果糖基葛根素抗血管生成活性和细胞周期阻滞活性的研究
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摘要
葛根素(Puerarin)是从豆科植物野葛及葛根中提取的一种异黄酮类单体化合物,文章旨在探究果糖基葛根素(PG)抗血管生成和细胞周期阻滞的能力,通过用不同浓度PG处理HUVECs,利用划痕实验、迁移实验和管腔形成实验检测PG的抗血管生成能力;用不同浓度PG处理MDA-MB-231细胞,用流式细胞仪检测PG的细胞周期阻滞能力;利用RT-PCR检测α-tubulin mRNA的表达水平;利用Western-blot检测α-tubulin,Cyclin A1,及CDK2的表达水平进行考察,结果显示PG对HUVECs的迁移和管腔形成有抑制作用,可以导致MDA-MB-231细胞S期阻滞,PG对MDA-MB-231细胞α-tubulin mRNA表达水平无影响,可以降低MDA-MB-231细胞CDK2的表达水平。表明了PG的抗血管生成活性和细胞周期阻滞能力与促进微管聚合的信号通路相关。
Puerarin,a kind of flavonoid compound,was extracted from Puerariae Radix.This paper aims to explore the ability of PG to resist angiogenesis and cell cycle arrest,HUVECs were treated with different concentrations of PG and scratch-wound healing assay,migration assay,and tube formation assay were used to detect the ability of PG to resist angiogenesis.MDA-MB-231 were treated with different concentrations of PG.The cell cycle arrest ability of PG was detected by flow cytometry.The α-tubulin mRNA expression level was detected by RT-PCR.The expression levels of α-tubulin,cyclin A1 and CDK2 were detected by Western-blot.The results showed that PG inhibited the migration and tube formation of HUVECs,can cause MDA-MB-231 cell S phase arrest and PG had no effect on the expression of α-tubulin mRNA in MDA-MB-231 cells.CDK2 levels were significantly reduced along with the increasing concentrations of PG.It indicated that the PG most certainly had anti-angiogenic activity and cell cycle arrest ability associated with signaling pathways that promoted microtubule polymerization.
Puerarin,a kind of flavonoid compound,was extracted from Puerariae Radix.This paper aims to explore the ability of PG to resist angiogenesis and cell cycle arrest,HUVECs were treated with different concentrations of PG and scratch-wound healing assay,migration assay,and tube formation assay were used to detect the ability of PG to resist angiogenesis.MDA-MB-231 were treated with different concentrations of PG.The cell cycle arrest ability of PG was detected by flow cytometry.The α-tubulin mRNA expression level was detected by RT-PCR.The expression levels of α-tubulin,cyclin A1 and CDK2 were detected by Western-blot.The results showed that PG inhibited the migration and tube formation of HUVECs,can cause MDA-MB-231 cell S phase arrest and PG had no effect on the expression of α-tubulin mRNA in MDA-MB-231 cells.CDK2 levels were significantly reduced along with the increasing concentrations of PG.It indicated that the PG most certainly had anti-angiogenic activity and cell cycle arrest ability associated with signaling pathways that promoted microtubule polymerization.
引文
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[2]Wang YC,Ma YY,Zheng Y,et al.In vitro and in vivo anticancer activity of a novel puerarin nanosuspension against colon cancer,with high efficacy and low toxicity[J].Inter J Pharma,2013,441:728-735.
[3]Budden R,Kuhl UG,Bahlsen J.Experiments on the toxic,sedative and muscle relaxant potency of various drug solvents in mice[J].Pharmacol Ther,1979,B5:467-474.
[4]He BF,Wu XM.China patent[P].201110119350.6.
[5]He BF,Wu XM.China patent[P].201210185733.8.
[6]Wu XM,He BF.An efficient novel glycosylation of flavonoid byβ-fructosidase resistant to hydrophilic organic solvents[J].Biores Technol,2013,129:659-662.
[7]Zhu JH,Wang XX,Chen JZ,et al.Effects of puerarin on number and activity of endothelial progenitor cells from peripheral blood[J].Acta Pharmacol Sin,2004,25(8):1045-1051.
[8]Folkman J.Tumor angiogenesis:therapeutic implications[J].Nature Engl J Med,1971,285:1182-1186.
[9]Lee OH.Sphingosine 1-phosphate induces angiogenesis,its angiogenic action and signaling mechanism in human umbilical vein endothelial cells[J].Biochem Biophys Res Commun,1999,264:743-750.
[10]Ashton AW.Inhibition of endothelial cell migration,intercellular communication,and vascular tube formation by thromboxane A(2)[J].J Biol Chem,1999,274:35562-35570.
[11]Meerloo J.Cell sensitivity assays:the MTT assay[J].Methods Mol Biol,2011,731:237-245.
[12]Cross MJ,Claesson WL.FGF and VEGF function in angiogenesis:signaling pathways,biological responses and therapeutic inhibition[J].Trends Pharmacol,2001,22:201-207.
[13]Crabtree DV,Ojima I,Geng X,Adler AJ.Tubulins in the primate retina:evidence that xanthophylls may be endogenous ligands for the paclitaxel-binding site[J].Bioorg Med Chem,2001,8:1967-1976.
[14]Wilson L,Panda D,Jordan MA.Modulation of microtubule dynamics by drugs:a paradigm for the actions of cellular regulators[J].Cell Struct Funct,1999,24:329-335.
[15]Mirco C,Andrei VP.Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer[J].Protein Express Purif,2003,32:83-88.
[16]Feichtigera H,Wielanda E,Armstrong VW.The acyl glucuronide metabolite of mycophenolic acid induces tubulin polymerization in vitro[J].Clin Biochem,2010,43:208-213.