成纤维细胞分泌胶原蛋白与5-氟尿嘧啶的时间抑制效应

Collagen secretion from fibroblasts and the interventional effect of 5-fluorouracil in a time-dependent manner

目的:观察5-氟尿嘧啶干预体外培养病理性瘢痕成纤维细胞增殖和胶原合成的作用及其时效关系。方法:实验于2005-05/2006-06在安徽医科大学微生物教研室完成。①原代细胞培养成纤维细胞:手术取4例增生性瘢痕、4例瘢痕疙瘩、4例正常皮肤组织,按文献方法进行成纤维细胞原代培养,待细胞融合后传代。②取4～6代传代细胞接种于96孔板,利用四唑盐比色法测定25g/L浓度5-氟尿嘧啶干预不同时间(1,2,5,10,15,30min)后3种体外培养成纤维细胞的增殖能力,统计得出最佳时效。③利用3H-脯氨酸掺入法检测经5-氟尿嘧啶干预不同时间后成纤维细胞的胶原蛋白分泌能力。结果:①原代培养成纤维细胞全部成活,形态良好,经5-氟尿嘧啶干预后细胞形态未发生明显变化。②四甲基偶氮唑盐法测3种不同来源成纤维细胞未干预时增殖能力,瘢痕疙瘩成纤维细胞>正常皮肤成纤维细胞(t=3.542,P<0.01),增生性瘢痕成纤维细胞>正常皮肤成纤维细胞(t=3.257,P<0.01);5-氟尿嘧啶干预成纤维细胞10min与5min比较,其对成纤维细胞的抑制效果增高(正常皮肤、增生性瘢痕、瘢痕疙瘩3种成纤维细胞10minA值依次为0.389±0.027,0.418±0.051,0.554±0.021;5minA值依次为0.537±0.033,0.589±0.074,0.783±0.015,q=3.80～4.18,P<0.05),与30min比较,差异无显著性。③干预10min3H-脯氨酸掺入量与空白对照组比较明显下降,差异有显著性(q=3.82～3.96,P<0.05),与5min干预组比较,差异均有显著性(q=3.96～4.14,P<0.05),与30min比较,差异无统计学意义。结论:5-氟尿嘧啶可有效抑制病理性瘢痕及正常皮肤来源的成纤维细胞体外增殖和胶原合成,干预10min时效应最佳。

AIM：To determine the interventional effect of 5-fluorouracil on the proliferation of fibroblasts of pathologic scars and collagen synthesis and the time effect. METHODS： The experiment was conducted in Microbes Department of Anhui Medical University between May 2005 and June 2006. （1）Fibroblasts culture from primary cells： Four cases of hypertrophic scars, four keloids, and four normal skins were harvested to culture fibroblasts according to the document method; cells passaged after fusing. （2）The fourth to sixth generation cells were collected and seeded on the 96-hole plate, and cell viability after interfered by 5-fluorouracil for different time （1, 2, 5, 10, 15 and 30 minutes） was evaluated by MTT assay to obtain the best time effect. （3）The technology of incorporation of ^3H-proline was used to detect the capability of fibroblasts to secrete collagen protein. RESULTS： （1）All the fibroblasts grew well and kept normal shape. Cell appearance kept the normal condition after interfered by 5-fiuorouracil. （2）MTT assay of the ability of proliferation of three kinds of fibroblasts before intervention showed that the ability of keloids was stronger than normal skins （t =3.542, P 〈 0.01 ）, and hypertrophic scars were stronger than normal skins （t=3.257, P〈 0.01）; the inhibition effect of intervention by 5-fiuorouracil for 10 minutes on fibroblasts was superior to that for 5 minutes （A value of normal skins, hypertrophic scars and keloids after interfered for 10 minutes： 0.389±0.027, 0.418±0.051, 0.554±0.021; for 5 minutes： 0.537±0.033, 0.589±0.074, 0.783±0.015, q = 3.80- 4.18, P 〈 0.05）; compared with that for 30 minutes, the differences were not significant. （3）After interfered by 5-fiuorouracil for 10 minutes, the incorporation of ^3H-proline was obviously decreased compared with the control group （q =3.82-3.96, P 〈 0.05）; compared with that for 5 minutes, the differences were statistically significant （q =3.96-4.14, P 〈 0.05）, while no obvious differences were found when compared with that for 30 minutes. CONCLUSION： 5-fiuorouracil can effectively inhibit the proliferation of fibroblasts from pathologic scars and normal skins and collagen protein secretion; 10 minutes is the best time for intervention.