摘要
试验旨在探讨5-氟尿嘧啶(5-Fluorouracil,5-FU)对体外培养的驴皮成纤维细胞增殖、凋亡的影响,为5-FU抑制驴皮成纤维细胞的增殖及驴皮伤口愈合机制的研究提供理论依据。试验采用低(10,50μg/mL)、中(100,250,500μg/mL)、高浓度(1000,1500,2000μg/mL)5-FU处理体外培养的驴皮成纤维细胞,用MTT法检测加药处理12,24,48,72,96 h的细胞增殖率,Caspase-3法检测加药处理0,4,8,24,48,72 h的细胞凋亡率,再通过RT-qPCR和ELISA检测添加不同浓度5-FU(10,100,1000μg/mL)对胶原蛋白合成相关基因表达及蛋白浓度的影响。结果表明:100μg/mL 5-FU处理成纤维细胞24 h增殖率开始显著降低(P<0.05),8 hCaspase-3活性显著升高(P<0.05);RT-qPCR结果显示,5-FU处理24 h可显著抑制胶原蛋白合成相关基因(TGF-β、Smad3、COL1A1、COL1A2、COL3A1)的表达(P<0.05),抑制程度呈剂量依赖性,但100μg/mL 5-FU处理后Smad7的表达量显著高于其他组(P<0.05);ELISA结果显示,100μg/mL 5-FU处理抑制TGF-β、COL1A1、COL3A1蛋白表达的效果最佳(P<0.05)。说明100μg/mL 5-FU处理24 h后,可在抑制体外培养的驴皮成纤维细胞过度增殖的同时,通过调控TGF-β/Smad信号通路,减少合成胶原蛋白相关基因的表达量和蛋白浓度。
The aim of this experiment was to investigate the effects of 5-Fluorouracil(5-FU)on proliferation and apoptosis of donkey skin fibroblasts in vitro to lay a theoretical foundation for studying the 5-FU inhibition on proliferation of fibroblasts and the mechanism of wound healing in donkey skin.In this experiment,donkey skin fibroblasts cultured in vitro were treated with low(10,50μg/mL),medium(100,250,500μg/mL)and high concentrations(1000,1500,2000μg/mL)of 5-FU,respectively,and the cell proliferation rates were measured by MTT method at 12,24,48,72,96 h after treatment;Caspase-3 method was used to detect the apoptosis rate at 0,4,8,24,48,72 h after treatment;and RT-qPCR and ELISA were used to detect the effect of 5-FU(10,100,1000μg/mL)on the and protein expression of collagen-synthesis-related genes.The results showed that in the treat of 100μg/mL the proliferation rate of fibroblasts began to decrease significantly at 24 h(P<0.05),and the Caspase-3 activity increased significantly at 8 h(P<0.05);RT-qPCR results showed that the expression of genes related to collagen synthesis(TGF-β,Smad3,Smad7,COL1A1,COL1A2,COL3A1)were significantly inhibited(P<0.05)at 24 h in a dose-dependent manner.However,the expression of Smad7 in 100μg/mL 5-FU group was significantly higher than that of other groups(P<0.05);ELISA results showed that the maximum inhibitory effect on protein expression of TGF-β,COL1A1,COL3A1(P<0.05)was obtained(100μg/mL 5-FU).It concluded that the excessive proliferation of fibroblasts could be inhibited,and the expression and protein concentration of collagen-synthesis-related genes could be reduced by regulating the TGF-β/Smad signaling pathway at 24 h after treatment when donkey skin fibroblasts in vitro was treated with 100μg/mL 5-FU.
作者
朱春灏
张媛
赵玉坤
曾维斌
王艳萍
ZHU Chunhao;ZHANG Yuan;ZHAO Yukun;ZENG Weibin;WANG Yanping(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China)
出处
《黑龙江动物繁殖》
2023年第2期1-6,共6页
Heilongjiang journal of animal reproduction
基金
国家自然科学基金项目(31760643)
石河子大学创新发展专项(CXFZ202007)。
