缺氧预处理脐带间充质干细胞诱导分化为内皮样细胞

Umbilical cord mesenchymal stem cells undergoing hypoxic preconditioning can differentiate into vascular endothelial-like cells

背景:糖尿病下肢血管病变发病率的升高,使如何通过改善下肢血管及增加新生血管生成成为关注的焦点,临床上已用人脐带间充质干细胞局部肌肉注射进行治疗,但是具体的治疗效果及机制尚不明确。目的:探讨缺氧预处理及氯化钴培养液对脐带间充质干细胞诱导分化为内皮样细胞的影响。方法:分离、培养脐带间充质干细胞,经不同浓度氯化钴模拟体内病理状态下的缺氧,通过ELISA检测细胞上清中碱性成纤维生长因子和血管内皮生长因子基因水平表达、MTT检测细胞增殖,选取最适氯化钴浓度,染色体进行氯化钴诱导前后安全性检测,用含10μg/L血管内皮生长因子、10μg/L的碱性成纤维细胞生长因子及体积分数10%胎牛血清的DMEM-LG/F12培养液向内皮样细胞方向诱导分化,鉴定诱导前及诱导后内皮样细胞表型CD31、假性血友病因子,通过三维血管形成模型的观察进行诱导前后脐带间充质干细血管形成能力检测。结果与结论:分离的脐带间充质干细经流式鉴定高表达脐带间充质干细相关表面标志。经含不同浓度氯化钴的培养液处理后,细胞增殖与作用时间呈正相关。根据碱性成纤维生长因子和血管内皮生长因子基因水平的表达,验证得出氯化钴浓度为200μmol/L为最适浓度。染色体检测显示氯化钴干预后的安全性可靠。诱导后CD31及假性血友病因子强阳性表达,三维血管成形观察显示脐带间充质干细诱导后可形成直径大小不等的管腔样结构,证实脐带间充质干细可诱导为内皮样细胞,且具有成血管的能力。

BACKGROUND： Incidence of diabetic lower extremity vascular disease is increasing, so how to improve blood vessels of the lower limbs and increase angiogenesis becomes the research focus. Umbilical cord mesenchymal stem ceils have been employed clinically via local intramuscular injection, but the specific therapeutic effect and mechanism are not clear. OBJECTIVE： To investigate the effects of hypoxic preconditioning and cobalt chloride medium on the differentiation of umbilical cord mesenchymal stem ceils into vascular endothelial-like cells in vitro. METHODS： Umbilical cord mesenchymal stem cells were isolated and cultured, and then treated with different concentrations of cobalt chloride. Enzyme linked immunosorbent assay was used to detect levels of basic fibroblast growth factor and vascular endothelial growth factor gene in cell supernatants, and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide to detect cell proliferation. The safety of umbilical cord mesenchymal stem cells before and after cobalt chloride induction was detected using chromosome. The umbilical cord mesenchymal stem cells were cultured in Dulbecco＇s modified Eagle＇s medium/Ham＇s nutrient mixture F-12 containing 10 tJg/L vascular endothelial growth factor, 10 pg/L basic fibroblast growth factor and 10% fetal bovine serum, which were induced to differentiate into vascular endothelial-like cells. Endothelial-like cell phenotype CD31 and von Willebrand factor were identified before and after induction, through the observation of three-dimensional model of angiogenesis, the angiogenic capacity of umbilical cord mesenchyma stem cells was determined.RESULTS AND CONCLUSION： Umbilical cord mesenchymal stem cells strongly expressed the surface marks. After the cobalt chloride induction, the proliferation of umbilical cord mesenchymal stem cells was positivelycorrelated with the time of induction. Based on the levels of vascular endothelial growth factor and basic fibroblast growth factor, the optimal concentration of cobalt chloride was 200 pmol/L. Chromosome detection showed the stability of cells after cobalt chloride induction. After induction, CD31 and yon Willebrand factor were strongly expressed. Three-dimensional observation showed umbilical cord mesenchymal stem cells could be induced to form the lumen-like structure with different diameter sizes, and umbilical cord mesenchymal stem cells could be induced to differentiate into endothelial-like cells, and have a angiogenic capacity.