金雀异黄素对体外培养的人皮肤成纤维细胞生物学活性和胶原合成的影响

Effects of genistein on biological activities of and collagen synthesis by human skin fibroblasts in vitro

目的观察金雀异黄素对体外培养的正常人皮肤成纤维细胞生长和胶原合成的影响。方法采用噻唑蓝（MTT）比色法检测不同浓度金雀异黄素对体外培养的人皮肤成纤维细胞存活力的影响，并绘制细胞的生长曲线；流式细胞仪检测细胞周期的变化；RT—PCR检测不同浓度金雀异黄素对成纤维细胞I型胶原mRNA表达的影响。结果0．03125，0．0625，0．125，0．25，0．5，1mg／L金雀异黄素作用24h时，成纤维细胞增殖率分别为97．7％，113．8％，132．5％，116．4％，94．5％和83．3％。当金雀异黄素质量浓度大于0．5mg／L，对成纤维细胞的增殖表现出抑制作用。浓度在0．0625～0．25mg／L之间时，对成纤维细胞的增殖表现出促进作用。0．0625、0．125、0．25mg／L金雀异黄素作用于成纤维细胞后，s期（41．15％±2．88％，61．89％±3．16％，48．18％±1．68％）和G2期（9．76％±3．99％，10．40％±0．54％，7．46％±2．47％）细胞明显高于对照组（s期为30．12％±0．60％，G2期为0．61％±0．16％），两组比较，差异均有统计学意义（P均〈0．05）；而G1期细胞（49．08％±3．58％，30．04％±1．89％，44．36％±3．92％）明显低于对照组（69．27％±0．73％），两组比较，差异均有统计学意义（P均〈0．05）；3个浓度组I型胶原mRNA的表达（0．4814±0．0138，0．5767±0．0291，0．5675±0．0272）均高于对照组（0．4101±0．0236），两组比较，差异均有统计学意义（P均〈0．01）。1mg／L和0．5mg／L的金雀异黄素作用成纤维细胞后，I型胶原mRNA的表达（0．1662±0．0165，0．2017±0．0203）低于对照组，两组比较，差异均有统计学意义（P均〈0．01）。结论一定浓度的金雀异黄素可以促进正常人皮肤成纤维细胞的增殖和生长，并促进成纤维细胞I型胶原mRNA的表达。

Objective To study the effects of genistein on the biological characteristics of and collagen synthesis by human skin fibroblasts in vitro. Methods Human skin fibroblasts （HSFBs） were obtained from the prepuce of healthy adolescents, and subjected to primary culture. After 5 to 15 passages of culture, HSFBs were treated with various concentrations （0.03125, 0.0625, 0.125, 0.25, 0.5, 1 mg/L） ofgenistein for different durations. The potential of cell proliferation was detected by MTT assay and growth curves were drawn for HSFBs. Flow cytometry （FCM） and RT-PCR were used to estimate cell cycle phases and mRNA expression of type I procollagen, respectively. Results The proliferation rate of HSFBs was 97.7%, 113.8%, 132.5%, 116.4%, 94.5% and 83.3% after treatment with genistein of 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 mg/L, respectively. The genistein of more than 0.5 mg/L displayed an inhibitive effect on the proliferation of HSFBs, while that between 0.0625 and 0.25 mg/L showed a promotive effect. After treatment with genistein at 0.0625,0.125 and 0.25 mg/L, the percentage of HSFBs in S phase and G2 phase significantly increased compared with untreated HSFBs （S phase： 41.15% ± 2.88%,61.89% ± 3.16%,48.18% ± 1.68% vs 30.12% ± 0.60%, P 〈 0.05; G2 phase： 9.76% ±3.99%, 10.40% ± 0.54%,7.46% ± 2.47% vs 0.61% ± 0.16%, P 〈 0.05）. Compared with the untreated HSFBs, the relative mRNA expression level of type I procollagen was increased with genistein of 0.0625, 0.125 and 0.25 mg/L （0.4814 ±0.0138,0.5767 ± 0.0291,0.5675 ± 0.0272 vs 0.4101 ± 0.0236, P 〈 0.01 ）,but decreased with genistein of 1 and 0.5 mg/L（0.1662 ± 0.0165 and 0.2017 ± 0.0203 vs 0.4101 ± 0.0236, P 〈 0.01 ）. Conelusion Certain concentrations of genistein could enhance the proliferation and growth of as well as mRNA expression of type I procollagen in HSFBs.