[1]高凡,王雨生,侯慧媛.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[J].眼科新进展,2016,36(5):401-405.[doi:10.13389/j.cnki.rao.2016.0108]
　GAO Fan,WANG Yu-Sheng,HOU Hui-Yuan.Effects of two hypoxia models on expression of matrix metalloproteinase in mesenchymal stem cells and their pro-angiogenesis action[J].Recent Advances in Ophthalmology,2016,36(5):401-405.[doi:10.13389/j.cnki.rao.2016.0108]
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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
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
36卷
期数:
2016年5期
页码:
401-405
栏目:
实验研究
出版日期:
2016-05-05

文章信息/Info

Title:
Effects of two hypoxia models on expression of matrix metalloproteinase in mesenchymal stem cells and their pro-angiogenesis action
作者:
高凡; 王雨生; 侯慧媛
710032　陕西省西安市，第四军医大学西京医院眼科、全军眼科研究所
Author(s):
GAO Fan ; WANG Yu-Sheng ;  HOU Hui-Yuan
Department of Ophthalmology , Eye Institute of Chinese PLA , Xijing Hospital , the Forth Military Medical University, Xi ’an 710032 , Shaanxi Province . China
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
Keywords:
hypoxia;  matrix metalloproteinases ;  mesenchymal stem cells ;  choroidal neovascularization
DOI:
10.13389/j.cnki.rao.2016.0108
文献标志码:
A
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（ｂｏｎｅｍａｒｒｏｗ-ｄｅｒｉｖｅｄｍｅｓｅｎｃｈｙｍａｌｓｔｅｍｃｅｌｌｓ，ＢＭＳＣｓ）中基质金属蛋白酶１３（ｍａｔｒｉｘｍｅｔａｌｌｏｐｒｏｔｅｉｎａｓｅ-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Ｃ５７ＢＬ／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（ＣｏＣｌ２）诱导化学缺氧。物理缺氧６ｈ、１２ｈ、２４ｈ和４８ｈ后检测ＭＭＰ-１３表达。选表达最高的处理时间，检测不同浓度ＣｏＣｌ２（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-ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ-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２４ｈ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－１ＣｏＣｌ２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－１ＣｏＣｌ２组中ＭＭＰ-１３表达与常氧组相同或稍低。三气培养箱构建的和ＣｏＣｌ２诱导的缺氧环境下培养２４ｈ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２４ｈ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、ＣｏＣｌ２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ＣｏＣｌ２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　ＣｏＣｌ２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 investigate the effects of physical and chemical hypoxia on the expression of matrix metalloproteinase-13 ( MMP-13 ) in bone marrow-derived mesenchymal stem cells ( BMSCs) and the tube formation of thesus macaque choroidretinal endothelial cells RF/6A which was induced by the condition media of BMSCs. Methods Mice BMSCs were cultured and identified. A tri-gas incubator was used for physical hypoxia. MMP-13 expression in BMSCs under physical hypoxia for 6 hours . 12 hours.24 hours and 48 hours was detected. CoC12 (0 Vmol . L-l , 100 ymol . L-1 , 200 ymol . 1’1 ,300 Vmol . L-l and 400 ymol . L-] ) was used for chemical hypoxia. MMP-13 expression was detected at the time point which the expression was the highest under physical hypoxia. Hypoxia-inducible factor-la ( HIF-Ie/) expression and BMSCs proliferation were detected in two hypoxia models. The pro-angiogenesis effect of condition media of BMSCs on RF/6A was confirmed by tube formation assay. Results The features of the cultured cells fit BMSCs. MMP-13 expression reached peak at 24 hours under physical hypoxia ( 3. 16 + 0. 24 ) . which was three times as that of normoxia ( 1. 00 + 0. 12)(P < 0. 01). MMP-13 expression with 100 Vmol . L-1 0r 200 Vmol . L-l CoC12 ( 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 ymol . L-] or 400 Vmol . L-l CoC12 were equal to or lower than that of normoxia. The relative HIF-Ie/ expression at 24 hours under physical and chemical hypoxia were 3. 40 + 0. 26 and 3. 12 + 0. 13 , respectively, which were all higher than that of normoxia ( 1. 00 + 0. 23 ) ( all P < 0. 01 ) , but no difference was found between two hypoxia models. The proliferative activity at 24 hours under physical and chemical hypoxia were l. 53 + 0. 04 ( OD value ) and l. 31 +0. 14 , respectively , which were all higher than that of normoxia ( 1. 04 + 0. 10) (P < 0. 05 ) . and the physical hypoxia was more than the chemical hypoxia. The total tube formation length of RF/6A under physical and chemical hypoxia were ( 5506 + 380 ) ym and ( 5 109 + 558 ) Vm , respectively , which were all higher than that of normoxia ( 3120 + 300) ym ( all P < 0. 01) ,but no difference was found between two hypoxia models (P > 0. 05 ) . Conclusion Both tri-gas incubator and CoC12 can successfully induce hypoxia of BMSCs and up-regulate the expression of MMP-13 in BMSCs to promote the angiogenesis. These results further suggest that hypoxia affects the development of angiogenesis via regulating MMPs expression in BMSCs.

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备注/Memo

备注/Memo:
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）
更新日期/Last Update: 2016-05-04