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目的探讨染料木黄酮对光老化成纤维细胞增殖作用的影响。方法经酶消化法分离培养真皮成纤维细胞,并进行细胞传代,免疫细胞化学鉴定细胞。实验分为3组:正常细胞对照组:未经处理的真皮成纤维细胞;8-甲氧补骨脂素(8-methoxypsoralan,8-MOP)/长波紫外线(ultraviolet A,UVA)对照组:用含50 ng/ml 8-MOP的培养基避光孵育细胞24 h,再以9 J/cm2UVA照射;8-MOP/UVA+染料木黄酮(0、1.25、2.5、5、10、20μg/ml)组:用含50 ng/ml8-MOP及各浓度染料木黄酮的培养基共同孵育细胞24 h,再以9 J/cm2UVA照射24 h。MTT法检测各组细胞增殖活力,流式细胞术检测各组细胞细胞周期,光镜下观察各组细胞分裂和分裂后表型,实时荧光定量PCR法检测各组细胞中基质金属蛋白酶-1、3水平。结果皮肤成纤维细胞阳性率达99%以上;8-MOP/UVA+染料木黄酮1.25、2.5μg/ml组细胞增殖率分别为8-MOP/UVA对照组的112.6%(P>0.05)和146.6%(P<0.05),染料木黄酮的光保护作用随浓度的升高而增强,然而随着浓度的继续升高,细胞增值率逐渐降低,当浓度达20μg/ml时,细胞增殖率仅为8-MOP/UVA对照组的19.4%(P<0.01),选择染料木黄酮的最佳保护浓度为2.5μg/ml;8-MOP/UVA对照组细胞细胞周期阻滞于S期,而8-MOP/UVA+染料木黄酮2.5μg/ml组细胞细胞周期则由S期进入G2/M期;正常细胞对照组、8-MOP/UVA组及8-MOP/UVA+染料木黄酮2.5μg/ml组分裂细胞表型占总细胞数目的比率分别为(99.7±0.58)%、(7.7±1.15)%、(57.7±3.51)%,各组间差异有统计学意义(P<0.01);8-MOP/UVA+染料木黄酮2.5μg/ml组细胞基质金属蛋白酶-1、3水平均明显低于8-MOP/UVA对照组,而高于正常细胞对照组,各组间差异均有统计学意义(P<0.05)。结论染料木黄酮可在一定程度上解除8-MOP/UVA所致光老化成纤维细胞的生长抑制作用。
Objective To investigate the effects of genistein on the proliferation of photoaging fibroblasts. Methods Dermal fibroblasts were isolated and cultured by enzyme digestion. The cells were passaged and identified by immunocytochemistry. The experiment was divided into three groups: normal control group: untreated dermal fibroblasts; 8-methoxypsoralan (8-MOP) / ultraviolet A (UVA) control group: Incubation of cells in dark for 24 h with 50 ng / ml 8-MOP medium followed by irradiation with 9 J / cm2 UVA; 8-MOP / UVA + genistein (0,1.25,2.5,5,10,20 μg / ml) : The cells were incubated with 50 ng / ml 8-MOP and genistein at different concentrations for 24 h and 24 h UVA at 9 J / cm2. The cell viability was measured by MTT assay. The cell cycle was detected by flow cytometry. The cell division and post-division phenotype were observed by light microscopy. The expression of matrix metalloproteinase-1, 3 levels. Results The positive rate of skin fibroblasts was over 99%. The proliferation rates of 8-MOP / UVA + genistein 1.25 and 2.5μg / ml groups were 112.6% (P> 0.05) and 146.6% (P <0.05). The photoprotective effect of genistein increased with the increase of concentration, however, with the increase of concentration, the cell proliferation rate decreased gradually. When the concentration reached 20μg / ml, the cell proliferation rate was only 8 -MOP / UVA control group was 19.4% (P <0.01), the optimal concentration of genistein was 2.5μg / ml. The cell cycle of 8-MOP / UVA control group was arrested in S phase, while the 8-MOP / UVA + genistein 2.5μg / ml group of cells from the S phase into the G2 / M phase; normal control group, 8-MOP / UVA group and 8-MOP / UVA + genistein 2.5μg / ml group of dividing cells (99.7 ± 0.58)%, (7.7 ± 1.15)% and (57.7 ± 3.51)%, respectively. There was a significant difference between the groups (P <0.01). The ratio of 8-MOP / UVA + Compared with 8-MOP / UVA control group, the level of MMP-1 and MMP-2 in 2.5μg / ml genistein group was significantly higher than that in normal control group (P <0.05 ). Conclusions Genistein can relieve the growth inhibition of photo-aged fibroblasts induced by 8-MOP / UVA to a certain extent.