5-氟尿嘧啶对体外培养瘢痕疙瘩成纤维细胞增殖和凋亡的实验研究
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摘要
目的:探讨5-氟尿嘧啶对体外培养的瘢痕疙瘩成纤维细胞增殖和凋亡方面的生物学影响。
方法:①成纤维细胞体外原代培养:手术取6例瘢痕疙瘩(Keloid,K)(耳垂、大腿各1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,按文献方法进行成纤维细胞原代培养,待细胞融合后传代,实验所用细胞均为处于4～8代对数生长期的瘢痕疙瘩成纤维细胞(Keloid fibroblast,KF);②将不同浓度5-氟尿嘧啶(5-fluorouracil,5-FU)(0.1,0.2,0.4,0.8,1.6 g/Li)对4～8代瘢痕疙瘩成纤维细胞分别进行干预24h,48h,72h,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(50%Inhibitiory concentration,IC_(50))值。再选用不超过IC_(50)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应用流式细胞术(Flow cytometry,FCM)分析法观察5-氟尿嘧啶干预后KF的细胞周期变化及细胞凋亡百分率;④应用Hoechst33258荧光染色法观察凋亡成纤维细胞的形态学特征;⑤应用蛋白免疫印迹技术(Western-blot)检测5-氟尿嘧啶对瘢痕疙瘩成纤维细胞Bcl-2、Bax蛋白表达的影响。
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。①0.1,0.2,0.4,0.8,1.6g/L 5-氟尿嘧啶分别干预24h,48h,72h后,各组间KF细胞抑制率两两比较差异均有显著性(P＜0.01)。并采用作图法求得三组不同干预时间下的半数抑制浓度IC_(50)分别为(0.816±0.041)g/L,(0.637±0.028)g/L,(0.430±0.032)g/L;②0.2,0.3,0.4,0.5,0.6 g/L 5-氟尿嘧啶干预48 h后,KF细胞凋亡率组间两两比较有差异(P＜0.05),与对照组比较,差异有显著性意义(P＜0.01);实验组与对照组细胞周期百分数比较,表现为G_0/G_1期停滞,S期减少,各组间两两比较有差异(P＜0.05);③各浓度5-氟尿嘧啶干预48 h后,Hoechst 33258荧光染色观察KF均有不同程度核浓缩,表明KF发生了不同程度的凋亡;④经不同浓度5-氟尿嘧啶干预KF 48 h后,实验组Bcl-2蛋白表达均低于对照组(P＜0.05),实验组Bax蛋白表达均高于对照组(P＜0.05)。
结论:本实验进一步揭示了5-氟尿嘧啶治疗瘢痕的作用机制,即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。应用低浓度、短周期注射5-氟尿嘧啶可能是治疗瘢痕疙瘩的一种新思路。
Objective:To explore biological effects of 5-fluorouracil on the proliferation and apoptosis of human keloid fibroblasts in vitro.
Methods: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. Fibroblasts were derived from four keloids.TheⅣ-Ⅷgeneration exponential phase of growth cell were used for experiment.②Keloid fibroblasts were stimulated with 5-fluorouracil(0.1,0.2,0.4,0.8,and 1.6 g/L) for24,48,and 72 hours.The inhibition ratio and 50%inhibitory concentration(IC_(50)) were calculated by MTT.In addition,0.2,0.3,0.4, 0.5 and 0.6 g/L 5-fluorouracil were prepared for the following experiment.③The cell apoptosis and cell cycle were measured by flow cytometry.④The cell morphological character was observed by Hoechst 33258 fluorescent staining.⑤The influence of 5-fluorouracil on Bcl-2 and Bax expressions were detected by western-blot.
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 24,48, and 72 hours,the inhibition ratios were significantly different between every two groups(P＜0.01).The IC_(50) partly was(0.816±0.041),(0.637±0.028),(0.430±0.032) g/L by mapping.②After 5-fiuorouracil(0.2,0.3,0.4,0.5,and 0.6 g/L) intervention for 48 hours, the keloid fibroblasts apoptosis rate had significant differences between every two groups (P＜0.05).Compared with the control group ceils,the differences were statistically significant(P＜0.01).Compared with the control group cells,the number of experiment cells at phase G_0/G_1 significantly increased and S decreased(P＜0.05).③By Hoechst 33258 fluorescent staining,the keloid fibroblasts interfered with 5-fluorouracil showed nuclear condensation and apoptosis at different extents.④There was a remarkable decrease of the Bcl-2 expression and a marked increase of the Bax expression in different concentrations 5-fluorouracil compared with the control group(P＜0.05 ).
Conclusion:The research revealed advanceedly the mechanism of action on 5-fluorouracil treated scars.5-fluorouracil could inhibit proliferation and induce apoptosis on human keloid flbroblasts in vitro.Low concentration and short-period injection with 5-fluorouracil would be a new consider of keloid treatment.
方法:①成纤维细胞体外原代培养:手术取6例瘢痕疙瘩(Keloid,K)(耳垂、大腿各1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,按文献方法进行成纤维细胞原代培养,待细胞融合后传代,实验所用细胞均为处于4～8代对数生长期的瘢痕疙瘩成纤维细胞(Keloid fibroblast,KF);②将不同浓度5-氟尿嘧啶(5-fluorouracil,5-FU)(0.1,0.2,0.4,0.8,1.6 g/Li)对4～8代瘢痕疙瘩成纤维细胞分别进行干预24h,48h,72h,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(50%Inhibitiory concentration,IC_(50))值。再选用不超过IC_(50)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应用流式细胞术(Flow cytometry,FCM)分析法观察5-氟尿嘧啶干预后KF的细胞周期变化及细胞凋亡百分率;④应用Hoechst33258荧光染色法观察凋亡成纤维细胞的形态学特征;⑤应用蛋白免疫印迹技术(Western-blot)检测5-氟尿嘧啶对瘢痕疙瘩成纤维细胞Bcl-2、Bax蛋白表达的影响。
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。①0.1,0.2,0.4,0.8,1.6g/L 5-氟尿嘧啶分别干预24h,48h,72h后,各组间KF细胞抑制率两两比较差异均有显著性(P＜0.01)。并采用作图法求得三组不同干预时间下的半数抑制浓度IC_(50)分别为(0.816±0.041)g/L,(0.637±0.028)g/L,(0.430±0.032)g/L;②0.2,0.3,0.4,0.5,0.6 g/L 5-氟尿嘧啶干预48 h后,KF细胞凋亡率组间两两比较有差异(P＜0.05),与对照组比较,差异有显著性意义(P＜0.01);实验组与对照组细胞周期百分数比较,表现为G_0/G_1期停滞,S期减少,各组间两两比较有差异(P＜0.05);③各浓度5-氟尿嘧啶干预48 h后,Hoechst 33258荧光染色观察KF均有不同程度核浓缩,表明KF发生了不同程度的凋亡;④经不同浓度5-氟尿嘧啶干预KF 48 h后,实验组Bcl-2蛋白表达均低于对照组(P＜0.05),实验组Bax蛋白表达均高于对照组(P＜0.05)。
结论:本实验进一步揭示了5-氟尿嘧啶治疗瘢痕的作用机制,即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。应用低浓度、短周期注射5-氟尿嘧啶可能是治疗瘢痕疙瘩的一种新思路。
Objective:To explore biological effects of 5-fluorouracil on the proliferation and apoptosis of human keloid fibroblasts in vitro.
Methods: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. Fibroblasts were derived from four keloids.TheⅣ-Ⅷgeneration exponential phase of growth cell were used for experiment.②Keloid fibroblasts were stimulated with 5-fluorouracil(0.1,0.2,0.4,0.8,and 1.6 g/L) for24,48,and 72 hours.The inhibition ratio and 50%inhibitory concentration(IC_(50)) were calculated by MTT.In addition,0.2,0.3,0.4, 0.5 and 0.6 g/L 5-fluorouracil were prepared for the following experiment.③The cell apoptosis and cell cycle were measured by flow cytometry.④The cell morphological character was observed by Hoechst 33258 fluorescent staining.⑤The influence of 5-fluorouracil on Bcl-2 and Bax expressions were detected by western-blot.
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 24,48, and 72 hours,the inhibition ratios were significantly different between every two groups(P＜0.01).The IC_(50) partly was(0.816±0.041),(0.637±0.028),(0.430±0.032) g/L by mapping.②After 5-fiuorouracil(0.2,0.3,0.4,0.5,and 0.6 g/L) intervention for 48 hours, the keloid fibroblasts apoptosis rate had significant differences between every two groups (P＜0.05).Compared with the control group ceils,the differences were statistically significant(P＜0.01).Compared with the control group cells,the number of experiment cells at phase G_0/G_1 significantly increased and S decreased(P＜0.05).③By Hoechst 33258 fluorescent staining,the keloid fibroblasts interfered with 5-fluorouracil showed nuclear condensation and apoptosis at different extents.④There was a remarkable decrease of the Bcl-2 expression and a marked increase of the Bax expression in different concentrations 5-fluorouracil compared with the control group(P＜0.05 ).
Conclusion:The research revealed advanceedly the mechanism of action on 5-fluorouracil treated scars.5-fluorouracil could inhibit proliferation and induce apoptosis on human keloid flbroblasts in vitro.Low concentration and short-period injection with 5-fluorouracil would be a new consider of keloid treatment.
引文
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