女贞子及其单体对人表皮黑素细胞黏附和迁移及肌动蛋白的影响
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摘要
目的
研究女贞子及其单体齐墩果酸、槲皮素、酪醇对人表皮黑素细胞黏附和迁移的调节作用,并观察其对黑素细胞内肌动蛋白细胞骨架结构及分布的影响。通过以上实验,为进一步研究女贞子治疗白癜风的作用机制提供一定的实验基础。
方法
体外分离培养人表皮黑素细胞,用不同浓度的女贞子及其单体齐墩果酸、槲皮素、酪醇作用于黑素细胞,XTT法测定细胞增殖情况,用经纤维连接蛋白FN包被的培养板测定细胞黏附率,采用Transwell微孔膜法观测细胞迁移情况,并用激光共聚焦显微镜观察经女贞子及其单体齐墩果酸、酪醇、槲皮素处理的黑素细胞内肌动蛋白细胞骨架的结构分布情况,采用荧光分析系统半定量分析细胞内荧光强度。
结果
1.经0.0375～0.3mg/ml女贞子作用的黑素细胞与对照组(未经药物处理的黑素细胞)相比,在FN上的黏附力随药物浓度升高而相应增强。当女贞子浓度达到0.6mg/ml时,细胞的黏附性开始下降。40μmol/L槲皮素作用后,可提高黑素细胞的黏附力,与对照组相比差异有显著性(P<0.05)。0.5～2 mmol/L酪醇可促进黑素细胞的黏附,以2mmol/L浓度作用最强(P<0.01)。试验浓度齐墩果酸对黑素细胞黏附无明显作用。
2.根据增殖实验结果选择无细胞毒性,并且在24h时不明显促进细胞增殖的最高浓度作为迁移实验的工作浓度,结果显示：对照组(不加药的完全培养基)穿过微孔滤膜的黑素细胞较少,零星散在分布；0.15 mg/ml女贞子组及2 mmol/L酪醇组穿过微孔滤膜的黑素细胞数较对照组显著增高(P<0.01); 12μmol/L齐墩果酸组穿过微孔滤膜的黑素细胞较对照组增多,差异具有显著性(P<0.05); 40μmol/L槲皮素组无显著促进细胞迁移的作用(P>0.05)。
3.经0.15 mg/ml女贞子、12μmol/L齐墩果酸、40μmol/L槲皮素、2 mmol/L酪醇处理的黑素细胞与对照组(未经药物处理的黑素细胞)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,胞内荧光强度均较对照组高,差异具有统计学意义。
结论
1.女贞子在体外可增强人表皮黑素细胞在FN上的黏附,并可促进黑素细胞的迁移。
2.酪醇可诱导黑素细胞在FN上的黏附,并促进黑素细胞的迁移；槲皮素可促进黑素细胞在FN上的黏附,而对黑素细胞迁移无明显作用；齐墩果酸具有诱导黑素细胞迁移的作用,但对黑素细胞的黏附无显著作用。提示齐墩果酸、槲皮素和酪醇可能是女贞子中诱导黑素细胞黏附和/或迁移的有效成分。
3.女贞子及其单体齐墩果酸、槲皮素、酪醇可能通过诱导黑素细胞内肌动蛋白细胞骨架的聚合,从而间接发挥它们促进黑素细胞黏附和/或迁移的作用。
Objective
To stuudy the effect of fructus ligustri lucidi and its monomer oleanolic acid, quercetin and tyrosol on the adhesion, migration and cellular actin of cultured human melanocyte, in order to further study of the mechanism of fructus ligustri lucidi in treating vitiligo.
Methods
To cultured human epidermal melanocytes in vitro, treated melanocyte with different concentrations of fructus ligustri lucidi and its monomer oleanolic acid, quercetin and tyrosol, the cell proliferation was detected by XTT assay, the cell adhesion rate was determined by FN-coated culture plates, the migration of melanocyte were detected by the method of transwell assay.We observed the structure and distribution of cellular actin cytoskeleton with confocal laser microscopy, and do a semi-quantitative analysis of cellular fluorescence intensity.
Result
1.At the range of 0.0375～0.3mg/ml of fructus ligustri lucidi, showed an increasing tendency of the adhesion of melanocyte with the corresponding increase of drug concentration compared with the control group(melanocyte without drugs treatment).When the concentration of fructus ligustri lucidi was at 0.6mg/ml,the adhesion rate of melanocytes began to decline. When the concentration of quercetin was at 40μmol/L, it could improve the adhesion of melanocytes, there was a significant difference compared with the control group (P<0.05). At the range of 0.5～2 mmol/L of tyrosol, the adhesion of melanocytes could be improved,and when the concentration of tyrosol was at 2 mmol/L, the effect was the strongest (P<0.01). There was no significant difference between the test concentrations of oleanolic acid and the control group.
2. According to the results of proliferation of melanocyte, we choice the working concentrations of transwell assay were the highest concentration with no cytotoxicity and without obvious stimulation of cell proliferation at 24h.Transwell assay revealed a promoting effect of both fructus ligustri lucidi and tyrosol on melanocyte migration(P<0.01),when the working concentrations of fructus ligustri lucidi and tyrosol was determined at 0.15 mg/ml and 2 mmol/L respectively;there was a significant difference of the count of melanocyte that through microfiltration membrane between the concentration of oleanolic acid at 12μmol/L and the control. (P<0.05);while the working concentration of quercetin at 40μmol/L had little effect on the migration of melanocyte.
3.More stress fibre fasciculus were formed by F-actin were found in the human epidermal melanocyte cell which was treated with Fructus ligustri lucidi(0.15 mg/ml), oleanolic acid (12μmol/L), quercetin (40μmol/L) and tyrosol (2 mmol/L) compared with the control group(melanocyte without drugs treatment). The stress fibre fasciculus were distributed inside the cell membrane and around the nucleus. Intracellular fluorescence intensity in this four groups was higher than the control group.
Conclusion
1. Fructus ligustri lucidi has a significant stimulatory effect on the adhesion and migration of human melanocyte.
2. Tyrosol has a significant stimulatory effect on the adhesion and migration of human melanocyte; quercetin can promote the adhesion of human melanocyte,but has no significant effect of the migration; oleanolic acid can induce the migration of melanocyte,but has little effect on melanocyte adhesion. Oleanolic acid, quercetin and tyrosol may be the active components of Fructus ligustri lucidi which have confirmative effect on adhesion and/or migration of human epidermal melanocyte.
3. Fructus ligustri lucidi and its monomer oleanolic acid, quercetin and tyrosol may indirectly stimulate the adhesion and migration of human melanocyte by promoting the actin cytoskeleton congregation in melanocyte in vitro.
研究女贞子及其单体齐墩果酸、槲皮素、酪醇对人表皮黑素细胞黏附和迁移的调节作用,并观察其对黑素细胞内肌动蛋白细胞骨架结构及分布的影响。通过以上实验,为进一步研究女贞子治疗白癜风的作用机制提供一定的实验基础。
方法
体外分离培养人表皮黑素细胞,用不同浓度的女贞子及其单体齐墩果酸、槲皮素、酪醇作用于黑素细胞,XTT法测定细胞增殖情况,用经纤维连接蛋白FN包被的培养板测定细胞黏附率,采用Transwell微孔膜法观测细胞迁移情况,并用激光共聚焦显微镜观察经女贞子及其单体齐墩果酸、酪醇、槲皮素处理的黑素细胞内肌动蛋白细胞骨架的结构分布情况,采用荧光分析系统半定量分析细胞内荧光强度。
结果
1.经0.0375～0.3mg/ml女贞子作用的黑素细胞与对照组(未经药物处理的黑素细胞)相比,在FN上的黏附力随药物浓度升高而相应增强。当女贞子浓度达到0.6mg/ml时,细胞的黏附性开始下降。40μmol/L槲皮素作用后,可提高黑素细胞的黏附力,与对照组相比差异有显著性(P<0.05)。0.5～2 mmol/L酪醇可促进黑素细胞的黏附,以2mmol/L浓度作用最强(P<0.01)。试验浓度齐墩果酸对黑素细胞黏附无明显作用。
2.根据增殖实验结果选择无细胞毒性,并且在24h时不明显促进细胞增殖的最高浓度作为迁移实验的工作浓度,结果显示：对照组(不加药的完全培养基)穿过微孔滤膜的黑素细胞较少,零星散在分布；0.15 mg/ml女贞子组及2 mmol/L酪醇组穿过微孔滤膜的黑素细胞数较对照组显著增高(P<0.01); 12μmol/L齐墩果酸组穿过微孔滤膜的黑素细胞较对照组增多,差异具有显著性(P<0.05); 40μmol/L槲皮素组无显著促进细胞迁移的作用(P>0.05)。
3.经0.15 mg/ml女贞子、12μmol/L齐墩果酸、40μmol/L槲皮素、2 mmol/L酪醇处理的黑素细胞与对照组(未经药物处理的黑素细胞)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,胞内荧光强度均较对照组高,差异具有统计学意义。
结论
1.女贞子在体外可增强人表皮黑素细胞在FN上的黏附,并可促进黑素细胞的迁移。
2.酪醇可诱导黑素细胞在FN上的黏附,并促进黑素细胞的迁移；槲皮素可促进黑素细胞在FN上的黏附,而对黑素细胞迁移无明显作用；齐墩果酸具有诱导黑素细胞迁移的作用,但对黑素细胞的黏附无显著作用。提示齐墩果酸、槲皮素和酪醇可能是女贞子中诱导黑素细胞黏附和/或迁移的有效成分。
3.女贞子及其单体齐墩果酸、槲皮素、酪醇可能通过诱导黑素细胞内肌动蛋白细胞骨架的聚合,从而间接发挥它们促进黑素细胞黏附和/或迁移的作用。
Objective
To stuudy the effect of fructus ligustri lucidi and its monomer oleanolic acid, quercetin and tyrosol on the adhesion, migration and cellular actin of cultured human melanocyte, in order to further study of the mechanism of fructus ligustri lucidi in treating vitiligo.
Methods
To cultured human epidermal melanocytes in vitro, treated melanocyte with different concentrations of fructus ligustri lucidi and its monomer oleanolic acid, quercetin and tyrosol, the cell proliferation was detected by XTT assay, the cell adhesion rate was determined by FN-coated culture plates, the migration of melanocyte were detected by the method of transwell assay.We observed the structure and distribution of cellular actin cytoskeleton with confocal laser microscopy, and do a semi-quantitative analysis of cellular fluorescence intensity.
Result
1.At the range of 0.0375～0.3mg/ml of fructus ligustri lucidi, showed an increasing tendency of the adhesion of melanocyte with the corresponding increase of drug concentration compared with the control group(melanocyte without drugs treatment).When the concentration of fructus ligustri lucidi was at 0.6mg/ml,the adhesion rate of melanocytes began to decline. When the concentration of quercetin was at 40μmol/L, it could improve the adhesion of melanocytes, there was a significant difference compared with the control group (P<0.05). At the range of 0.5～2 mmol/L of tyrosol, the adhesion of melanocytes could be improved,and when the concentration of tyrosol was at 2 mmol/L, the effect was the strongest (P<0.01). There was no significant difference between the test concentrations of oleanolic acid and the control group.
2. According to the results of proliferation of melanocyte, we choice the working concentrations of transwell assay were the highest concentration with no cytotoxicity and without obvious stimulation of cell proliferation at 24h.Transwell assay revealed a promoting effect of both fructus ligustri lucidi and tyrosol on melanocyte migration(P<0.01),when the working concentrations of fructus ligustri lucidi and tyrosol was determined at 0.15 mg/ml and 2 mmol/L respectively;there was a significant difference of the count of melanocyte that through microfiltration membrane between the concentration of oleanolic acid at 12μmol/L and the control. (P<0.05);while the working concentration of quercetin at 40μmol/L had little effect on the migration of melanocyte.
3.More stress fibre fasciculus were formed by F-actin were found in the human epidermal melanocyte cell which was treated with Fructus ligustri lucidi(0.15 mg/ml), oleanolic acid (12μmol/L), quercetin (40μmol/L) and tyrosol (2 mmol/L) compared with the control group(melanocyte without drugs treatment). The stress fibre fasciculus were distributed inside the cell membrane and around the nucleus. Intracellular fluorescence intensity in this four groups was higher than the control group.
Conclusion
1. Fructus ligustri lucidi has a significant stimulatory effect on the adhesion and migration of human melanocyte.
2. Tyrosol has a significant stimulatory effect on the adhesion and migration of human melanocyte; quercetin can promote the adhesion of human melanocyte,but has no significant effect of the migration; oleanolic acid can induce the migration of melanocyte,but has little effect on melanocyte adhesion. Oleanolic acid, quercetin and tyrosol may be the active components of Fructus ligustri lucidi which have confirmative effect on adhesion and/or migration of human epidermal melanocyte.
3. Fructus ligustri lucidi and its monomer oleanolic acid, quercetin and tyrosol may indirectly stimulate the adhesion and migration of human melanocyte by promoting the actin cytoskeleton congregation in melanocyte in vitro.
引文
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