A型肉毒毒素引起人增生性瘢痕成纤维细胞的凋亡

Botulinum toxin type A induces apoptosis of human hypertrophic scar fibroblasts

背景:A型肉毒毒素临床上可以治疗增生性瘢痕,体外细胞培养研究发现可以抑制增生性瘢痕成纤维细胞的增殖。目的:观察A型肉毒毒素对人增生性瘢痕成纤维细胞增殖的抑制作用,以及对人增生性瘢痕成纤维细胞凋亡的影响。方法:通过消化法分离培养出人增生性瘢痕成纤维细胞,分别用不同浓度的A型肉毒毒素对细胞的生长增殖过程进行干预,通过MTT染色,于酶联免疫检测仪570nm测定吸光度来研究细胞生长增殖情况,计算抑制率。通过Hoechst33342及PI染色检测人增生性瘢痕成纤维细胞凋亡情况,并计算凋亡率。结果与结论:人增生性瘢痕成纤维细胞生长过程中呈梭形,细胞生长旺盛,细胞融合成单层,细胞排列成高度一致性。经A型肉毒毒素处理后,细胞增殖速度明显减慢,细胞数量减少,细胞排列方向散乱。MTT染色后吸光度减弱,随A型肉毒毒素浓度增加,吸光度明显减弱,与对照细胞比较,差异有显著性意义(P<0.05),半数抑制率出现在0.4IU/L。在荧光显微镜下,人增生性瘢痕成纤维细胞经Hoechst33342和PI染色后细胞核呈现蓝色,细胞核为光滑的圆形或椭圆形外观。A型肉毒毒素干预后细胞核致密浓缩,染色不均匀,折光性增强,核膜皱缩,部分细胞核碎裂,出现碎块,有凋亡小体出现。随着A型肉毒毒素浓度的增加细胞凋亡率逐渐增高,与对照细胞比较差异有显著性意义(P<0.05),半数凋亡率在0.4IU/L。说明A型肉毒毒素可以抑制人增生性瘢痕成纤维细胞的增殖,其主要通过引起细胞凋亡的途径来抑制成纤维细胞的增殖。

BACKGROUND： Botulinum toxin type A can be used to treat human hypertrophic scar clinically and inhibit the proliferation of human hypertrophic scar fibroblasts in vitro.OBJECTIVE： To investigate the effect of botulinum toxin type A on the proliferation and apoptosis of human hypertrophic scar fibroblasts. METHODS： Human hypertrophic scar fibroblasts were isolated and cultured using digestion method. The cells were treated with botulinum type A at different concentrations. After 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide dyeing, the cell solutions were detected with enzyme-linked immunosorbent detector at 570 nm. The inhibitory rate was calculated. The cultured cells were stained with hoechst 33342 and PI. The apoptotic cells were observed under fluorescence microscopy and the apoptotic rate was calculated. RESULTS AND CONCLUSION： The hypertrophic scar fibroblasts represented the spindle-like character with uniform composition and strong proliferation. After treated with botulinum toxin type A, the cells grew slowly, the cell amount reduced and the arrangement of the cells was scattered. The absorbance after 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide dyeing decreased. There was a statistically significant difference compared with the control group （P 〈 0.05）. The half inhibitory rate was 0.4 lUlL. After hoechst 33342 and PI dyeing, the nuclei became blue and showed a smooth round or oval appearance. After treated with botulinum toxin type A, the nuclei were condensed with uneven dyeing. Some of the nuclei were fragmented and apoptotic bodies appeared. The apoptotic rate was increased with increasing concentrations of botulinum toxin type A. There was a statistically significant difference between the control group and the treated group （P 〈 0.05）. The half apoptotic rate was 0.4 lUlL. These findings indicate that botulinum toxin type A can cause apoptosis of the human hypertrophic scar fibroblasts, thus inhibiting the proliferation of the cells.