积雪苷体外对瘢痕疙瘩成纤维细胞增殖及结缔组织生长因子表达的影响

目的 探讨积雪苷体外对瘢痕疙瘩成纤维细胞增殖和对结缔组织生长因子（CTGF）表达的影响。方法 取手术切除的瘢痕疙瘩组织作成纤维细胞的原代培养，加入不同浓度积雪苷，观察细胞的形态变化，MTT法检测细胞活性，免疫组化和Western印迹法检测积雪苷对瘢痕疙瘩成纤维细胞结缔组织生长因子表达的影响。结果 细胞形态学观察显示，经不同浓度积雪苷处理的成纤维细胞呈现明显的抑制及凋亡征象。积雪苷在1 ～ 100 mg/L范围内，在24、48、72 h时积雪苷浓度与细胞活性抑制率均呈正相关，r分别为0.95、0.90和0.92，P值均 ＜ 0.01；且各浓度不同时间段细胞活性抑制率间单因素方差分析，P值均 ＜ 0.01。瘢痕疙瘩成纤维细胞中CTGF呈强阳性表达，而经积雪苷处理瘢痕疙瘩成纤维细胞48 h后，CTGF表达有所减弱，每100个成纤维细胞中CTGF表达阳性细胞数均值未加药组为73个，1 mg/L积雪苷组为54个，10 mg/L积雪苷组为46个，未加药组与1 mg/L、10 mg/L积雪苷组比较，差异均有统计学意义（t值分别为4.34和6.26，P值均 ＜ 0.01）；1 mg/L积雪苷组与10 mg/L积雪苷组比较，差异亦有统计学意义（t = 1.95，P ＜ 0.05）。Western印迹显示，积雪苷作用48 h后，成纤维细胞中CTGF的表达量比未加药的细胞明显减弱，且表达量随药物剂量的加大有递减趋势。结论 积雪苷能有效抑制瘢痕疙瘩成纤维细胞的增殖和结缔组织生长因子的表达。

In vitro effects of asiaticoside on the proliferation of and connective tissue growth factor expression by keloid-derived fibroblasts

Objective To investigate the in vitro effect of asiaticoside on the proliferation of and connective tissue growth factor （CTGF） expression by keloid-derived fibroblasts． Methods Tissue samples from patients with keloid were obtained for primary culture of fibroblasts. After 3 to 7 passages, the fibroblasts were incubated with different concentrations （100, 10, 1, 0.1, 0.01 mg/L） of asiaticoside or dimethyl sulfoxide （DMSO） for 24, 48 and 72 hours followed by the determination of cell viability by methyl thiazolyl tetrazolium （MTT） assay. Immunohistochemistry and Western blot were carried out to quantify the expression of CTGF in the fibroblasts at 48 hours after treatment with different concentrations of asiaticoside. The morphology of fibroblasts was observed before and after the treatment with asiaticoside. Results As morphological observation showed, different concentrations of asiaticoside induced an obvious apoptosis and growth inhibition in fibroblasts. The growth of fibroblasts was suppressed by asiaticoside of 1－100 mg/L in a dose-dependent manner （r = 0.95, 0.90, 0.92 for 24-, 48- and 72-hour treatment respectively, all P < 0.01）, and one-factor analysis of variance revealed statistical differences in the growth inhibition rate in fibroblasts between different treatment durations for each tested concentration of asiaticoside （all P < 0.01）. There was a strong expression of CTGF in untreated fibroblasts, which was weakened by the treatment with asiaticoside for 48 hours. The number of CTGF-positive fibroblasts per 100 cells was 73 in untreated fibroblasts, significantly higher than that in fibroblasts treated with asiaticoside at 1 mg/L （54, t = 4.34, P < 0.01） and 10 mg/L （46, t = 6.26, P < 0.01）, and statistical differences were observed between the fibroblasts treated with asiaticoside at 1 mg/L and 10 mg/L （t = 1.95, P < 0.05）. Western blot also showed that the expression of CTGF was inhibited by 48-hour treatment with asiaticoside, and the inhibitive effect displayed a trend to increase with the rise in the concentration of asiaticoside. Conclusion Asiaticoside can effectively inhibit the proliferation of and CTGF expression by fibroblasts in vitro.