女贞子乙醇提取物及酪醇对人表皮黑素细胞黏附和迁移的影响

Effects of ethanol extract of glossy privet fruit and its monomer tyrosol on the adhesion and migration ofhuman epidermal melanocytes

目的探讨女贞子乙醇提取物及其单体酪醇对人表皮黑素细胞黏附和迁移的调节作用。方法体外分离培养人表皮黑素细胞，XTT法测定细胞增殖情况，用经FN包被的培养板测定细胞黏附率，Transwell微孔膜法观测细胞迁移情况，激光共聚焦显微镜观察经中药处理的细胞内肌动蛋白细胞骨架的结构分布，半定量分析细胞内荧光强度。结果0．0375～0．6mg／ml女贞子乙醇提取物均可促进黑素细胞在FN上的黏附（P〈0．05或P〈0．01）；0．5～2mmol／L酪醇可促进黑素细胞黏附（P〈0．05或P〈0．01）。0．15mg／ml女贞子乙醇提取物及2mmol／L酪醇无明显细胞毒性，在24h时不明显促进细胞增殖，选择此浓度为工作浓度，女贞子乙醇提取物及酪醇穿过微孔滤膜的黑素细胞数分别为43．7和51．0个，显著高于对照组的20．3个（P〈0．01）；两种药物工作浓度作用的黑素细胞与对照组相比，胞内可见较多呈束状的应力纤维，并集中分布于细胞膜内侧和细胞核周围，胞内荧光强度均高于对照组（P〈0．01）。结论女贞子乙醇提取物及其单体酪醇在体外可促进黑素细胞的黏附及迁移，其机制可能与诱导黑素细胞内肌动蛋白细胞骨架的聚合有关。

Objective To study the regulatory effect of ethanol extract of glossy privet fruit and its monomer tyrosol on the adhesion and migration of human epidermal melanocytes. Methods Epidermal melanocytes were isolated from human foreskin, and subjected to a primary euhure. After 3-5 passages, the melanocytes were treated with various concentrations of ethanol extract of glossy privet fruit （0.0375-0.6 mg/ml） and tyrosol （0.125-2 mmol/L） for 24-72 hours. The XTF colorimetric assay was carried out to evaluate the proliferation of melanocytes, fibronectin （FN）-coated culture plates were used to evaluate the adhesion activity of melanocytes, and Transwell assay was conducted to assess the migration activity of melanoeytes. Confoeal laser microscopy was utilized to observe the structure and distribution of actin cytoskeleton in melanocytes, and cellular fluorescence intensity was determined by a semi-quantitative analysis. Statistical analysis was carried out by using unpaired t test. Results The adhesion activity of melanoeytes to FN was significantly enhanced by the ethanol extract of 0.0375-0.6 mg/ml from glossy privet fruit （P 〈 0.05 or 0.01 ）, and by tyrosol of 0.5-2 mmol/L （P 〈 0.05 or 0.01 ）. As xTr assay showed, neither the ethanol extract of 0.15 mg/ml nor tyrosol of 2 mmol/L had cytotoxicity or promotive effect on cell proliferation. Hence, 0.15 mg/ml and 2 mmol/L were determined as the working concentrations of ethanol extract and tyrosol respectively. The number of cells migrating through micropore membranes per high-power field （× 200） was 43.7 and 51.0 in melanoeytes treated with the ethanol extract of 0.15 mg/ml and tyrosol of 2 mmol/L, respectively, significantly higher than that in untreated melanoeytes （20.3, both P 〈 0.01 ）. Compared with untreated melanocytes, those treated with the ethanol extract of 0.15 mg/ml and those with tyrosol of 2 mmol/L showed higher intracellular fluorescence intensity （P 〈 0.01） and more stress fiber bundles which congregated inside the cell membrane and around the nuclei. Conclusions The ethanol extract of glossy privet fruit and its monomer tyrosol can promote the adhesion and migration of human melanocytes in vitro, likely by promoting the congregation of aetin cytoskeleton in meianocytes.