方法：采用波形蛋白(Vimentin)和角蛋白(CK)免疫细胞化学染色鉴定原代培养的HPFs。分别设置对照组、DOX低、中、高浓度(50、100、200mg/L)组，应用CCK-8法和划痕实验检测各组HPFs活力及迁移能力，利用细胞三维培养及过碘酸-雪夫(PAS)染色观察各组细胞VM生成情况，并通过Western blot法检测各组细胞基质金属蛋白酶(MMP)-9及血管内皮生长因子(VEGF)的表达情况。

结果：免疫细胞化学染色结果显示，细胞呈梭形且Vimentin阳性表达，CK阴性表达，符合成纤维细胞特性。与对照组相比，DOX组细胞活力、迁移能力、VM密度及MMP-9和VEGF蛋白的表达均显著降低(P<0.05)，DOX各浓度组组间相比均有差异(P<0.05)。相关性分析提示HPFs形成的VM密度与MMP-9和VEGF蛋白表达水平呈显著正相关(r=0.949、0.960，均P<0.05)。

结论：DOX可通过减少MMP-9及VEGF的表达抑制HPFs的活力及迁移能力，减少VM的形成，提示MMP-9和VEGF可能是翼状胬肉形成VM的分子机制。

METHODS: Primary cultured HPFs were identified by Vimentin and CK through immunocytochemical staining. HPFs were divided into control group and DOX group including low, medium and high concentrations(50, 100, 200mg/L). The activity and migration of HPFs were detected by cell counting kit-8(CCK-8)and wound healing assay. The density of VM was observed by three-dimensional cell culture and periodic acid schiff(PAS)staining and compared the differences of VM formation in each group. Western blot was used to analyze the expression of matrix metalloproteinase-9(MMP-9)and vascular endothelial growth factor(VEGF).

RESULTS:Immunocytochemical staining results showed that the cells were spindle shaped, meanwhile, they were positive for Vimentin and negative for CK, which were consistent with the characteristics of fibroblasts. Compared with the control group, the cell activity, mobility, VM density and the expression of MMP-9 and VEGF proteins in the DOX group were significantly decreased(P<0.05). Compared among different concentrations of DOX groups, the differences were statistically significant(P<0.05). Correlation analysis indicated that VM density formed by HPFs was significantly positively correlated with the protein expression of MMP-9 and VEGF(r=0.949, 0.960, all P<0.05).

CONCLUSION: DOX can inhabit HPFs activity, migration, VM density by reducing the expression of MMP-9 and VEGF, suggesting that MMP-9 and VEGF may be the molecular mechanisms of VM formation in pterygium.