Zhao Zhi-Li, Yang Rong-Ya, Gu Ting-Min, Wang Chi, Sui Zhi-Fu, Chang Dong-Qing
Aesthetic and Plastic Surgery Center, Dermatology Diagnosis and Treatment Center of PLA, General Hospital of Beijing Military Command, Beijing 100700, China.
Zhonghua Yi Xue Za Zhi. 2009 Jun 9;89(22):1577-81.
To investigate the possibilities of human mesenchymal stem cells (hMSCs) migrating toward the oxidative stress injuries of endothelial cells.
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 x 10(5) 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).
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].
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.
探讨人间充质干细胞（hMSCs）向氧化应激损伤的内皮细胞迁移的可能性。
从人骨髓中体外分离培养hMSCs，采用特定培养基诱导P3代hMSCs分化为成骨细胞、脂肪细胞和内皮细胞，鉴定其多向分化能力。通过流式细胞术（FCM）和免疫组织化学检测P3代hMSCs的标志物抗原。然后建立hMSCs向氧化应激损伤的内皮细胞迁移的细胞模型，即将1×10⁵个hMSCs接种于Transwell上室，与接种于Transwell下室的ECV-304细胞间接共培养，并在培养基中加入3% H₂O₂（终浓度0.01 ml/ml）损伤1 h，损伤的ECV-304细胞+hMSCs组（n = 8）作为实验组；同时，将hMSCs与未损伤的ECV-304细胞在Transwell小室中间接共培养，ECV-304细胞+hMSCs组（n = 8）和hMSCs单培养组（n = 8）在Transwell小室中作为对照组。各组培养12 h后，对Transwell上室中迁移的hMSCs进行苏木精-伊红（HE）染色，并在倒置相差显微镜下计数。为了解hMSCs迁移至氧化应激损伤内皮细胞的原因，采用酶联免疫吸附测定（ELISA）法检测H₂O₂处理1 h的ECV-304细胞组（H₂O₂处理组）或未用H₂O₂处理组（对照组）细胞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）的浓度。
多向分化实验表明，培养的P3代hMSCs可在体外诱导分化为成骨细胞、脂肪细胞和内皮细胞。FCM和免疫组织化学检测显示，hMSCs中CD29、CD44、CD90和CD106表达阳性，而CD31、CD34、CD45和CD49b表达阴性。损伤的ECV-3０4细胞+hMSCs组hMSCs向氧化应激损伤的ECV-304细胞的体外迁移作用平均为（8.00±0.22）个细胞/高倍视野（HP），显著高于ECV-304细胞+hMSCs组[（0.20±0.05）个细胞/HP，P<0.01]和hMSCs单培养组[（0.00±0.00）个细胞/HP，P<0.01]。H₂O₂处理组细胞上清液中MCP-1和VCAM-1的浓度显著高于对照组[（69.2±3.5）ng/ml比（62.5±3.6）ng/ml，P<0.05；（114.0±7.5）ng/ml比（97.2±5.0）ng/ml，P<0.01]。
内皮细胞的氧化应激损伤可趋化hMSCs向损伤部位迁移，其机制可能与氧化应激损伤释放一定浓度的趋化因子导致MCP-1和VCAM-1升高有关。
