不同缺氧状态下骨髓间充质干细胞表达血管内皮生长因子的差异

Difference of vascular endothelial growth factor expression in bone marrow mesenchymal stem cells under various hypoxic conditions

背景:骨髓间充质干细胞在植入心肌缺血/梗死区域后,面临缺氧缺血微环境,其如何通过旁分泌细胞因子来发挥重建侧支微循环、修复心肌的作用,是目前存在争论的焦点之一。目的:探讨骨髓间充质干细胞在不同时程缺氧环境下血管内皮生长因子的表达规律。设计、时间及地点:细胞学体外实验,于2008-03/11在南昌大学第二附属医院分子医学实验中心完成。材料:清洁级新西兰大白兔2只,由南昌大学医学院实验动物中心提供。方法:常规差速贴壁法分离培养兔骨髓间充质干细胞。细胞缺氧微环境在Anaero Pack密闭方盒中产生,盒中置入一次性缺氧催化袋GENbox anaer(可吸收O2,产生CO2),指示条监测氧的耗竭,根据缺氧培养时程分为缺氧0,6,12,24h组。主要观察指标:RT-PCR法检测血管内皮生长因子mRNA的表达,Western Blot法检测血管内皮生长因子蛋白的表达。结果:血管内皮生长因子mRNA及蛋白的表达水平均为缺氧0h组<6h组<12h组<24h组(P<0.01)。结论:缺氧24h以内的培养环境下,骨髓间充质干细胞表达血管内皮生长因子的水平呈缺氧时程依赖性增加。

BACKGROUND： Following transplantation, bone marrow mesenchymal stem cells （BMSCs） were in a microenvironment of hypoxia and ischemia in the ischemia/infarct myocardium. How to reestablish collateral microenvironment and repair myocardium by paracrine cytokine received more attention at present. OBJECTIVE： To investigate the regularity of vascular endothelial growth factor （VEGF） expression in BMSCs that under different time course of hypoxia. DESIGN, TIME AND SETTING： The in vitro cytological study was performed at the Molecular Medicine Experimental Center, Second Affiliated Hospital, Nanchang University from March to November 2008. MATERIALS： Two clean New Zealand rabbits were supplied by the Experimental Animal Center, Medical College, Nanchang University. METHODS： Rabbit BMSCs were conventionally harvested by differential adhesion method. Cell hypoxia microenvironment was established in the AnaeroPack closed box that was filled with hypoxic catalysis bag GENbox anaer （absorbing 02, producing CO2）. Instructions monitored the exhaustion of oxygen. There were 0-hour, 6-hour, 12-hour, and 24-hour hypoxia groups. MAIN OUTCOME MEASURES： VEGF mRNA expression was determined by RT-PCR. VEGF protein expression was detected by Western blot analysis. RESULTS： The ranking of VEGF mRNA and VEGF protein expression was： 0-hour hypoxia group 〈 6-hour hypoxia group 〈 12-hour hypoxia group 〈 24-hour hypoxia group （P 〈 0.01 ）. CONCLUSION： Within 24 hours of hypoxia, the VEGF expression in BMSCs is up-regulated along with the prolongation of the hypoxic culture time.