低氧预处理通过激活低氧诱导因子1α增强老年人骨髓间充质干细胞促血管新生能力  

Hypoxic pretreatment enhancing the angiogenesis of aged human bone marrow mesenchymal stem cells through hypoxia inducible factor-1α

作　　者：宋慧芳 谈佳音[2] 刘阳 张卫国[2] 郭蕊[3] SONG Hui-fang;TAN Jia-yin;LIU Yang;ZHANG Wei-guo;GUO Rui(Department of Hepatological Surgery of the First Hospital,Shanxi Medical University;Department of Anatomy,Shanxi Medical University;Morphology Laboratory,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第一医院肝胆外科 [2]山西医科大学人体解剖学教研室 [3]山西医科大学形态学实验室,太原030001

出　　处：《解剖学报》2022年第1期35-41,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金(81702239);山西省自然科学基金(201801D221397,201701D221252)。

摘　　要：目的探讨低氧预处理对老年人骨髓间充质干细胞(hBMSCs)促血管新生能力的影响,为改善老年患者自体干细胞移植治疗缺血性心肌损伤疗效提供支持。方法老年hBMSCs经过低氧预处理后,倒置相差显微镜下摄片观察细胞形态,流式细胞术检测细胞表面标志物,并进行成骨、成脂诱导分化检测其分化潜能。随后收集青年hBMSCs常氧培养基(norCM),老年hBMSCs常氧和低氧培养基(hypoCM),分别为norCM-young、norCM-old和hypoCM-old 3组条件培养基,未加干细胞的等体积培养基为阴性对照(control)组。各组条件培养基分别处理人脐静脉内皮细胞(HUVECs),用CCK-8法检测细胞存活率,用管状结构形成实验检测体外血管新生能力。BCA法检测各组条件培养基总蛋白含量,Western blotting技术检测细胞中及条件培养基中低氧诱导因子1α(HIF-1α)含量。结果低氧预处理对老年hBMSCs形态、表面标志物及分化潜能影响不显著(P>0.05,n≥3)。HypoCM-old组与norCM-old组相比,低氧-复氧培养条件下HUVECs的存活率显著提高(P<0.05,n=5),HUVECs细胞管状结构形成能力也显著提高(P<0.01,n=5)。HypoCM-old组总蛋白量显著高于norCM-old组(P<0.05,n=3),hypoCM-old组hBMSCs中HIF-1α表达量显著高于norCM-old组(P<0.05,n=3),同时hypoCM-old组培养基中HIF-1α的含量显著高于norCM-old组(P<0.01,n=3)。结论低氧预处理的老年hBMSCs通过旁分泌促进体外HUVECs存活和管状结构的形成,而其促进作用与HIF-1α的活化相关。Objective To investigate the effect of hypoxic pretreatment on the angiogenesis of aged human bone marrow mesenchymal stem cells(hBMSCs),so to provide experimental support for more effective autologous stem cell transplantation therapy in aged patients with ischemic myocardial injury.Methods The aged hBMSCs were cultured in a hypoxic incubator,and then the cell morphology was observed under inverted phase-contrast microscope,the surface markers were detected by flow cytometry,and the differentiation potential was detected by osteogenic and adipogenic differentiation.Subsequently,the conditioned medium(CM)of young hBMSCs under normoxic culture(norCM),the conditioned media of aged hBMSCs under normoxic and hypoxic culture(hypoCM)were collected respectively.They were named as norCM-young,norCM-old and hypoCM-old.The equal volume of medium,which was not treated with stem cells,was set as control group.Human umbilical vein endothelial cells(HUVECs)were treated with 4 conditioned media,the cell survival rate was detected by CCK-8 assay,and the tube formation experiment was used to detect the angiogenesis ability in vitro.The BCA method was used to detect the total protein concentration of the conditioned medium of each group,and the Western blotting was used to detect the expression of hypoxia inducible factor-1α(HIF-1α)in the cells and the conditioned media.Results There was not significant effect of hypoxic pretreatment on the morphology,surface markers and differentiation ability of aged hBMSCs(P>0.05.n≥3).Compared with the norCM-old group,hypoCM-old group significantly improved the survival of HUVECs under hypoxia-reoxygenation condition(P<0.05,n=5),and the tube formation ability of it(P<0.01,n=5).The total protein concentration of hypoCM-old group was significantly higher than that of norCM-old group(P<0.05,n=3).The expression of HIF-1αin hBMSCs of hypo-old group was significantly higher than that of nor-old group(P<0.05,n=3),while the content of HIF-1αin conditioned medium of hypoCM-old group was significan

关 键 词：低氧预处理 低氧诱导因子1Α 老龄 骨髓间充质干细胞 血管新生 免疫印迹法 人 

分 类 号：R322[医药卫生—人体解剖和组织胚胎学]