Mesenchymal Stem Cells Promote Diabetic Corneal Epithelial Wound Healing Through TSG-6-Dependent Stem Cell Activation and Macrophage Switch
Mesenchymal Stem Cells Promote Diabetic Corneal Epithelial Wound Healing Through TSG-6-Dependent Stem Cell Activation and Macrophage Switch
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间充质干细胞通过 TSG-6 依赖性干细胞激活和巨噬细胞开关促进糖尿病角膜上皮伤口愈合
DOI：
10.1167/iovs.17-21506
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发表时间：
2017-08-01
影响因子：
4.4
通讯作者：
Zhou, Qingjun
中科院分区：
文献类型：
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作者：
Di, Guohu;Du, Xianli;Zhou, Qingjun
目的.探讨骨髓间充质干细胞（BM-MSCs）在1型糖尿病小鼠角膜上皮创伤愈合中的作用及机制。用结膜下注射BM-MSC或重组肿瘤坏死因子-α刺激的基因/蛋白-6（TSG-6）治疗糖尿病小鼠。用荧光素染色法检测角膜上皮愈合率。RT-PCR和Western blot检测TSG-6 mRNA和蛋白表达水平。流式细胞术和免疫荧光染色分析白细胞和巨噬细胞的浸润情况。通过短发夹RNA（shRNA）敲除，在培养的角膜缘上皮干/祖细胞、巨噬细胞和糖尿病小鼠中进一步评价了TSG-6的作用。局部MSC移植显著促进糖尿病角膜上皮伤口愈合，伴随角膜TSG-6表达升高，角膜上皮细胞增殖增加，炎症反应减弱。此外，在培养的人角膜缘上皮干/祖细胞中，TSG-6增强集落形成效率，刺激有丝分裂增殖，并上调DNp 63的表达水平。此外，在糖尿病小鼠角膜和体外巨噬细胞培养中，TSG-6减轻白细胞浸润，并促进募集的巨噬细胞极化为抗炎M2表型，吞噬能力增加。此外，TSG-6的shRNA敲低可显著抑制MSC移植促进上皮干/祖细胞活化和巨噬细胞极化。本研究首次证明了MSCs分泌的TSG-6通过激活角膜上皮干/祖细胞和加速M2巨噬细胞极化促进糖尿病小鼠角膜上皮伤口愈合。
PURPOSE. To explore the role and mechanism of bone marrow-derived mesenchymal stem cells (BM-MSCs) in corneal epithelial wound healing in type 1 diabetic mice.METHODS. Diabetic mice were treated with subconjunctival injections of BM-MSCs or recombinant tumor necrosis factor-a-stimulated gene/protein-6 (TSG-6). The corneal epithelial wound healing rate was examined by fluorescein staining. The mRNA and protein expression levels of TSG-6 were measured by quantitative RT-PCR and Western blot. The infiltrations of leukocytes and macrophages were analyzed by flow cytometry and immunofluoresence staining. The effect of TSG-6 was further evaluated in cultured limbal epithelial stem/progenitor cells, macrophages, and diabetic mice by short hairpin RNA (shRNA) knockdown.RESULTS. Local MSC transplantation significantly promoted diabetic corneal epithelial wound healing, accompanied by elevated corneal TSG-6 expression, increased corneal epithelial cell proliferation, and attenuated inflammatory response. Moreover, in cultured human limbal epithelial stem/progenitor cells, TSG-6 enhanced the colony-forming efficiency, stimulated mitogenic proliferation, and upregulated the expression level of DNp63. Furthermore, in diabetic mouse cornea and in vitro macrophage culture, TSG-6 alleviated leukocyte infiltration and promoted the polarization of recruited macrophages to anti-inflammatory M2 phenotypes with increased phagocytotic capacity. In addition, the promotion of epithelial stem/progenitor cell activation and macrophage polarization by MSC transplantation was largely abrogated by shRNA knockdown of TSG-6.CONCLUSIONS. This study provided the first evidence of TSG-6 secreted by MSCs promoting corneal epithelial wound healing in diabetic mice through activating corneal epithelial stem/progenitor cells and accelerating M2 macrophage polarization.