两种缺氧模型对小鼠骨髓间充质干细胞中基质金属蛋白酶表达及其对促血管形成能力的影响  

Effects of two hypoxia models on expression of matrix metalloproteinase in mesenchymal stem cells and their pro-angiogenesis action

作　　者：高凡[1] 王雨生[1] 侯慧媛[1] 

机构地区：[1]第四军医大学西京医院眼科、全军眼科研究所,陕西省西安市710032

出　　处：《眼科新进展》2016年第5期401-405,409,共6页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:81200708;81070748;81200151);第四军医大学西京医院优秀人才助推计划(2014-2016);国家重点基础研究发展计划(编号:2011CB510200)~~

摘　　要：目的观察物理和化学缺氧模型中小鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)中基质金属蛋白酶13(matrix metalloproteinase-13,MMP-13)的表达及其对猴脉络膜-视网膜内皮细胞RF/6A管腔形成能力的影响。方法取C57 BL/6J小鼠骨髓,培养鉴定BMSCs。采用三气培养箱模拟物理缺氧及氯化钴(Co Cl2)诱导化学缺氧。物理缺氧6h、12 h、24 h和48 h后检测MMP-13表达。选表达最高的处理时间,检测不同浓度Co Cl2(0μmol·L^(-1)、100μmol·L^(-1)、200μmol·L^(-1)、300μmol·L^(-1)及400μmol·L^(-1))处理后MMP-13表达。检测缺氧后BMSCs中缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达及BMSCs增殖能力,观察其条件培养基诱导的RF/6A管腔形成情况。结果细胞经鉴定符合BMSCs特征。物理缺氧24 h后,MMP-13蛋白表达量达高峰(3.16±0.24),较常氧组(1.00±0.12)增加3倍(P<0.01)。100μmol·L^(-1)和200μmol·L^(-1)CoC l2组(1.60±0.09、1.64±0.24)中MMP-13表达较常氧组(1.00±0.20)均增加(均为P<0.05),而300μmol·L^(-1)和400μmol·L^(-1)CoC l2组中MMP-13表达与常氧组相同或稍低。三气培养箱构建的和CoC l2诱导的缺氧环境下培养24 h后,BMSCs中HIF-1α蛋白表达较常氧组(1.00±0.23)明显增加,相对表达量分别为3.40±0.26和3.12±0.13(均为P<0.01),而两种缺氧模型间无明显差异。在培养24 h时,物理缺氧组(1.53±0.04)和化学缺氧组(1.31±0.14)均较常氧组(1.04±0.10)细胞增殖能力增强(均为P<0.05),且物理缺氧促增殖效果更明显。三气培养箱模拟的物理缺氧组、CoC l2诱导的化学缺氧组和常氧组RF/6A管腔形成总长度分别为(5506±380)μm、(5109±558)μm和(3120±300)μm,三气培养箱模拟的物理缺氧组和CoC l2诱导的化学缺氧组较常氧组均明显增加(均为P<0.01),而两种缺氧模型之间差异则无统计学意义(P>0.05)。结论 CoC l2和三气培养箱均可建立缺氧模型,促进BMSCs表达MMP-13,提高其促血管形成�Objective To investigate the effects of physical and chemical hypoxia on the expression of m atrix m etalloproteinase-1 3（ MMP-1 3） in bone m arrow-derived m esenchym al stem cells（ BMSCs） and the tube form ation of rhesus m acaque choroid-retinal endothelial cells RF /6 A which was induced by the condition m edia of BMSCs.Meth od s Mice BMSCs were cultured and identified. A tri-gas incubator was used for physical hypoxia. MMP-1 3 expression in BMSCs under physical hypoxia for 6 hours,1 2hours,2 4 hours and 4 8 hours was detected. Co Cl2（ 0 μmol· L^-1,100 μmol· L^-1,2 0 0 μm ol· L^-1,300 μmol·L^-1and 4 0 0 μm ol· L^-1） was used for chemical hypoxia. MMP-1 3 expression was detected at the tim e point which the expression was the highest under physical hypoxia. H ypoxia-inducible factor-1 α（ H IF-1 α） expression and BMSCs proliferation were detected in two hypoxia m odels. The pro-angiogenesis effect of condition m edia of BMSCs on RF /6 A was confirm ed by tube form ation assay. R esu lts The features of the cultured cells fit BMSCs. MMP-1 3 expression reached peak at 2 4hours under physical hypoxia（ 3. 1 6 ± 0. 2 4）,which was three tim es as that of norm oxia（ 1. 0 0 ± 0. 1 2）（ P〈 0. 0 1）. MMP-1 3 expression with 1 0 0 μm ol · L^-1or 2 0 0μmol· L^-1Co Cl2（ 1. 60 ± 0. 09,1. 64 ± 0. 24） were all higher than that of normoxia（ 1. 00 ± 0. 20）（ all P〈 0. 05）,but MMP-13 expression with 300 μmol·L^-1or 4 0 0μmol· L^-1Co Cl2 were equal to or lower than that of norm oxia. The relative H IF-1 α expression at 2 4 hours under physical and chem ical hypoxia were 3. 4 0 ± 0. 2 6 and 3. 1 2± 0. 1 3,respectively,which were all higher than that of norm oxia（ 1. 0 0 ± 0. 2 3）（ all P〈 0. 0 1）,but no difference was found between two hypoxia m odels. The proliferative activity at 2 4 hours under physical and chem ical hypoxia were 1. 5 3 ± 0. 0 4（ OD value） and 1. 3 1 ± 0. 1 4,respectively,which were all higher than that of norm oxia（ 1. 0 4± 0

关 键 词：缺氧 基质金属蛋白酶 间充质干细胞 脉络膜新生血管 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]