Di Guohu, Du Xianli, Qi Xia, Zhao Xiaowen, Duan Haoyun, Li Suxia, Xie Lixin, Zhou Qingjun
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China.
Invest Ophthalmol Vis Sci. 2017 Aug 1;58(10):4344–4354. doi: 10.1167/iovs.17-21506.
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.
Diabetic mice were treated with subconjunctival injections of BM-MSCs or recombinant tumor necrosis factor-α-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.
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 ΔNp63. 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.
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.
探讨骨髓间充质干细胞（BM-MSCs）在1型糖尿病小鼠角膜上皮伤口愈合中的作用及机制。
对糖尿病小鼠进行结膜下注射BM-MSCs或重组肿瘤坏死因子-α刺激基因/蛋白-6（TSG-6）治疗。通过荧光素染色检查角膜上皮伤口愈合率。采用定量RT-PCR和蛋白质免疫印迹法检测TSG-6的mRNA和蛋白表达水平。通过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）敲低在培养的角膜缘上皮干细胞/祖细胞、巨噬细胞和糖尿病小鼠中进一步评估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，同时角膜TSG-6表达升高，角膜上皮细胞增殖增加，炎症反应减轻。此外，在培养的人角膜缘上皮干细胞/祖细胞中，TSG-6提高了集落形成效率，刺激有丝分裂增殖，并上调了ΔNp63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减轻了白细胞浸润，并促进募集的巨噬细胞向具有增强吞噬能力的抗炎M2表型极化。此外，TSG-6的shRNA敲低在很大程度上消除了MSC移植对上皮干细胞/祖细胞激活和巨噬细胞极化的促进作用。
本研究首次证明MSCs分泌的TSG-6通过激活角膜上皮干细胞/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。
