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目的研究朝藿定C(Epimedin C)诱导C3H小鼠胚胎间充质干细胞(murine embryonic mesenchymal stem cells,C3H/10T1/2)内皮样分化作用。方法体外培养C3H/10T1/2细胞,MTT法和结晶紫法检测不同浓度下朝藿定C对细胞的毒性作用;显微镜下观察朝藿定C诱导后的细胞形态变化;采用流式细胞仪检测朝藿定C对细胞周期分布的影响;采用半定量PCR检测血管内皮细胞标志物CD31、CD34、血管内皮细胞锌指1(vascular endothelial zinc finger 1,Vezf1)、血管生成素1(angiopoietin 1,Ang1)、血管生成素2(angiopoietin 2,Ang2)mRNA的表达;采用免疫细胞化学染色法检测血小板内皮黏附分子-1(platelet-endothelial cell adhesion molecule-1,CD31)、细胞外-5’-核苷酸酶(ecto-5’-nucleotidase,CD73)、内皮细胞特异性分子-1(endothelial cell specific molecule-1,ESM-1)、整合素β5(integrinβ5)的表达。结果朝藿定C在浓度1~30μmol/L时,不影响C3H/10T1/2细胞的存活率。30μmol/L处理24 h后,对细胞周期无明显影响。朝藿定C能诱导C3H/10T1/2细胞向血管内皮细胞分化,使细胞呈明显漩涡状排列。PCR检测结果显示,与干预前比较,经诱导5天后的C3H/10T1/2细胞血管内皮细胞标志因子、CD34、Vezf1、Ang1、Ang2 mRNA水平明显增加(P<0.05,P<0.01);细胞免疫化学染色结果显示诱导5天后的C3H/10T1/2细胞血管内皮细胞标志性蛋白CD31、CD73、ESM-1均呈阳性表达,与对照组比较差异有统计学意义(P<0.01,P<0.05)。结论朝藿定C能诱导C3H/10T1/2细胞向血管内皮样细胞分化。
Objective To investigate the endothelium-like differentiation of C3H / 10T1 / 2 cells induced by Epimedin C in C3H mice. Methods C3H / 10T1 / 2 cells were cultured in vitro. MTT assay and crystal violet assay were used to detect the effects of epimedin C on the cells. The morphological changes of cells treated with epimedin C were observed under a microscope. Flow cytometry Epimedin C on the cell cycle distribution; using semi-quantitative PCR detection of vascular endothelial cell markers CD31, CD34, vascular endothelial zinc finger 1 (Vezf1), angiopoietin 1 (Ang1 ), Angiopoietin 2 (Ang2) mRNA were detected by immunohistochemical method. The expressions of platelet-endothelial cell adhesion molecule-1 (CD31), extracellular 5’-nucleoside Expression of ecto-5’-nucleotidase (CD73), endothelial cell-specific molecule-1 (ESM-1) and integrinβ5. Results Epimedia C at a concentration of 1 ~ 30μmol / L, did not affect the survival rate of C3H / 10T1 / 2 cells. After treatment with 30μmol / L for 24 h, there was no significant effect on cell cycle. Epimedin C can induce C3H / 10T1 / 2 cells to differentiate into vascular endothelial cells, so that cells were significantly swirling arrangement. The results of PCR showed that the levels of Vascular Endothelial Cell Marker, CD34, Vezf1, Ang1 and Ang2 mRNA in C3H / 10T1 / 2 cells increased significantly after 5 days of induction (P <0.05, P <0.01) The results of chemical staining showed that the expression of markers CD31, CD73 and ESM-1 of C3H / 10T1 / 2 cells were positive after 5 days induction compared with the control group (P <0.01, P <0.05) . Conclusion Epimedin C can induce the differentiation of C3H / 10T1 / 2 cells into vascular endothelial cells.