Corneal stromal stem cells (CSSC) reduce corneal inflammation, prevent fibrotic scarring, and regenerate transparent stromal tissue in injured corneas. These effects rely on factors produced by CSSC to block the fibrotic gene expression. This study investigated the mechanism of the scar-free regeneration effect. Primary human CSSC (hCSSC) from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferon-γ and lipopolysaccharides, or to M2 anti-inflammatory phenotype by interleukin-4, in a Transwell system. The time-course expression of human transforming growth factor β3 (hTGFβ3) and hTGFβ1 were examined by immunofluorescence and qPCR. TGFβ3 knockdown for > 70% in hCSSC [hCSSC-TGFβ3(si)] was achieved by small interfering RNA transfection. Naïve CSSC and hCSSC-TGFβ3(si) were transplanted in a fibrin gel to mouse corneas, respectively, after wounding by stromal ablation. Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined. hTGFβ3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition. Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFβ3 at days 1 and 3 post-injury, along with the reduced expression of mouse inflammatory genes (CD80, C-X-C motif chemokine ligand 5, lipocalin 2, plasminogen activator urokinase receptor, pro-platelet basic protein, and secreted phosphoprotein 1). By day 14, hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes (fibronectin, hyaluronan synthase 2, Secreted protein acidic and cysteine rich, tenascin C, collagen 3a1 and α-smooth muscle actin), and the injured corneas remained clear. However, hCSSC-TGFβ3(si) lost these anti-inflammatory and anti-scarring functions, and the wounded corneas showed intense scarring. This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFβ3, inducing a scar-free tissue response.

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转化生长因子β3介导角膜基质干细胞疗法的抗瘢痕形成作用

角膜基质干细胞（CSSC）可以减少角膜发炎，防止纤维化瘢痕形成并在受伤的角膜中再生透明的基质组织。这些作用依赖于CSSC产生的因子来阻断纤维化基因的表达。本研究探讨了无疤痕再生作用的机理。将来自供体角膜缘的原代人CSSC（hCSSC）培养至第3代，并与通过干扰素γ和脂多糖处理诱导为M1促炎表型或通过诱导M2抗炎表型的小鼠巨噬细胞RAW264.7细胞共培养Transwell系统中的interleukin-4。通过免疫荧光和qPCR检测人转化生长因子β3（hTGFβ3）和hTGFβ1的时程表达。TGFβ3抑制> hCSSC [hCSSC-TGFβ3（si）]中的70％是通过小分子干扰RNA转染实现的。初次CSSC和hCSSC-TGFβ3（si）在通过基质消融术受伤后分别通过纤维蛋白凝胶移植到小鼠角膜。检查了角膜的清晰度以及小鼠炎症和纤维化基因的表达。在M1条件下与RAW细胞共培养时，hCSSC上调了hTGFβ3。将hCSSC移植到受伤的小鼠角膜后，在损伤后的第1天和第3天，hTGFβ3显着上调，同时小鼠炎症基因（CD80，CXC趋化因子配体5，脂蛋白2，纤溶酶原激活物尿激酶受体，促血小板凝集素）的表达降低。碱性蛋白和分泌的磷蛋白1）。到第14天时，hCSSC治疗显着降低了纤维化和瘢痕组织基因（纤连蛋白，透明质酸合酶2，分泌酸性蛋白和富含半胱氨酸，腱生蛋白C，胶原蛋白3a1和α平滑肌肌动蛋白），受伤的角膜仍清晰可见。然而，hCSSC-TGFβ3（si）失去了这些抗炎和抗瘢痕形成的功能，受伤的角膜显示出强烈的疤痕。这项研究表明，hCSSC的角膜再生作用是由TGFβ3介导的，可诱导无疤痕的组织反应。