Study on alantolactone-induced differentiation of mesenchymal stem cells into vascular cells

摘要

为了从传统药物中高效筛选有促进血管生成活性的药物成份,我们构建了以血管内皮生长因子(Vascular endothelial growth factor,VEGF)基因启动子作为药物作用靶点的HEK293细胞模型,该细胞中,VEGF基因启动子通过响应药物分子的刺激而调控荧光素酶报告基因的表达.这一细胞模型使我们能从大量药物单体分子中快速高效地初步筛选出有诱导血管生成的活性成分.我们进一步利用大鼠骨髓间充质干细胞(rMSC)对初步筛选出的土木香内酯进行了活性研究,以辛伐他汀为阳性对照探究土木香内酯对rMSC表达血管相关细胞标记分子——VEGF和α-平滑肌肌动蛋白(α-Smooth muscle actin,α-SMA)的影响,从而判断土木香内酯对rMSC向血管内皮细胞和血管平滑肌细胞诱导分化的作用.结果显示,0.1μM、1μM、3μM和5μM的土木香内酯均能上调HEK293模型细胞中荧光素酶的表达,与空白对照组比较有显著性差异,其中3μM土木香内酯的作用优于相同浓度的辛伐他汀(P=0.036),其它浓度的土木香内酯作用与辛伐他汀相当,没有显著性差异;1μM土木香内酯处理rMSCs 3天后,VEGF和α-SMA基因的mRNA相对表达量较空白对照组均有显著性差异,但是5μM土木香内酯作用弱于相同浓度的辛伐他汀(P<0.05);免疫荧光法检测1μM土木香内酯诱导rMSCs 3天后,原位表达的α-SMA和VEGF蛋白(FITC标记)的荧光亮度均比空白对照组强,和5μM的辛伐他汀无差异.上述结果显示了我们构建的血管活性药物分子高效筛选体系的可靠性,并从分子和蛋白水平证实了土木香内酯的血管诱导功能.%To promote efficient screening of active angiogenic drugs from traditional medicines, we constructed a human embryonic kidney-293 cell model using vascular endothelial growth factor (VEGF) gene promoter as the drug target. In this model, VEGF gene promoter may regulate the expression of the luciferase reporter gene by responding to the stimulation of drug molecules. This cell model allows rapid and efficient screening of vascular-inducing active components from several drug monomer molecules. Furthermore, we used rat bone marrow mesenchymal stem cells (rMSCs) to conduct a preliminary study on the activity of alantolactone. Using simvastatin as a positive control, we investigated the effects of alantolactone on the expression of vascular-related cell marker molecules such as VEGF andα-smooth muscle actin (α-SMA) in rMSCs. According to our results, 0.1, 1, 3 and 5μM of alantolactone upregulated the transcriptional luciferase gene activity of VEGF promoter, and a significant difference from that in the control group was observed. Among them, 3μM of alantolactone showed the better effect than that of 3μM of simvastatin (P=0.036) and other concentrations of alantolactone and simvastatin showed similar effects. Compared with that in the control group, rMSCs induced with 1μM alantolactone for 3 days showed a significant increase in the relative mRNA expressions of VEGF andα-SMA genes. However, these effect of 5μM alantolactone were weaker than those of 5μM simvastatin (P<0.05);rMSCs treated with 1μM alantolactone for 3 days showed brighter green fluorescence (FITC marker) ofα-SMA and VEGF in situ expression than that observed in the control group and similar fluorescence intensity than that of simvastatin group in an immunoradiometric assay. The above results demonstrate the reliability of the highly efficient system for screening of active drug molecules and confirmed the vascular induction function of alantolactone at the gene and protein levels.