血小板活化因子致角膜上皮伤口迟愈合的实验研究

目的　利用兔去上皮角膜模型 ,研究血小板活化因子 (PAF)对角膜伤口愈合的作用及其分子生物学机制。方法　离体角膜上做正中直径 7mm圆形去上皮角膜伤口。去上皮角膜分为 3组 ,即对照、PAF及BN (PAF拮抗剂 )组 ,培养 4 8h后 ,行角膜上皮染色观察伤口愈合状况。分别对兔角膜上皮 (RCE)和角膜基质 (RCK)细胞进行体外传代培养 ,RCE和RCK细胞经PAF和 (或 )BN处理 ,培养 2 4h ,提纯RNA。应用RT PCR及核酸杂交技术分别检测肝细胞生长因子 (HGF)、角质形成生长因子 (KGF)及表皮生长因子 (EGF)基因在RCK和RCE细胞及HGF受体 (HGF R)基因在RCE细胞中的表达强度。分别应用CyQUANT荧光结合和Boyden小房技术检测PAF对RCE细胞黏附、增殖和迁徙的影响。结果　PAF (10 0nmol/L )明显抑制角膜上皮伤口愈合 ,4 8h对照、PAF和BN组角膜上皮未愈合面积经电脑图像分析分别为 :(6 0± 1.5 )U、(5 8 0± 7 0 )U和 (5 0± 1 0 )U。PAF明显增强RCE细胞黏附作用 ,对照、PAF和BN组每 96孔板贴附细胞数荧光光度平均值分别为 :36 96± 372、790 8± 6 71和 3487± 32 4。RT PCR结果显示 :PAF使HGFmRNA在RCK的表达强度降低 4 .1倍 ,同时明显减弱HGF R在RCE细胞中的表达 ,核酸杂交实验证实PCR结果。结论PAF明显增强RCE细胞的黏附作用 ,

Corneal epithelial wound healing is delayed by platelet activating factor treatment

OBJECTIVE: To investigate the role of PAF in corneal epithelial wound healing and its mechanisms. METHODS: Rabbit corneal epithelial (RCE) and keratocyte (RCK) cells were cultured and used at passage 2 to 3. For the in vitro wound healing experiments, a 7 mm central corneal de-epithelialization was performed in rabbit corneas. Corneas were incubated in the presence of 100 nmol/L PAF; 4 micro mol/L of the PAF antagonist BN was added 30 min before PAF. The area of the cornea uncovered by the epithelium was measured after 48 h using an imaging system. Cell adhesion, proliferation and migration were analyzed using CyQUANT fluorescence binding assay and Boyden chamber respectively. RNA was extracted and RT-PCR and Northern blot analyses were performed using HGF, KGF, EGF and HGF-R primers and probes. RESULTS: Forty-eight hours after the wound, the uncovered area (in arbitrary units) for control corneas was (6.0 +/- 1.5) U. PAF dramatically inhibited corneal epithelial wound closure, and the uncovered area was (58.0 +/- 7.0) U. The PAF antagonist completely abolished the effect of PAF with values similar to controls (5.0 +/- 1.0) U. PAF significantly increased the cell adhesion among RCE cells, while at the concentration of 100 nmol/L, PAF had no effect on the proliferation and migration of RCE cells. RT-PCR results revealed that PAF at 100 nmol/L decreased the HGF mRNA expression 4.1 times in RCK and HGF-R 3.5 times in RCE at 24 h compared to control group, while PAF had no effect on the expression of KGF and EGF in RCE cells. Northern blot data confirmed these results. CONCLUSIONS: Our results suggest that PAF causes significant delay of corneal epithelial wound healing. This effect may partially be due to the increased RCE cell adhesion and suppressed expression of HGF gene in RCK and HGF-R. The PAF antagonist BN can completely abolish the effect of PAF and enhance the corneal wound repair.