[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 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).

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.