齐墩果酸对瘢痕疙瘩成纤维细胞生物学功能的影响

Effect of oleanolic acid on biological function of keloid fibroblasts

目的探讨齐墩果酸对瘢痕疙瘩成纤维细胞的生长和迁移的生物学功能的影响。方法收集北部战区总医院整形外科9例患者手术切除的瘢痕疙瘩组织标本,并进行体外培养成纤维细胞。采用不同浓度的齐墩果酸处理成纤维细胞,实验分成3组:对照组加入0.9%NaCl;5μmol/L齐墩果酸组加入5μmol/L齐墩果酸;10μmol/L齐墩果酸组加入10μmol/L齐墩果酸。3组均进行噻唑蓝实验(MTT)检测细胞的增殖状况;流式细胞实验检测细胞周期;膜联蛋白V碘化丙啶(AV-PI)染色检测细胞的凋亡;Transwell实验检测齐墩果酸对细胞的迁移作用;蛋白质印迹法和Real-time PCR实验分别检测相关蛋白表达和mRNA水平。所有实验均设置3个复孔。3组间数据进行方差分析比较,两两比较使用LSD-t检验,P<0.05为差异有统计学意义。结果3组MTT结果发现齐墩果酸能够抑制细胞的增殖,作用24 h时,5μmol/L和10μmol/L齐墩果酸组细胞增殖(吸光度A值)分别为0.66±0.02、0.46±0.02,对照组为0.78±0.00,3组间比较F=114.4,P<0.001;5、10μmol/L齐墩果酸组分别与对照组比较,差异有统计学意义(t=5.94、P<0.001,t=15.60、P<0.001)。流式细胞实验结果发现,细胞周期G1/S期的转导受到阻滞,5μmol/L和10μmol/L齐墩果酸组G1期细胞百分比分别为(72.17±2.04)%和(82.02±1.18)%,与对照组[(62.47±4.95)%]比较差异有统计学意义(t=3.14、P=0.030,t=6.38、P<0.001)。AV-PI染色发现5μmol/L齐墩果酸组(0.9%)和10μmol/L齐墩果酸组(3.4%)细胞的凋亡比例比对照组(0.4%)明显增加,3组间比较F=119.6,P<0.001,5、10μmol/L齐墩果酸组分别与对照组比较差异有统计学意义(t=4.39、P<0.001,t=13.44、P<0.001)。Transwell实验发现5μmol/L齐墩果酸组(57.13±2.65)和10μmol/L齐墩果酸组(42.15±2.55)细胞的迁移数量比对照组(72.27±3.32)明显下调,3组间比较F=101.3、P<0.001,5、10μmol/L齐墩果酸组分别与对照组比较差异有统计学意义(t=6.50、P<0.001,t=14.41、P<0.001)。蛋白质印迹法实验发现齐墩果酸可以抑制细胞周期素D1(Cyclin D1)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和基质金属蛋白酶-2(MMP2)的表达:5μmol/L齐墩果酸与对照组比较t=8.70、P<0.001,t=5.00、P=0.040,t=12.41、P<0.001,t=10.46、P<0.001;10μmol/L齐墩果酸与对照组比较t=31.61、P<0.001,t=23.17、P<0.001,t=12.11、P<0.001,t=44.52、P<0.001。Real-time PCR反应发现Cyclin D1、Bcl-2、Bax、MMP2的mRNA表达水平也受到了抑制,5μmol/L齐墩果酸与对照组比较t=5.42、P<0.001,t=3.11、P=0.040,t=16.11、P<0.001,t=11.71、P<0.001;10μmol/L齐墩果酸与对照组比较t=51.78、P<0.001,t=30.89、P<0.001,t=10.64、P<0.001,t=17.10、P<0.001。结论齐墩果酸作用于瘢痕疙瘩成纤维细胞24 h后能够抑制瘢痕疙瘩成纤维细胞的增殖和迁移并诱导其凋亡,10μmol/L的齐墩果酸作用强于5μmol/L,齐墩果酸可以用于防治瘢痕疙瘩。

Objective To investigate the effects of oleanolic acid on the growth and migration of keloid fibroblasts.Methods Keloid tissue samples from 9 patients in the Department of Plastic Surgery of General Hospital of Northern Theater were collected and fibroblasts were cultured in vitro.Fibroblasts were treated with different concentrations of oleanolic acid and divided into three groups:control group added 0.9%NaCl;5μmol/L oleanolic acid group added 5μmol/L oleanolic acid;10μmol/L oleanolic acid group added 10μmol/L oleanolic acid.MTT assay was used to detect cell proliferation;flow cytometry was used to detect cell cycle.Annexin V propidium iodide(AV-PI)staining was used to detect cell apoptosis.Transwell assay was used to detect the migration of oleanolic acid.Western blotting and real-time PCR were used to detect the expression of related proteins and mRNA activity.Each group was made in triplicate.Analysis of variance was used to compare the data among the three groups.LSD-t test was used for pairwise comparison,and P<0.05 was considered to be statistically significant.Results MTT result showed that oleanolic acid could inhibit the proliferation of cells.After 24 hours,the proliferation of cells in 5μmol/L oleanolic acid group and 10μmol/L oleanolic acid group were 0.660±0.020 and 0.460±0.020,respectively,compared with 0.780±0.001 in the control group,F=114.4,P<0.001.Compared with the control group,the difference was statistically significant(t=5.94,P<0.001,t=15.60,P<0.001);flow cytometry showed that the cell cycle G1/S phase transduction was blocked,5μmol/L oleanolic acid group and 10μmol/L oleanolic acid group were significantly inhibited.The percentage of G1 phase cells in the 5μmol/L oleanolic acid group was significantly higher than that in the control group(t=3.14,P=0.030,t=6.38,P<0.001).AⅤ-PI staining showed that the number of apoptotic cells in the 5μmol/L oleanolic acid group(0.9%)and 10μmol/L oleanolic acid group(3.4%)was significantly higher than that in the control group(0.4%),and the difference among the three groups was F=119.6,P<0.001.Transwell assay showed that the migration number of cells in 5μmol/L oleanolic acid group(57.13±2.65)and 10μmol/L oleanolic acid group(42.15±2.55)was significantly lower than that in control group(72.27±3.32),F=101.3,P<0.001.Compared with the control group,the difference was statistically significant(t=6.50,P<0.001,t=14.41,P<0.001).Western blotting showed that oleanolic acid could inhibit the expression of Cyclin D1,Bcl-2,Bax and MMP2.Compared with the control group,5μmol/L oleanolic acid t=8.70,P<0.001,t=5.00,P=0.040,t=12.41,P<0.001,t=10.46,P<0.001;compared with the control group,10μmol/L oleanolic acid t=31.61,P<0.001,t=23.17,P<0.001,t=12.11,P<0.001,t=44.52,P<0.001.Real-time PCR reaction showed that the mRNA activity levels of Cyclin D1,Bcl-2,Bax,MMP2 were also inhibited.Compared with the control group,5μmol/L oleanolic acid t=5.42,P<0.001,t=3.11,P=0.040,t=16.11,P<0.001,t=11.71,P<0.001;compared with the control group,10μmol/L oleanolic acid t=51.78,P<0.001,t=30.89,P<0.001,t=10.64,P<0.001,t=17.10,P<0.001.Conclusions Oleanolic acid(5μmol/L and 10μmol/L)can inhibit the proliferation and migration of keloid fibroblasts and induce apoptosis of keloid fibroblasts after treating keloid fibroblasts for 24 hours,which can inhibit the growth of keloid and be used for the prevention and treatment of keloid.