discuss the mechanism of action of bone marrow mesenchymal stem cells (BMSCs) in retinal photoreceptor cell injury. Methods 24SD rats were divided into normal group (n=6), light injury group (n=6, subretinal injection of aseptic saline 4μl), PBS subretinal injection group (n=6, subretinal injection of PBS 4μi, and the solute was BMSCs) and BMSCs subretinal transplantation group (n=6, ubretinal transplantation of 2× 105BMSCs).Except the normal group, the other groups were irradiated by 24h, after 10d, the operation was performed, and after 14d eyeballs were removed after the operation,and the primary and passage of rat BMSCs were cultured, frozen sections were made and HE staining was performed, and the number of outer nuclear layers were calculated. Then, the expression of BMSCs surface markers in rats were detected by the Immunofluorescence method,and the cell apoptosis were detected by TUNEL method. Results Compared with the normal group, the number of outer nuclear layer in BMSCs group was decreased significantly, and the percentage of apoptosis cells was increased signⅢcantly. Compared with the light injury group and PBS group, the number of outer nuclear layer in BMSCs group were increased significantly, and the percentage of apoptosis cells were decreased significantly. The BMSCs cells were not expressed MAP2, CDllb, Rhodopsin, Calretintin in the subretinal space, and some of them were positive for Nestin expression. At the same time, the expression of bFGF in the retina of DAPI positive BMSCs cells was not expressed in BDNF and CNIF. Conclusion The BMSCs subretinal transplantation, which can reduce the apoptosis of retinal photoreceptor cells, the principle of action may be to play a role in the secretion of bFGF through the subretinal space.