Schlegel Franziska, Appler Marco, Halling Michelle, Smit Francis Edwin, Mohr Friedrich-Wilhelm, Dhein Stefan, Dohmen Pascal Maria
Department of Cardiac Surgery, Leipzig Heart Center, University of Leipzig, Leipzig, Germany.
Department of Cardiac Surgery, Heart Center Rostock, University of Rostock, Rostock, Germany.
Med Sci Monit Basic Res. 2017 Aug 17;23:285-294. doi: 10.12659/msmbr.905081.
BACKGROUND The aims of this study were to compare the morphological, biochemical, and functional properties of reprogrammed bone marrow stem cell (BMSC)-derived arterial endothelial cells (AECs) and venous endothelial cells (VECs), following adenosine triphosphate (ATP)-stimulation in a mini pig animal model. MATERIAL AND METHODS Bone marrow aspiration was performed in six adult mini pigs. Harvested mononuclear cells were isolated, cultured, and treated with vascular endothelial growth factor (VEGF) (16 µg/ml). Transformed cells were characterized using immunofluorescence staining for CD31 and von Willebrandt factor (vWF) and expression of endothelial nitric oxide synthase (eNOS). Cell release of nitric oxide (cNO) was measured using spectrophotometry. Matrigel assays were used to investigate angiogenesis in transformed BMSCs. RESULTS Reprogrammed BMSCs in culture showed a typical cobblestone-like pattern of growth. Immunofluorescence staining was positive for CD31 and vWF expression. Expression of eNOS, using immunofluorescence staining and Western blot, showed no difference between the reprogrammed BMSCs and VECs. Spectrophotometric examination following stimulation with 10mmol/l ATP, showed comparable cNO release for reprogrammed BMSCs (10.87±1.76 pmol/106 cells/min) and VECs (13.23±2.16 pmol/10^6 cells/min), but reduced cNO release for AECS (3.44±0.75 pmol/10^6 cells/min). Matrigel assay for angiogenesis showed vascular tube formation of differentiated BMSC endothelial cells (grade 3.25). BMSCs cultured without VEGF did not demonstrate vascular tube formation. CONCLUSIONS The findings of this study showed that eNOS expression and release of NO could be used to show that BMSCs can be reprogrammed to functional VECs and AECs.
背景 本研究的目的是在小型猪动物模型中，比较经三磷酸腺苷（ATP）刺激后，重编程骨髓干细胞（BMSC）来源的动脉内皮细胞（AEC）和静脉内皮细胞（VEC）的形态学、生物化学和功能特性。
材料与方法 对6只成年小型猪进行骨髓穿刺。分离收获的单核细胞，进行培养并用血管内皮生长因子（VEGF）（16μg/ml）处理。使用针对CD31和血管性血友病因子（vWF）的免疫荧光染色以及内皮型一氧化氮合酶（eNOS）的表达来鉴定转化细胞。使用分光光度法测量细胞一氧化氮（cNO）释放。使用基质胶试验研究转化的BMSC中的血管生成。
结果 培养的重编程BMSC呈现典型的鹅卵石样生长模式。免疫荧光染色显示CD31和vWF表达呈阳性。使用免疫荧光染色和蛋白质印迹法检测，重编程的BMSC和VEC之间eNOS的表达无差异。用10mmol/l ATP刺激后的分光光度检查显示，重编程的BMSC（10.87±1.76 pmol/10^6细胞/分钟）和VEC（13.23±2.16 pmol/10^6细胞/分钟）的cNO释放相当，但AEC的cNO释放减少（3.44±0.75 pmol/10^6细胞/分钟）。血管生成的基质胶试验显示分化的BMSC内皮细胞形成血管管（3.25级）。未用VEGF培养的BMSC未显示血管管形成。
结论 本研究结果表明，eNOS表达和NO释放可用于表明BMSC可重编程为功能性VEC和AEC。
