骨髓间充质干细胞在激光损伤大鼠视网膜下分化的观察

Observation of bone marrow mesenchymal stem cells after subretinally transplanted into laser-injured rat retina

目的 鉴定骨髓间充质干细胞 (MSC)在体外不诱导的条件下移植于掺钕钇铝石榴石(Nd :YAG)激光损伤大鼠视网膜后的定位以及蛋白表达情况。方法 体外培养大鼠MSCs ,用荧光染料 4′ ,6 二脒 2 苯基吲哚 (DAPI)标记MSCs,视网膜下移植后分别于 10、2 0、35和 5 0d处死动物 ,做DAPI荧光观察 ,并在阳性的连续切片上作神经元核 (NeuN) ,神经元特异性烯醇化酶 (NSE) ,胶质纤维酸性蛋白 (GFAP)和角蛋白 (CK)免疫组化染色及HE染色。分别于损伤前及损伤后即刻、1～ 7周检测损伤移植组、损伤组和损伤注射生理盐水组的视网膜电图 (ERG)b波分析。结果 培养的MSC集落生长迅速 ,均一性好 ;DAPI染色观察表明 ,10d时移植细胞多集中于移植部位周围 ,但分布散乱 ;2 0d时可见阳性细胞分布范围扩大 ,分布于视网膜视锥、视干细胞层、双极细胞层及节细胞层 ;到35d阳性细胞范围进一步扩大 ,且细胞向损伤部位迁移 ;5 0d观察损伤范围与 35d相比扩大不明显。免疫组织化学染色发现阳性区域的细胞在NeuN、NSE、GFAP和CK的表达有不均一性。检测到的阳性区域视网膜未见到玫瑰花结样结构 ,但某些区域的细胞排列不整齐 ,在某些部位有细胞层增生。HE染色表明移植组损伤的恢复好于损伤对照组 ,ERGb波检测表明

Objective Bone marrow mesenchymal stem cells (MSCs) develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of retina. Eye was known as an immunologically privileged organ. Because of its special structure, it′s difficult for blood cells to enter retina. This article is to trace bone marrow mesenchymal stem cell (MSCs) after subretinally transplanted into Nd: YAG laser-injured rat retinal without in vitro differentiation induction. Method 4′, 6-diamidino-2-phenylindole (DAPI)-labeled MSCs were used to trace the change of MSCs after transplantation on day 10, 20, 35 and 50. The sequenced sections were used for Immunohistochemitry identification for neuronal nuclei (NeuN), neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP) and pancytokeratin (CK). Electroretinogram (ERG) b waves were recorded for each eye of the laser injured group, the laser injured transplanted group and the laser injured with saline injection control group before, right after or at every end of 1 to 7 weeks. Results On day 10, the DAPI positive cells were mainly crowded around the transplanted site. On day 20 the positive area enlarged and scattered into RPE layer, photoreceptor layer, bipolar layer and ganglion cell layer. Then the positive area enlarged more widely on day 35 and more cells could be found migrate to the lesion site. The positive area didn′t enlarge much on day 50 than on day 35. No formation of rosettes was found during the observation. But the expression of NeuN?NSE?GFAP and CK was not uniform as the normal retina. Cell proliferation was still found. But HE staining showed that the lesion in the transplanted group was better than that of the control group. Correspondingly ERG b-wave value at week 5 was higher than the control group. Conclution From these cells, a proportion of the cells regenerated from bone marrow can be differentiated into retina in vivo. Although the MSCs-derived cells could not precisely express neuro-like proteins, they could help recover lesion and ERG b-wave value.