Suppression of alkali-induced oxidative injury in the cornea by mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface
Suppression of alkali-induced oxidative injury in the cornea by mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface
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DOI：
10.1016/j.exer.2013.10.002
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发表时间：
2013-11-01
影响因子：
3.4
通讯作者：
Holan, Vladimir
中科院分区：
文献类型：
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作者：
Cejkova, Jitka;Trosan, Peter;Holan, Vladimir
本研究旨在探讨兔骨髓间充质干细胞（MSCs）是否能有效降低兔角膜碱诱导的氧化应激。将碱（0.15 N NaOH）应用于右眼角膜，然后用自来水冲洗。在第一组兔子中，受伤的角膜没有得到治疗。第二组在损伤后立即将MSC应用于损伤的角膜表面，并缝合眼睑两天。然后拆除缝线。在第三组中，在损伤后立即将接种有MSC的角膜基质支架（以及在第四组中仅纳米纤维）转移到角膜上，并缝合眼睑。两天后，将眼睑缝合线与支架一起移除。在损伤后第4、10或15天处死兔，使用超声测厚仪和实时PCR对角膜进行化学、形态学、中央角膜厚度（作为角膜水合的指标）的检查。结果表明，在未处理的损伤角膜中，上皮中出现丙二醛（MDA）和硝基酪氨酸（NT）（脂质过氧化和氧化应激的重要标志物）的表达。抗氧化剂醛脱氢酶3A 1（ALDH 3A 1）在角膜上皮中减少，特别是在浅表部分，其中凋亡细胞死亡（通过活性半胱天冬酶-3检测）较高。(In对照角膜上皮MDA和NT不存在，ALDH 3A 1在上皮的所有层中高度存在。细胞凋亡是偶发的）。在受伤的未治疗的角膜进一步角膜紊乱发展：基质金属蛋白酶9（MMP 9）和促炎细胞因子的表达，是高的。在实验结束时（第15天），损伤的未处理角膜血管化，并且角膜基质中存在大量炎性细胞。血管内皮生长因子（VEGF）表达和巨噬细胞数量较高。在仅用MSC支架覆盖的损伤角膜（没有MSC）或仅用MSC治疗的损伤角膜（在没有支架的情况下转移）中获得的结果与在未治疗的损伤角膜中发现的结果没有显著差异。相比之下，在用MSC在角膜基质支架上处理的损伤角膜中，ALDH 3A 1表达在上皮中保持高水平（如在对照角膜中），并且所采用的其他免疫组织化学标记物的阳性表达非常低（MMP 9）或不存在（NT、MDA、促炎细胞因子），也与对照角膜类似。与未处理的损伤角膜相比，用MSC处理的损伤角膜中角膜新生血管和角膜基质中炎性细胞的浸润被显著抑制。碱损伤后出现的中央角膜厚度增加以及角膜混浊仅在用MSC在支架上治疗的损伤角膜中在10天的过程中恢复到正常水平。促炎细胞因子的基因表达与其免疫组化表达相一致。总之，MSC的骨支架保护形成有毒的过氧亚硝酸盐（检测NT残留），降低凋亡细胞死亡，减少基质金属蛋白酶和促炎细胞因子的产生。这导致角膜炎症和新生血管减少，并显着加速角膜愈合。(C)2013爱思唯尔有限公司保留所有权利。
The purpose of this study was to investigate whether rabbit bone marrow-derived mesenchymal stem cells (MSCs) effectively decrease alkali-induced oxidative stress in the rabbit cornea. The alkali (0.15 N NaOH) was applied on the corneas of the right eyes and then rinsed with tap water. In the first group of rabbits the injured corneas remained untreated. In the second group MSCs were applied on the injured corneal surface immediately after the injury and eyelids sutured for two days. Then the sutures were removed. In the third group nanofiber scaffolds seeded with MSCs (and in the fourth group nanofibers alone) were transferred onto the corneas immediately after the injury and the eyelids sutured. Two days later the eyelid sutures were removed together with the nanofiber scaffolds. The rabbits were sacrificed on days four, ten or fifteen after the injury, and the corneas were examined immunohistochemically, morphologically, for the central corneal thickness (taken as an index of corneal hydration) using an ultrasonic pachymeter and by real-time PCR. Results show that in untreated injured corneas the expression of malondialdehyde (MDA) and nitrotyrosine (NT) (important markers of lipid peroxidation and oxidative stress) appeared in the epithelium. The antioxidant aldehyde dehydrogenase 3A1 (ALDH3A1) decreased in the corneal epithelium, particularly in superficial parts, where apoptotic cell death (detected by active caspase-3) was high. (In control corneal epithelium MDA and NT are absent and ALDH3A1 highly present in all layers of the epithelium. Cell apoptosis are sporadic). In injured untreated cornea further corneal disturbances developed: The expressions of matrix metalloproteinase 9 (MMP9) and proinflammatory cytokines, were high. At the end of experiment (on day 15) the injured untreated corneas were vascularized and numerous inflammatory cells were present in the corneal stroma. Vascular endothelial growth factor (VEGF) expression and number of macrophages were high. The results obtained in injured corneas covered with nanofiber scaffolds alone (without MSCs) or in injured corneas treated with MSCs only (transferred without scaffolds) did not significantly differ from the results found in untreated injured corneas. In contrast, in the injured corneas treated with MSCs on nanofiber scaffolds, ALDH3A1 expression remained high in the epithelium (as in the control cornea) and positive expression of the other immunohistochemical markers employed was very low (MMP9) or absent (NT, MDA, proinflammatory cytokines), also similarly as in the control cornea. Corneal neovascularization and the infiltration of the corneal stroma with inflammatory cells were significantly suppressed in the injured corneas treated with MSCs compared to the untreated injured ones. The increased central corneal thickness together with corneal opalescency appearing after alkali injury returned to normal levels over the course of ten days only in the injured corneas treated with MSCs on nanofiber scaffolds. The expression of genes for the proinflammatory cytokines corresponded with their immunohistochemical expression. In conclusion, MSCs on nanofiber scaffolds protected the formation of toxic peroxynitrite (detected by NT residues), lowered apoptotic cell death and decreased matrix metalloproteinase and proinflammatory cytokine production. This resulted in reduced corneal inflammation as well as neovascularization and significantly accelerated corneal healing. (C) 2013 Elsevier Ltd. All rights reserved.