摘要
目的探讨成人骨髓间充质干细胞(bone marrow mesenchymal stem cells BMSCs)在体外缺氧环境下对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖和血管形成能力的影响及其可能机制。方法密度梯度离心法收集分离成人骨髓血MSCs并进行体外培养扩增,传代至4代进行实验,流式细胞仪鉴定MSCs表面标志。BMSCs缺氧培养0h(对照组)、12h、24h和48h后,RT-PCR检测SDF-1和VEGF基因表达,ELISA法检测细胞上清液中SDF-1和VEGF蛋白含量。HUVECs传代培养后分成三组进行实验:对照组、BMSCsCMN-HUVECs组,BMSCsCMH-HUVECs组,MTT检测三组细胞增殖能力,体外血管形成实验分析三组细胞在Matrigel上管腔样结构形成情况。结果 (1)BMSCs呈旋涡状长梭形即纤维母细胞样生长;(2)人BMSCs阳性表达CD29、CD44和CD90,而CD34、CD45和CD106为阴性;(3)BMSCs缺氧培养后SDF-1和VEGF在mRNA和蛋白水平表达均较常氧培养显著增高(P均<0.05);(4)BMSCsCMH明显提高HU-VECs增殖能力(P<0.05),显著增加HUVECs在Matrigel上形成管腔样结构的能力(P<0.05)。结论人BMSCs在缺氧环境下通过旁分泌SDF-1和VEGF提高血管内皮细胞增殖和管腔样结构形成能力,促进血管新生。
Objective To investigate the effects of BMSCs on proliferation and angiogenesis of HU- VECs and its possible mechanism in hypoxia. Methods BMSCs separated by density gradient eentrifugation were cultured, expanded and used at passage 4. Their phenotypic characterizations were indentified by flow cytometry. After BMSCs exposed to hypoxia for Oh （control）,12h,24h and 48h, intracellular levels of SDF-1 and VEGF mRNA were assayed by RT-PCR, and the secretion of SDF-1 and VEGF was quantitatively analyzed by ELISA. HUVECs were cultured, passaged and divided into 3 groups： control, BM- SCs^CMN- HUVECs, BMSCsCMH-HUVECs. Proliferation of HUVECs were detected by MTT. Tube-like structure formation in Matrigel were analyzed as the assay of angiogenesis in vitro. Results （1） Under in- verted phase-contrast microscopy, BMSCs exhibited fibroblast-like morphology; （2） BMSCs expressed CD29,CD44 and CD90, but not CD34,CD45 and CD106. （3） Both mRNA and protein levels of SDF-1 and VEGF increased significantly compared with those of the control group （P〈0.05） ; （4） BMSCs^CMH significantly increased the proliferation of HUVECs and the number of tube-like structures in Matrigel（P〈0. 05）. Conclusion Human BMSCs improve proliferation and tube-like structures formation of Vascular endothelial cells and promote angiogenesis in hypoxia, through paracrined SDF-1 and VEGF.
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2012年第1期84-88,共5页
Chinese Journal of Histochemistry and Cytochemistry
基金
国家自然科学基因(81060100)
