成纤维细胞在角膜创伤愈合反应中迁移、分化和凋亡。

Fibrocyte migration, differentiation and apoptosis during the corneal wound healing response to injury.

机构信息

Cole Eye Institute, Cleveland Clinic, Cleveland, OH, United States.

University of Sao Paulo, Sao Paulo, Brazil.

出版信息

Exp Eye Res. 2018 May;170:177-187. doi: 10.1016/j.exer.2018.02.018. Epub 2018 Feb 24.

Abstract

The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (αSMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ αSMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also αSMA+ myofibroblasts. At 21 days after PTK, few GFP+ αSMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature αSMA+ myofibroblasts. Most GFP+ CD45+ αSMA+ myofibroblasts that did develop subsequently underwent apoptosis-likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGFβ requisite for myofibroblast survival.

摘要

本研究旨在确定骨髓来源的成纤维细胞是否在基质产生瘢痕的损伤后迁移到角膜,并分化为α-平滑肌肌动蛋白 (αSMA)+肌成纤维细胞。表达绿色荧光蛋白 (GFP) 骨髓细胞的嵌合小鼠发生纤维化(混浊)产生不规则光动力角膜切削术(PTK)。使用多重免疫组织化学(IHC)检测 GFP 和纤维母细胞标记物(CD34、CD45 和波形蛋白),以检测纤维母细胞浸润角膜和 GFP+αSMA+肌成纤维细胞的形成。使用激活的 caspase-3、GFP 和 CD45 的免疫组织化学检测纤维母细胞和其他正在发生凋亡的造血细胞。手术后 14 天,PTK 治疗的小鼠角膜出现中度混浊,并且在手术后 21 天恶化并持续存在。GFP+CD34+CD45+纤维母细胞,可能还有其他 CD34+和/或 CD45+造血和干细胞/祖细胞,在手术后一天内渗透到角膜并在基质中大量存在。纤维母细胞和其他骨髓来源的细胞在手术后四天和七天逐渐减少。PTK 后四天,5%的 GFP+细胞表达激活的 caspase-3。PTK 后 14 天,超过 50%的 GFP+CD45+细胞也是 αSMA+肌成纤维细胞。PTK 后 21 天,基质中很少有 GFP+αSMA+细胞存活,超过 95%的存活细胞表达激活的 caspase-3,表明它们正在发生凋亡。在不规则 PTK 后 4 至 21 天发育的 GFP+CD45+SMA+细胞可能来自纤维母细胞。然而,在 C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J 嵌合小鼠的不规则 PTK 后,超过 95%的纤维母细胞和其他造血细胞在成熟的 αSMA+肌成纤维细胞形成之前发生凋亡。随后形成的大多数 GFP+CD45+αSMA+肌成纤维细胞也经历了凋亡-可能是由于上皮基底膜再生和剥夺上皮衍生的 TGFβ,这是肌成纤维细胞存活所必需的。

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