人瘢痕疙瘩成纤维细胞相关基因表达及青蒿琥酯的作用  

Expressions of the related genes in fibroblasts of human keloid and the effect of artesunate

作　　者：肖亮[1] 贺光照[1] 余泽浩[1] 唐小清[1] 任玉涵[2] 

机构地区：[1]重庆医科大学附属第一医院烧伤整形外科,重庆400016 [2]重庆医科大学附属第一医院感染科,重庆400016

出　　处：《中华烧伤杂志》2013年第2期185-190,共6页Chinese Journal of Burns

摘　　要：目的 验证瘢痕疙瘩Fb中是否存在钙/钙调蛋白依赖性丝氨酸蛋白激酶（CASK）和分化抑制因子1（ID1）的异常表达，观察青蒿琥酯对二者的影响。方法 收集笔者单位患者手术后废弃的瘢痕疙瘩样本15个和正常皮肤组织样本12个，采用组织微粒贴壁法行Fb原代培养，取第3~8代细胞用于实验。免疫荧光染色观察2种Fb中CASK和ID1的表达，瘢痕疙瘩Fb用不同浓度青蒿琥酯作用不同时间，通过噻唑蓝比色法确定药物的半数抑制浓度（IC50）并作为后续实验药物干预浓度。选取正常皮肤Fb设为正常对照组（添加培养液处理），收集瘢痕疙瘩Fb分为瘢痕对照组（添加培养液处理）及瘢痕给药组（添加含IC50青蒿琥酯的培养液处理），流式细胞术检测各组Fb细胞周期和凋亡的变化，RT-PCR、蛋白质印迹法检测各组Fb中CASK和ID1的基因与蛋白表达情况。对数据行单因素方差分析及LSD-t检验。结果 瘢痕疙瘩和正常皮肤Fb中均存在CASK和ID1表达，选择75 mg/L青蒿琥酯为后续实验干预浓度。（1）G0/G1期和G2/M期的细胞百分比3组之间比较，差异有统计学意义（F值分别为118.064、163.840，P值均小于0.01）。其中瘢痕给药组G0/G1期为（91.4±1.4）%，明显高于瘢痕对照组的（80.7±0.3）%和正常对照组的（82.4±0.6）%（t值分别为12.740、9.872，P值均小于0.05）；G2/M期为（6.9±0.3）%，明显低于瘢痕对照组的（13.7±0.3）%和正常对照组的（12.7±0.8）%（t值分别为43.702、12.276，P值均小于0.05）。（2）早晚期细胞凋亡率3组之间比较，差异均有统计学意义（F值分别为61.879、4710.862，P值均小于0.01）。其中瘢痕给药组早期凋亡率为（7.1±1.0）%，明显高于瘢痕对照组的（2.6±0.4）%和正常对照组的（2.7±0.3）%（t值分别为7.974、7.767，P值均小于0.05）；晚期凋亡率为（14.9±0.3）%，明显高于瘢痕对照组的（2.3±0.3）%和�Objective To verify whether abnormal expression of calcium/calmodulin dependent serine protein kinase （CASK） and inhibitors of differentiation 1 （ID1） exist in Fb of keloid, and to observe the effect of artesunate on two genes. Methods Fifteen samples of keloid and 12 samples of normal skin tissue （discarded） excised from patients admitted to our hospital were collected. Tissue particle adherent method was used in the primary culture of Fb, and cells from the third to the eighth passage were used for test. Expressions of CASK and ID1 in Fb harvested from both sources were observed with immunofluorescence staining. Fb of keloid were stimulated with artesunate in various concentration for different time, and the median inhibitory concentration （IC50） was determined with the MTT colorimetric assay, which served as the intervention concentration of artesunate. Fb of normal skin were set as normal control group （NC, treated with medium solution）. Fb of keloid were divided into scar control group （SC, treated with medium solution） and scar administration group （SA, treated with artesunate in IC50）. The cycle and apoptosis of Fb were detected with flow cytometric assay, and the nucleic acid and protein expressions of CASK and ID1 of Fb in each group were determined with RT-PCR and Western blotting. Data were processed with one-way analysis of variance and LSD-t test.ResultsExpressions of CASK and ID1 were detected in two kinds of Fb. The concentration of 75 mg/L was selected as the intervention concentration of artesunate. （1） There were statistically significant differences among the three groups in the percentages of cells in G0/G1 phase and G2/M phase （with F values respectively 118.064 and 163.840, P values all below 0.01）. The percentage of cells in G0/G1 phase of group SA was （91.4±1.4）%, which was significantly higher than that of group SC and group NC [respectively （80.7±0.3）% and （82.4±0.6）%, with t values respectively 12.740 and 9.872, P values all below 0.05]

关 键 词：瘢痕疙瘩 成纤维细胞 青蒿素类 钙 钙调蛋白依赖性丝氨酸蛋白激酶 分化抑制因子1 

分 类 号：R6[医药卫生—外科学]