甘草查尔酮A对小鼠黑色素瘤B16F10细胞体外迁移能力的影响

Effects of Licochalcone A on Invasion and Metastasis in Mouse Melanoma B16F10 Cells

目的：探讨甘草中黄酮类化合物甘草查尔酮A（licochalcone A,LCA）对小鼠黑色素瘤B16F10细胞株体外迁移能力的影响。方法：取对数生长期B16F10细胞按8 000个/孔接种于96孔板中,磺酰罗丹明B（SRB）法测定LCA（0,5,10,15,20μmol·L-^1）作用24 h对细胞活力影响;取对数生长期B16F10细胞按1×10^5个/孔接种于24孔板中,划痕实验检测LCA（0,5,10,15μmol·L^-1）作用24 h细胞体外迁移能力;取对数生长期B16F10细胞按5×10^5个/瓶接种于细胞培养瓶中,LCA（0,5,10,15μmol·L^-1）作用24 h后,明胶酶谱法检测细胞培养上清基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）活性;ELISA检测MMP-2和MMP-9蛋白含量;实时荧光定量PCR（Q-PCR）和半定量PCR（RT-PCR）法检测MMP-2和MMP-9基因表达水平。结果：5-15μmol·L-1的LCA对小鼠黑色素瘤B16F10细胞作用24 h,对细胞活力没有显著的影响;划痕试验结果说明,LCA可剂量依赖性地降低B16F10细胞的体外迁移能力;明胶酶谱结果显示,LCA可明显抑制B16F10细胞分泌MMP-2和MMP-9的活性;ELISA试验表明,LCA可明显降低MMP-2和MMP-9蛋白表达;Q-PCR和RT-PCR结果显示,LCA可明显降低MMP-2和MMP-9的mRNA基因表达量。结论：甘草查尔酮A能抑制B16F10小鼠黑色素瘤细胞体外转移侵袭作用,其机制可能与抑制MMP-2和MMP-9表达有关。

Objective： The purpose of this study is to investigate the effects of licochalcone A （LCA） on the invasion and metastasis in mouse melanoma B16F10 cells. Method： The cell proliferation rate was evaluated using sulforhodamine B （SRB） method. And the anti-metastasis ability was observed using scratch wound assay. The activated of matrix metal proteinase-2 （MMP-2） and matrix metal proteinase-9 （MMP-9） were evaluated using gelatin zymography. The expression in protein level of MMP-2 and MMP-9 were detected using enzyme-elinked iemmunosorbent essay （ELISA）. The mRNA expression of MMP-2 and MMP-9 were assessed using quantitative real-time polymerase chain reaction （PCR） and reverse transcription-PCR. Result： There was no significant effect on proliferation rate after treated with LCA 5-15 μmol·L^-1 in B16F10 cells. LCA attenuated B16F10 cells migration in a concentration-dependent manner. LCA was able to reduce the activities of MMP-2 and MMP-9, down-regulated the expression of MMP-2 and MMP-9 both in mRNA and protein levels. Conclusion： LCA attenuated mouse melanoma B16F10 cells migration by inhibition of MMP-2 and MMP-9 expression.