Nakano Yukiko, Oyamada Masahito, Dai Ping, Nakagami Takuo, Kinoshita Shigeru, Takamatsu Tetsuro
Departments of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, Japan.
Invest Ophthalmol Vis Sci. 2008 Jan;49(1):93-104. doi: 10.1167/iovs.07-0255.
To explore connexin43 (Cx43) knockdown as an efficient treatment for corneal endothelial injury in an in vivo rat corneal scrape injury model.
Scrape injury was induced in the corneal endothelium, and immunolabeling (ZO-1, alpha-SMA, Cx43) was performed to analyze changes in Cx43 expression during wound healing. Single injection of Cx43 antisense oligodeoxynucleotide (AS-ODN), small interfering RNA (siRNA), or adenovirus (CMV-Cx43-mRFP1) was applied into the anterior chamber simultaneously with the injury, and wound closure was examined by immunolabeling (ZO-1, Cx43) and propidium iodide staining. Corneal endothelium proliferation on day 1 after injury was studied by Ki67-immunolabeling. Cx43-knockdown treatment was performed also without injury, and its effect on Cx43 expression and Ki67 immunolabeling was examined. The postinjury appearance of myofibroblasts in Cx43 AS-ODN- and sense-ODN-treated corneas was compared by alpha-SMA-immunolabeling.
Complete wound closures were observed in five of six corneas on day 3 after injury with either Cx43 AS-ODN or siRNA treatment, whereas no complete closure was observed on day 3 in the control corneas (S-ODN, zero of six; or nonsense siRNA, zero of six). Consistently, Cx43 overexpression using adenovirus delayed wound closure. Cx43 knockdown increased the number of Ki67-positive proliferating cells on day 1, whereas it decreased the number of alpha-SMA-positive myofibroblasts on day 5. Cx43 knockdown without injury decreased Cx43 expression and induced endothelial proliferation in vivo.
These results show that Cx43 knockdown induces corneal endothelium proliferation but inhibits endothelial-mesenchymal transition/transformation after injury, suggesting that Cx43 knockdown is a new therapeutic approach for acceleration of wound closure and for prevention of retrocorneal fibrous membrane formation.
在大鼠角膜刮伤损伤的体内模型中，探索下调连接蛋白43（Cx43）作为角膜内皮损伤的有效治疗方法。
诱导角膜内皮刮伤，并进行免疫标记（紧密连接蛋白1（ZO-1）、α-平滑肌肌动蛋白（α-SMA）、Cx43）以分析伤口愈合过程中Cx43表达的变化。在损伤同时向前房单次注射Cx43反义寡脱氧核苷酸（AS-ODN）、小干扰RNA（siRNA）或腺病毒（CMV-Cx43-mRFP1），并通过免疫标记（ZO-1、Cx43）和碘化丙啶染色检查伤口闭合情况。通过Ki67免疫标记研究损伤后第1天角膜内皮的增殖情况。也在无损伤情况下进行Cx43敲低处理，并检查其对Cx43表达和Ki67免疫标记的影响。通过α-SMA免疫标记比较Cx43 AS-ODN和正义寡核苷酸（S-ODN）处理的角膜中损伤后肌成纤维细胞的出现情况。
用Cx43 AS-ODN或siRNA处理后，在损伤后第3天，六只角膜中有五只观察到完全伤口闭合，而在对照角膜中（S-ODN，六只中零只；或无义siRNA，六只中零只）在第3天未观察到完全闭合。一致地，使用腺病毒过表达Cx43会延迟伤口闭合。Cx43敲低在第1天增加了Ki67阳性增殖细胞的数量，而在第5天减少了α-SMA阳性肌成纤维细胞的数量。无损伤情况下Cx43敲低降低了Cx43表达并在体内诱导了内皮增殖。
这些结果表明，Cx43敲低可诱导角膜内皮增殖，但抑制损伤后内皮-间充质转化/转变，提示Cx43敲低是加速伤口闭合和预防角膜后纤维膜形成的一种新的治疗方法。
