Jester J V, Petroll W M, Barry P A, Cavanagh H D
Department of Ophthalmology, University of Texas, Southwestern Medical Center at Dallas 75235-9057, USA.
Invest Ophthalmol Vis Sci. 1995 Apr;36(5):809-19.
The purpose of this study was to correlate the temporal expression of alpha-smooth muscle specific actin (alpha-SM actin), a molecular marker for myofibroblast transformation, with corneal wound contraction.
After full-thickness, central corneal injury in rabbit eyes, the anterior width of the wound (wound gape) was measured in the same animals using in vivo confocal microscopy. In addition, animals were sacrificed at various times after injury for the determination of alpha-SM actin expression by immunofluorescent microscopy using a mouse monoclonal antibody specific for human alpha-actin. Antibody specificity was confirmed by Western blot analysis of normal and wound fibroblasts. Expression of alpha-SM actin also was related spatially to f-actin and the wound margin by co-localization with phalloidin and DTAF (5([4,6-dichlorotriazin-2yl]amino)fluorescein), a fluorescent marker bound to the wound margin.
Wound contraction was most evident from days 7 to 42, when wound gape progressively decreased from 574 +/- 120 microns to 250 +/- 61 microns. Thereafter, the wound remained stable to day 84 (304 +/- 58 microns). Expression of alpha-SM actin directly correlated with wound contraction--appearing across the wound at day 7, the full thickness of the wound at day 14, and the posterior wound at day 28. alpha-SM actin was localized exclusively to phalloidin-stained, f-actin microfilament bundles or stress fibers within wound healing fibroblasts, and the disappearance of alpha-SM actin correlated with the concomitant disappearance of stress fibers at days 28 to 42. Staining of the wound margin with DTAF confirmed that the expression of alpha-SM actin was limited to fibroblasts within the wound.
The expression of alpha-SM actin was directly correlated to corneal wound contraction, appearing at the initiation of and disappearing at the completion of the contraction process. Furthermore, the exclusive expression of alpha-SM actin by fibroblasts present only within the wound suggests that local environmental factors unique to the wound may play an important role in myofibroblast transformation.
本研究旨在将α-平滑肌特异性肌动蛋白（α-SM肌动蛋白）（一种肌成纤维细胞转化的分子标志物）的时间表达与角膜伤口收缩相关联。
在兔眼进行全层中央角膜损伤后，使用体内共聚焦显微镜在同一动物中测量伤口的前部宽度（伤口裂口）。此外，在损伤后的不同时间处死动物，使用针对人α-肌动蛋白的小鼠单克隆抗体通过免疫荧光显微镜确定α-SM肌动蛋白的表达。通过对正常和成纤维细胞进行蛋白质免疫印迹分析来确认抗体特异性。α-SM肌动蛋白的表达还通过与鬼笔环肽和DTAF（5([4,6-二氯三嗪-2基]氨基)荧光素）共定位在空间上与f-肌动蛋白和伤口边缘相关，DTAF是一种与伤口边缘结合的荧光标记物。
伤口收缩在第7天至42天最为明显，此时伤口裂口从574±120微米逐渐减小至250±61微米。此后，伤口在第84天（304±58微米）保持稳定。α-SM肌动蛋白的表达与伤口收缩直接相关——在第7天出现在伤口处，第14天出现在伤口全层，第28天出现在伤口后部。α-SM肌动蛋白仅定位于伤口愈合成纤维细胞中经鬼笔环肽染色的f-肌动蛋白微丝束或应力纤维，并且α-SM肌动蛋白的消失与第28天至42天应力纤维的同时消失相关。用DTAF对伤口边缘进行染色证实α-SM肌动蛋白的表达仅限于伤口内的成纤维细胞。
α-SM肌动蛋白的表达与角膜伤口收缩直接相关，在收缩过程开始时出现，在收缩过程完成时消失。此外，仅存在于伤口内的成纤维细胞对α-SM肌动蛋白的特异性表达表明伤口特有的局部环境因素可能在肌成纤维细胞转化中起重要作用。
