间充质干细胞衍生的外泌体通过VEGF依赖性机制改善视网膜色素上皮细胞中的蓝光刺激和视网膜激光损伤。
Mesenchymal stem cells-derived exosomes ameliorate blue light stimulation in retinal pigment epithelium cells and retinal laser injury by VEGF-dependent mechanism.
作者信息
He Guang-Hui, Zhang Wei, Ma Ying-Xue, Yang Jing, Chen Li, Song Jian, Chen Song
机构信息
Tianjin Eye Hospital; Tianjin Key Lab of Ophthalmology and Visual Science; Tianjin Eye Institute; Clinical College of Ophthalmology, Tianjin Medical University, Tianjin 300020, China.
出版信息
Int J Ophthalmol. 2018 Apr 18;11(4):559-566. doi: 10.18240/ijo.2018.04.04. eCollection 2018.
AIM
To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor-A (VEGF-A) in blue light injured human retinal pigment epithelial (RPE) cells and laser-induced choroidal neovascularization (CNV) in rats.
METHODS
Exosomes were isolated from hUCMSCs and characterized by transmission electron microscope and Western blot. MSCs-derived exosomes were cultured with RPE cells exposed to blue light. The mRNA and protein expression of VEGF-A were determined by real time-polymerase chain reaction (PCR) and Western blot, respectively. Immunofluorescence assay was used for the detection of the expression level of VEGF-A. We injected different doses of MSCs-derived exosomes intravitreally to observe and compare their effects in a mouse model of laser-induced retinal injury. The histological structure of CNV in rats was inspected by hematoxylin-eosin (HE) staining and fundus fluorescein angiography. The expression of VEGF-A was detected by immunohistochemistry.
RESULTS
Exosomes exhibited the typical characteristic morphology (cup-shaped) and size (diameter between 50 and 150 nm). The exosomes marker, CD63, and hUCMSCs marker, CD90, showed a robust presence. , MSCs-derived exosomes downregulated the mRNA(Exo-L: =6.485, 7.959, 9.286; Exo-M: =7.517, 10.170, 13.413; Exo-H: =10.317, 12.234, 14.592, <0.05) and protein (Exo-L: =2.945, 4.477, 6.657; Exo-M: =4.713, 6.421, 8.836; Exo-H: =6.539, 12.194, 12.783; <0.05) expression of VEGF-A in RPE cells after blue light stimulation. , we found that the MSCs-derived exosomes reduced damage, distinctly downregulated VEGF-A (Exo-H: =0.957, 1.382; <0.05), and gradually improved the histological structures of CNV for a better visual function (Exo-L: 0.346, Exo-M: 3.382, Exo-H: 8.571; <0.05).
CONCLUSION
MSCs-derived exosomes ameliorate blue light stimulation in RPE cells and laser-induced retinal injury downregulation of VEGF-A.
目的
观察人脐带血间充质干细胞（hUCMSCs）来源的外泌体对蓝光损伤的人视网膜色素上皮（RPE）细胞中血管内皮生长因子-A（VEGF-A）表达及大鼠激光诱导脉络膜新生血管（CNV）的影响。
方法
从hUCMSCs中分离外泌体，通过透射电子显微镜和蛋白质免疫印迹进行鉴定。将MSCs来源的外泌体与暴露于蓝光的RPE细胞共培养。分别通过实时聚合酶链反应（PCR）和蛋白质免疫印迹法测定VEGF-A的mRNA和蛋白表达。采用免疫荧光法检测VEGF-A的表达水平。向玻璃体内注射不同剂量的MSCs来源的外泌体，在激光诱导的视网膜损伤小鼠模型中观察并比较其效果。通过苏木精-伊红（HE）染色和眼底荧光血管造影检查大鼠CNV的组织结构。采用免疫组织化学法检测VEGF-A的表达。
结果
外泌体呈现典型的特征形态（杯状）和大小（直径在50至150nm之间）。外泌体标志物CD63和hUCMSCs标志物CD90均有显著表达。MSCs来源的外泌体下调了蓝光刺激后RPE细胞中VEGF-A的mRNA（Exo-L：=6.485，7.959，9.286；Exo-M：=7.517，10.170，13.413；Exo-H：=10.317，12.234，14.592，<0.05）和蛋白（Exo-L：=2.945，4.477，6.657；Exo-M：=4.713，6.421，8.836；Exo-H：=6.539，12.194，12.783；<0.05）表达。我们发现MSCs来源的外泌体减少了损伤，显著下调了VEGF-A（Exo-H：=0.957，1.382；<0.05），并逐渐改善了CNV的组织结构，以获得更好的视觉功能（Exo-L：0.346，Exo-M：3.382，Exo-H：8.571；<0.05）。
结论
MSCs来源的外泌体改善了RPE细胞中的蓝光刺激和激光诱导的视网膜损伤——VEGF-A的下调。
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