朝藿定C诱导C3H小鼠胚胎间充质干细胞向内皮样细胞分化的研究  

Epimedin C Induced Mesenchymal Stem Cells C3H /10T1/2 to Differentiate into Endothelioid Cells in Vitro: an Experimental Study

作　　者：赵岩[1] 高建莉[1] 陈素红[2] 吕圭源[1] 

机构地区：[1]浙江中医药大学药物研究所,杭州310053 [2]温州医科大学中药研究院,浙江325035

出　　处：《中国中西医结合杂志》2015年第3期343-348,共6页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学海外合作项目(No.81228024);国家自然科学基金青年项目(No.81302989)

摘　　要：目的研究朝藿定C（Epimedin C）诱导C3H小鼠胚胎间充质干细胞（murine embryonicm esenchymal stem cells, C3 H/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’-nu-cleotidase，CD73）、内皮细胞特异性分子-1（endothelial cell specific molecule-1，ESM-1）、整合素β5（integrin β5）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24h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门OTl／2细胞血管内皮细胞标志因子、CD34、Vezf1、Ang1、Ang2mRNA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 study the endothelioid differentiation effect of Epimedin C on murine embryonic mesenchymal stem cells （C3H/10T1/2）. Methods C3H/10T1/2 cells were cultivated in vitro. The cytotoxicity of Epimedin C at different concentrations was determined by MTT assay and crystal vio- let assay. Morphological changes were observed under microscope after treated with Epimendin C. The effect of Epimendin C on the cell cycle distribution was determined by flow cytometry, mRNA expression levels of endothelial markers, such as CD31, CD34, vascular endothelial zinc finger 1 （Vezf1）, angiopoi- etin 1 （Ang1）, and angiopoietin 2 （Ang2） were detected by semi-quantitative PCR. Protein expression levels of platelet endothelial adhesive molecule 1 （CD31）, ecto-5＇-nucleotidase （CD73）, endothelial cell specific molecule-1 （ESM-1）, and integrin 135 were determined by immunocytochemical （IHC） staining. Results Epimedin C could not affect the survival rate of C3H/10Tlf2 cells at 1 -30 μmol/L. Its cell cycle distribution was not significantly changed after treated by 30 μmol/L Epimedin C for 24 h. C3H/10T1/2 cells were differentiated to vascular endothelial cells by Epimedin C treatment, with significant morphological changes （whirlpool-like structure）. PCR results indicated that mRNA levels of classic endothelial mark- ers, namely CD34, Vezfl, Angl, and Ang2 were significantly increased in C3H/10Tlf2 cells after treated with Epimedin C for 5 days （P 〈0.05, P 〈0.01 ）. Protein expression levels of CD31, CD73, and ESM-1 were also positively expressed after treated with Epimedin C for 5 days, showing statistical difference when compared with those of the control group （P 〈0.01, P 〈0.05）. Conclusion Epimendin C could in- duce C3H/10T1/2 cells to differentiate into endothelioid cells.

关 键 词：朝藿定C 胚胎间充质干细胞 诱导分化 内皮样细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]