血管内皮细胞氧化应激损伤对骨髓间质干细胞趋化作用的体外观察

目的 探讨氧化应激损伤血管内皮细胞定向趋化人骨髓间质干细胞(hMSC)的可能性.方法 体外分离和培养hMSC,分别加入不同条件培养基进行成脂肪细胞、成骨细胞和成血管内皮细胞诱导分化;以免疫组织化学染色和流式细胞仪检测hMSC抗原表达.利用人脐静脉内皮细胞系ECV-304细胞建立氧化应激损伤趋化hMSC的细胞模型:在Transwell培养板下腔接种5×105 ECV-304细胞并用3%H2O2(终浓度为0.01 ml/ml)处理1 h后,上腔内接种1×105 hMSC,为损伤细胞+hMSC组;同时设未损伤细胞+hMSC组(Transwell板下腔接种未经H2O2处理的ECV-304细胞,上腔内接种hMSC)和单纯hMSC组(Transwell板下腔不接种ECV-304细胞)作为对照.培养12 h后苏木精染色,倒置相差显微镜下计数各组细胞Transwell上腔迁移的hMSC数.另以酶联免疫吸附试验检测H2O2处理1 h的ECV-304细胞(H2O2处理组)和未予H2O2处理ECV-304细胞(对照组)培养上清液中单核细胞趋化蛋白1(MCP-1)和血管细胞黏附分子1(VCAM-1)浓度.结果 hMSC经加入不同条件培养基诱导后可以分化为脂肪细胞、成骨细胞和血管内皮细胞.免疫组织化学染色和流式细胞仪检测显示hMSC CD29、CD44、CD90和CD106抗原呈阳性表达,CD31、CD34、CD45和CD49b抗原呈阴性表达.损伤细胞+hMSC组发生迁移的hMSC数为(8.00±0.22)个/高倍视野,明显高于未损伤细胞+hMSC组[(0.20±0.05)个/高倍视野,P<0.01]和单纯hMSC组[(0.00±0.00)个/高倍视野,P<0.01].H2O2处理组细胞培养上清液中MCP-1和VCAM-1浓度分别为(69.2±3.5)、(114.0±7.5)ng/ml,均明显高于对照组[(62.5±3.6)ng/ml,P<0.05;(97.2±5.0)ng/ml,P<0.01].结论 血管内皮细胞氧化应激损伤可趋化hMSC定向迁移到损伤血管,其机制可能与氧化应激损伤导致的趋化因子MCP-1和VCAM-1浓度升高有关.

Migratory and chemoattractant responses of mesenchymal stem cells to oxidative stress injury of endothelial cell in vitro

Objective To investigate the possibilities of human mesenchymal stem cells (hMSCs) migrating toward the oxidative stress injuries of endothelial cells. Methods hMSCs were isolated and cultured from human marrow in vitro and the multipotential differentiation of P3 hMSCs identified by specific medium induced to differentiate into osteoblasts, adipocytes and endothelial cells. And the marker antigen of P3 hMSCs was detected by flow cytometry (FCM) and immunohistochemistry. Then a cellular model of hMSCs migrating toward the oxidative stress injuries of endothelial cells was created, i.e. 1×105 hMSCs were seeded in Transwell upper chamber, indirectly co-cultured with ECV-304 cells seeded in the Transwell inferior chamber and was injured by adding 3% H2O2 into the medium (final concentration of 0.01 ml/ml) for 1 h, the injured ECV-304 cells + hMSCs group (n=8), as experimental group, and in the mean time, hMSCs indirectly co-cultured with uninjured ECV-304 cells in Transwell chamber, ECV-304 cells + hMSCs group (n=8) and hMSCs monoculture group (n =8) in Transwell chamber as control groups. After a 12-h culture in all groups, the migrating hMSCs in Transwell upper chamber were HE-stained and counted under an inverted phase contrast microscope. To understand the reason why hMSCs migrated to the oxidative stress injured endothelial cells, ELISA was employed to measure the concentration of monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) of cellular supernatant in ECV-304 cells with H2O2 1-h treating group (H2O2 treatment group) or without H2O2 treating group (control group). Results The multipotential differentiation experiment demonstrated that the cultured P3 hMSCs can be induced to differentiate in vitro into osteoblasts, adipocytes and endothelial cells. And the expressions of CD29, CD44, CD90 and CD106 were positive in hMSCs while CD31, CD34, CD45 and CD49b negative by using FCM and immunohistochemistry. And the effects of hMSCs upon in vitro movement toward oxidative stress injuries of ECV-304 cells were averaged (8.00±0.22) cells/HP in the injured ECV-304 cells +hMSCs group, significantly higher than those of the ECV-304 cells + hMSCs group[(0.20±0.05) cells/ HP, P<0.01] and the hMSCs monoculture group [(0.00±0.00) cells/HP, P<0.01). The concentrations of MCP-1 and VCAM-1 in cellular supernatant of the H2O2 treatment group were significantly higher than those of the control group [(69.2±3.5) ng/ml vs (62.5±3.6) ng/ml, P<0.05; (114.0±7.5) ng/ml vs (97.2±5.0) ng/ml, P<0.01]. Conclusions The oxidative stress injuries of endothelial cells chemoattracted the hMSCs toward the injured site and its mechanism may be correlated with releasing a certain concentration of chemoattractant factor to result in the elevations of MCP-1 and VCAM-1 by oxidative stress injury.