5-氟尿嘧啶对瘢痕疙瘩成纤维细胞增殖与凋亡机制影响的初步探讨

目的拟观察5-氟尿嘧啶对瘢痕疙瘩成纤维细胞Smad7和β转化生长因子（transforming growth factor-β，TGF-β）Ⅰ型受体以及细胞凋亡相关基因Bcl-2和Bax蛋白表达的影响。方法（1）瘢痕疙瘩组织成纤维细胞原代培养，取4～6代传代细胞加入5个不同浓度（10，20，40，80，160μmol／L）5-氟尿嘧啶干预24，48，72h。（2）利用四甲基偶氮唑盐法（MTT）测定瘢痕疙瘩成纤维细胞的增殖能力。（3）应用蛋白免疫印迹法（Western-blot）检测各组瘢痕疙瘩成纤维细胞中Smad7和TGF-βI型受体的表达及对Bcl-2、Bax蛋白表达的影响。结果（1）MTT实验中，当5-氟尿嘧啶浓度为10，20μmol／L作用24h时，未发现成纤维细胞死亡，与空白对照组相比差异无统计学意义（P〉0．05）；其他浓度下作用各组成纤维细胞死亡现象差异均有统计学意义（P〈0．01）。（2）免疫印迹法实验结果如下：①与空白对照组相比，TGF-βI组Smad7表达明显减弱，TGF-βI型受体表达则显著增强，差异有统计学意义（P〈0．01）。②加入5-氟尿嘧啶干预后可显著增强Smad7的表达，差异有统计学意义（P〈0．01）。③经不同浓度5-氟尿嘧啶干预后，实验组Bcl-2蛋白表达均低于对照组，差异有统计学意义（P〈0．05）。但实验组Bax蛋白表达均高于对照组（P〈0．05），TGF-βI型受体表达无明显影响，差异均无统计学意义（P〉O．05）。结论5-氟尿嘧啶对瘢痕疙瘩成纤维细胞具有抑制增殖和诱导凋亡的作用。

Mechanism of the proliferation and apoptosis of keloid fibroblasts induced by 5-fluouracil:a preliminary study

Objective To investigate the of 5-fluorouracil effects on the expression of Smad7,TGF-β receptorⅠ,Bcl-2 and Bax in keloid fibroblasts.Methods After primary culture of keloid fibroblasts,4-6 passages of cells were inoculated in 5 different concentrations of 5-fluorouracil(10,20,40,80,160μmol/L)for 24,48 and 72 hours.Proliferative ability of keloid fibroblasts was detected by MTT assay.Expression of Smad 7,TGF-βreceptorⅠ,Bcl-2 and Bax in keloid fibroblasts was measured by Western blot.Results During MTT,5-fluorouracil did not affect cell viability at 24 hour at the concentration of 10 and 20 μmol/L.Compared with the control group,no significant difference was detected(P＞0.05).At other concentrations,fibroblast death was visible in each group(P＜0.01).Western blot analysis showed that the expression of Smad7 significantly decreased and the expression of TGF-β receptor Ⅰ significantly increased in the TGF-β1 group compared with the blank control group(P＜0.0 1).5-fluorouracil could significantly enhance the expression of Smad7(P＜0.01).There was a remarkable decrease of the Bcl-2 expression and marked increase of the Bax expression in different concentrations of 5-fluorouracil compared with the control group(P＜0.05).But,5-fluorouracil did not show any effect on the synthesis of TGF-β receptor Ⅰ.Conclusion 5-fluorouracil could inhibit proliferation and induce apoptosis on human keloid fibroblasts in vitro.