Mayo Courtney, Ren Ruiyi, Rich Celeste, Stepp Mary Ann, Trinkaus-Randall Vickery
Departments of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Invest Ophthalmol Vis Sci. 2008 Oct;49(10):4384-91. doi: 10.1167/iovs.08-1688. Epub 2008 May 23.
Previously, the authors demonstrated that BzATP, a P2X(7) receptor agonist, enhanced corneal epithelial migration in vitro. The goal here was to characterize the role of the P2X(7) receptor in the repair of in vivo corneal epithelial debridement wounds and in the structural organization of the corneal stroma.
Epithelial debridement was performed on P2X(7) knockout (P2X(7)(-/-)) and wild-type (WT) mice, and eyes were harvested after 16 hours. Corneas were stained with Richardson vital stain, and the wound area was recorded. Corneas were fixed and prepared for light microscopic, immunohistochemical, and electron microscopic analysis. Cuprolinic blue staining was performed to analyze stromal proteoglycans (PGs). Real-time PCR was performed to examine the expression of stromal collagens.
P2X(7) was present in the WT corneal epithelium but was not detected in P2X(7)(-/-) mice. Pannexin-1, a protein demonstrated to interact with P2X(7), was absent from the wound edge in P2X(7)(-/-). This was associated with a trend toward delayed corneal reepithelialization. Stromal ultrastructure and collagen alignment were altered in P2X(7)(-/-), and collagen fibrils had smaller diameters with a larger interfibrillar distances. Expression of collagen alpha1(I) and alpha3(v) was reduced. There were 30% fewer sulfated PGs along fibrils in the P2X(7)(-/-) stroma.
In the absence of the P2X(7) receptor, the expression of proteins in the corneal epithelium was altered and wound healing was compromised. Loss of receptor resulted in morphologic changes in the stroma, including changes in alignment of collagen fibrils, decreased expression of collagen, and smaller fibrils with fewer PGs per fibril.
此前，作者证明P2X(7)受体激动剂BzATP可增强体外角膜上皮细胞迁移。本研究的目的是确定P2X(7)受体在体内角膜上皮清创伤口修复及角膜基质结构组织中的作用。
对P2X(7)基因敲除（P2X(7)(-/-)）小鼠和野生型（WT）小鼠进行上皮清创，16小时后摘取眼球。用理查森活体染色法对角膜进行染色，并记录伤口面积。将角膜固定后进行光镜、免疫组化和电镜分析。采用铜啉蓝染色法分析基质蛋白聚糖（PGs）。通过实时定量PCR检测基质胶原蛋白的表达。
WT角膜上皮中存在P2X(7)，但在P2X(7)(-/-)小鼠中未检测到。Pannexin-1是一种已证明与P2X(7)相互作用的蛋白质，在P2X(7)(-/-)小鼠的伤口边缘不存在。这与角膜再上皮化延迟的趋势相关。P2X(7)(-/-)小鼠的基质超微结构和胶原排列发生改变，胶原纤维直径较小，纤维间距离较大。胶原蛋白α1(I)和α3(v)的表达降低。P2X(7)(-/-)小鼠基质中沿纤维的硫酸化PGs减少30%。
在缺乏P2X(7)受体的情况下，角膜上皮中蛋白质的表达发生改变，伤口愈合受到损害。受体缺失导致基质形态学改变，包括胶原纤维排列改变、胶原蛋白表达降低以及纤维直径变小且每条纤维含有的PGs减少。
