Department of Ophthalmology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China.
Mol Vis. 2023 May 20;29:58-67. eCollection 2023.
To evaluate the relationship between basement membrane (BM) regeneration and the spatiotemporal expression of TGF-β1 during wound healing in rabbits with corneal perforating injury.
Forty-two rabbits were randomly allocated into 7 experimental groups, with 6 rabbits per group at each time point. The central cornea of the left eye was injured with 2.0 mm trephine to establish the perforating injury model. Six rabbits that received no treatment were used as controls. The cornea was evaluated at 3 days, 1-3 weeks, and 1-3 months after injury with a slit lamp for haze levels. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed to quantify the relative expression of TGF-β1 and α-SMA mRNA. Immunofluorescence (IF) was used to assess TGF-β1 and alpha-smooth actin (α-SMA) expression and localization. BM regeneration was assessed using transmission electron microscopy (TEM).
After injury, dense haze appeared at 1 month and then gradually faded. The relative expression of TGF-β1 mRNA peaked at 1 week and then decreased until 2 months. The relative α-SMA mRNA expression reached its peak at 1 week, then reached a small peak again at 1 month. IF results showed that TGF-β1 was initially detected in the fibrin clot at 3 days and then in the entire repairing stroma at 1 week. TGF-β1 localization gradually diminished from the anterior region to the posterior region at 2 weeks to 1 month, and it was nearly absent at 2 months. The myofibroblast marker α-SMA was observed in the entire healing stroma at 2 weeks. Localization of α-SMA gradually disappeared from the anterior region at 3 weeks to 1 month, remaining only in the posterior region at 2 months and disappearing at 3 months. Defective epithelial basement membrane (EBM) was first detected at 3 weeks after injury, then gradually repaired, and was nearly regenerated at 3 months. A thin and uneven Descemet's membrane (DM) was initially detected at 2 months after injury, then gradually regenerated to some extent, but remained abnormal at 3 months.
In the rabbit corneal perforating injury model, EBM regeneration was observed earlier than DM. At 3 months, complete EBM regeneration was observed, while the regenerated DM was still defective. TGF-β1 was distributed throughout the entire wound area in the early stages and then decreased from the anterior to the posterior region. α-SMA exhibited a similar temporospatial expression to TGF-β1. EBM regeneration may play a key role in low expression of TGF-β1 and α-SMA in the anterior stroma. Meanwhile, incomplete DM regeneration may contribute to the sustained expression of TGF-β1 and α-SMA in the posterior stroma.
评价兔角膜穿孔伤愈合过程中基底膜（BM）再生与转化生长因子-β1（TGF-β1）时空表达的关系。
42 只兔子随机分为 7 个实验组，每组 6 只兔子。左眼中央角膜用 2.0mm 环钻造成穿孔伤模型。不做任何处理的 6 只兔子作为对照组。裂隙灯检查混浊度评估角膜损伤后 3 天、1-3 周和 1-3 个月的情况。实时定量聚合酶链反应（qRT-PCR）用于定量检测 TGF-β1 和α-SMA mRNA 的相对表达。免疫荧光（IF）用于评估 TGF-β1 和α-平滑肌肌动蛋白（α-SMA）的表达和定位。透射电镜（TEM）用于评估 BM 再生。
损伤后 1 个月出现密集混浊，然后逐渐消退。TGF-β1mRNA 的相对表达在 1 周时达到峰值，然后下降至 2 个月。α-SMA mRNA 的相对表达在 1 周时达到峰值，然后在 1 个月时再次达到小峰值。IF 结果显示，TGF-β1 于第 3 天在纤维蛋白凝块中首次检测到，然后在第 1 周在整个修复基质中检测到。TGF-β1 的定位从第 2 周的前区逐渐向 1 个月的后区减少，2 个月时几乎消失。在第 2 周可观察到肌成纤维细胞标志物α-SMA 存在于整个愈合基质中。3 周时，α-SMA 的定位从前区逐渐向 1 个月的后区消失，仅在 2 个月时在后区残留，3 个月时消失。损伤后第 3 周首次检测到上皮基底膜（EBM）缺陷，然后逐渐修复，3 个月时几乎再生。受伤后 2 个月时首次检测到薄而不均匀的德斯梅特膜（DM），随后在一定程度上再生，但 3 个月时仍异常。
在兔角膜穿孔伤模型中，EBM 的再生早于 DM。3 个月时，观察到完整的 EBM 再生，而再生的 DM 仍存在缺陷。TGF-β1 在早期分布于整个伤口区域，然后从前区向后区减少。α-SMA 的时空表达与 TGF-β1 相似。EBM 再生可能在低表达的 TGF-β1 和前基质中的α-SMA 中起关键作用。同时，不完全的 DM 再生可能导致 TGF-β1 和α-SMA 在后部基质中的持续表达。
