5-氟尿嘧啶对体外培养瘢痕疙瘩成纤维细胞的抑制及半数抑制浓度

目的：国内外一些学者使用传统抗代谢药物5-氟尿嘧啶防止纤维化，在治疗瘢痕疙瘩方面取得了一定的临床疗效，但对其药物作用机制以及临床药物应用的浓度尚无确切认定。实验拟进一步验证5-氟尿嘧啶对体外培养瘢痕疙瘩成纤维细胞增殖的抑制以及半数抑制浓度。 方法：实验于2006—09／2007—07在安徽医科大学微生物教研室及安徽医科大学附属医院中心实验室完成。①实验材料：6例瘢痕疙瘩（耳垂、大腿各l例、胸前、上臂各2例）均为安徽医科大学附属医院整形外科手术病例，均无系统性疾病和激素、其他药物注射史，对用于实验的所取组织患者均知情同意。治疗方案经医院医学伦理委员会批准。②实验方法：成纤维细胞体外原代培养，待细胞融合后传代，实验所用细胞均为处于4-8代对数生长期的成纤维细胞。将不同浓度5-氟尿嘧啶（O．1，0．2，0．4，0．8，1．6g／L）分别对4～8代瘢痕疙瘩成纤维细胞进行干预72h。再选用接近IC50的5个浓度：0．2，0．3，0．4，0．5，0．6g／L，作为余下实验浓度。③实验评估：采用四甲基偶氮唑盐比色法检测各浓度药物对成纤维细胞的抑制率，计算5-氟尿嘧啶对瘢痕疙瘩成纤维细胞的半数抑制浓度（IC50）值。应用流式细胞术分析法观察5-氟尿嘧啶干预后瘢痕疙瘩成纤维细胞的细胞周期变化及细胞凋亡百分率；应用Hoechst33258荧光染色法观察凋亡成纤维细胞的形态学特征。 结果：6例瘢痕疙瘩标本原代培养成纤维细胞成活良好，均用于实验，进入结果分析。①O．1，0．2，0．4，0．8，1．6g／L 5-氟尿嘧啶干预72h后，细胞抑制率组间两两比较差异均有显著性（P〈O．01）。作图得半数抑制浓度IC50为（0．400±0．032）g／L。②0．2，0．3，0．4，0．5，0．6g／L5-氟尿嘧啶干预48h后，细胞凋亡率组间两两比较差?

Inhibitory effect of 5-fluorouracil on in vitro cultured human keloid fibroblasts and determination of 50% inhibitory concentration of 5-fluorouracil

AIM： 5- fluorouracil is always used to prevent fibrosis, which has achieved certain curative effect on keloid in clinic. However, its mechanism and application dosage in clinic are still uncertain. This study explored the inhibitory effects and concentration of 5-fluorouracil on the proliferation of human keloid fibroblasts in vitro.
METHODS： The experiment was performed at Microbes Department of Anhui Medical University and the center laboratory of Affiliated Hospital of Anhui Medical University from September 2006 to July 2007. ①Keloids of ear lobe, thigh, chest and upper arm were derived from 6 cases of Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University. All the patients did not have any systemic disease or drug injection like hormone. The informed consent was obtained from the patients, and the experiment was admitted by the Hospital Ethics Committee. ②Fibroblasts were cultured in vitro, and the Ⅳ-Ⅶ generation cells at exponential phase of growth were used for experiment. Keloid fibroblasts were stimulated with 5-fluorouracil （0.1, 0.2, 0.4, 0.8, and 1.6 g/L） for 72 hours. In addition, 0.2, 0.3, 0.4, 0.5 and 0.6 g/L 5-fluorouracil were prepared for the following experiment. ③The inhibition ratio and 50% inhibitory concentration （IC50） were calculated by MTT. The cell apoptosis and cell cycle were measured by flow cytometry. The cell morphological character was observed by Hoechst 33258 fluorescent staining.
RESULTS： Six keloid specimens were primarily cultured well, and all were included in the final analysis. ①After 5-fluorouracil （0.1, 0,2, 0.4, 0.8, and 1.6 g/L） intervention for 72 hours, the inhibition ratio were significantly different between every two groups （P 〈 0.01）. The IC50 was （0.400±0.032） g/L by mapping. ②After 5-fluorouracil （0.2, 0.3, 0.4, 0.5, and 0.6 g/L） intervention for 48 hours, the apoptosis rate had significant differences between every two groups （P 〈 0.05）. Compared with the control group cells