Kamei M, Kawasaki A, Tano Y
The Eye Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Microsc Res Tech. 1998 Sep 1;42(5):311-6. doi: 10.1002/(SICI)1097-0029(19980901)42:5<311::AID-JEMT2>3.0.CO;2-S.
To investigate changes in retinal pigment epithelial (RPE) cells during wound healing, we evaluated the deposition of newly synthesized extracellular matrix (ECM) over time during wound healing in rat RPE cultures. We also estimated the effect of growth factors on the healing rate and ECM synthesis. After preparing rat RPE cell sheet cultures, we made round 1-mm defects in the cultures. Fibronectin, laminin, and collagen IV synthesis were evaluated with immunocytochemistry every 12 hours after wounding. S-phase cell distribution was analyzed every 12 hours by 5-bromodeoxyuridine uptake. We added either platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor- beta2 (TGF-beta2) to cultures at concentrations of 1, 10, and 100 ng/mL and immunocytochemically analyzed the effects on ECM and estimated the rate of wound closure. Although approximately 50% closure was achieved 24 hours after wounding, fibronectin deposits first appeared at that time. Laminin and collagen IV were first detected at 36 hours and fibronectin staining had extended toward the wound center. S-phase cells were distributed in concentric rings that moved centripetally over time and corresponded to the leading edge of the area stained with anti-ECM antibodies. TGF-beta2 enhanced ECM deposition, but EGF and PDGF did not. TGF-beta2 decreased the healing rate in a dose-dependent manner, whereas PDGF promoted wound closure. EGF enhanced closure at the highest concentration only. In summary, wound healing in RPE may be initiated when cells at the wound edge slide or migrate toward the wound center, which is followed by cell proliferation and then ECM synthesis. ECM components may be produced in a specific sequence during healing. TGF-beta2 may promote RPE cell differentiation, and PDGF may enhance proliferation during wound healing of the RPE.
为了研究伤口愈合过程中视网膜色素上皮（RPE）细胞的变化，我们评估了大鼠RPE细胞培养物在伤口愈合过程中随时间新合成的细胞外基质（ECM）的沉积情况。我们还估计了生长因子对愈合速率和ECM合成的影响。制备大鼠RPE细胞片培养物后，我们在培养物中制造了直径1毫米的圆形缺损。受伤后每12小时用免疫细胞化学方法评估纤连蛋白、层粘连蛋白和IV型胶原的合成。通过5-溴脱氧尿苷摄取每12小时分析一次S期细胞分布。我们以1、10和100 ng/mL的浓度向培养物中添加血小板衍生生长因子（PDGF）、表皮生长因子（EGF）或转化生长因子-β2（TGF-β2），并用免疫细胞化学方法分析其对ECM的影响并估计伤口闭合速率。尽管受伤后24小时实现了约50%的闭合，但此时纤连蛋白沉积物首次出现。层粘连蛋白和IV型胶原在36小时首次检测到，纤连蛋白染色向伤口中心延伸。S期细胞呈同心环分布，随着时间向心移动，与抗ECM抗体染色区域的前沿相对应。TGF-β2增强了ECM沉积，但EGF和PDGF没有。TGF-β2以剂量依赖的方式降低愈合速率，而PDGF促进伤口闭合。EGF仅在最高浓度时增强闭合。总之，RPE中的伤口愈合可能在伤口边缘的细胞向伤口中心滑动或迁移时开始，随后是细胞增殖，然后是ECM合成。ECM成分可能在愈合过程中按特定顺序产生。TGF-β2可能促进RPE细胞分化，而PDGF可能在RPE伤口愈合过程中增强增殖。
