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

发布时间：2018-02-04 04:47

本文关键词： 缺氧基质金属蛋白酶间充质干细胞脉络膜新生血管　出处：《眼科新进展》2016年05期 　论文类型：期刊论文

【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(P0.01)。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P0.05),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P0.01),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P0.05),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P0.01),而两种缺氧模型之间差异则无统计学意义(P0.05)。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,提高其促血管形成能力,提示缺氧可能调控BMSCs表达MMPs进而影响新生血管发生发展。
[Abstract]:Objective to observe the bone marrow-derived mesenchymal stem cells of mouse bone marrow mesenchymal stem cells in physical and chemical hypoxia models. Matrix metalloproteinase 13 matrix metalloproteinase-13. The expression of MMP-13 and its effect on the formation of RF/6A lumen in choroidal and retinal endothelial cells of monkey. Methods the bone marrow of C57 BL/6J mice was harvested. BMSCs was used to culture and identify BMSCs. Three air incubators were used to simulate physical anoxia and cobalt chloride Co Cl2) to induce chemical hypoxia, and physical hypoxia for 6 h and 12 h. After 24 h and 48 h, the expression of MMP-13 was detected. The highest treatment time was selected to detect Co Cl2(0 渭 mol 路L ~ (-1). 100 渭 mol 路L ~ (1) ~ (1) (~ (2) 渭 mol 路L ~ (-1)). The expression of MMP-13 was detected after treatment with 300 渭 mol 路L ~ (-1) and 400 渭 mol 路L ~ (-1), and the expression of hypoxia inducible factor-1 伪 (HIF-1 伪) in BMSCs after hypoxia was detected. Hypoxia-inducible factor-1 伪. The expression of HIF-1 伪 and the proliferative ability of BMSCs were observed. The formation of RF/6A lumen induced by conditioned medium was observed. Results the cells were identified to accord with the characteristics of BMSCs, and the cells were exposed to physical hypoxia for 24 h. The expression of MMP-13 protein reached the peak of 3.16 卤0.24). Compared with normal oxygen group (1. 00 卤0. 12), it was increased by 3 times (P0.01U. 100 渭 mol 路L ~ (-1)) and 200 渭 mol 路L ~ (-1) (P < 0. 01) and #number0# 渭 mol 路L ~ (-1) (P < 0. 01). 1.60 卤0.09. The expression of MMP-13 in 1.64 卤0.24 was significantly higher than that in normal oxygen group (P0.05). 300 渭 mol 路L ~ (-1) and 400 渭 mol 路L ~ (-1)). The expression of MMP-13 in the CoC L1 group was the same or slightly lower than that in the normoxic group. The expression of HIF-1 伪 protein in BMSCs was significantly higher than that in normal oxygen group (1.00 卤0.23). The relative expression levels were 3.40 卤0.26 and 3.12 卤0.13 (both P 0.01), but there was no significant difference between the two hypoxia models. The proliferation ability of the cells in the physical hypoxia group (1.53 卤0.04) and the chemical hypoxia group (1.31 卤0.14) was higher than that in the normal oxygen group (1.04 卤0.10) (P 0.05). The effect of physical hypoxia on proliferation was more obvious. The total length of RF/6A lumen formation induced by CoC L1 was 5506 卤380 渭 m in chemical hypoxia group and normoxic group, respectively. 5109 卤558 渭 m and 3120 卤300 渭 m respectively. The physical hypoxia group and the chemical hypoxia group induced by CoC l2 were significantly increased compared with the normal oxygen group (P0.01). However, there was no significant difference between the two hypoxia models (P 0.05). Conclusion both CoC l2 and three air incubators can establish hypoxia models and promote the expression of MMP-13 by BMSCs. It is suggested that hypoxia may regulate the expression of MMPs in BMSCs and affect the development of neovascularization.
【作者单位】：第四军医大学西京医院眼科、全军眼科研究所;
【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)~~
【分类号】：R77
【正文快照】： 骨髓间充质干细胞(bone marrow-derived mesen-骨髓贴壁法取C57BL/6J小鼠骨髓细胞。采用脱颈chymal stem cells,BMSCs)具有取材方便、自体移植椎方法处死小鼠,体积分数75%酒精浸泡5 min,剔不排斥等优点,已成为细胞及基因治疗的首选种除肌肉和结缔组织后,取出完整股骨和胫骨。

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