| Introduction: Mesenchymal stem cells (MSCs) are a type of non-hematopoietic stem cells which reside in the bone marrow. According to recent researches, these cells possess multipotentiality, usually restricted to the tissues in which they reside, and a capacity of inducing immune tolerance and immune suppression. Moreover, MSCs are relatively easy to isolate and replicate extensively in vitro. Transplantation of MSCs have already been used in treatment of ischemic and injury diseases, while it was found in clinical application that few MSCs rested and differentiated in local tissue and were inadequate to meet the need of tissue repair. Thus, how to enhance the migration and settlement of MSCs to the damaged tissue becomes a critical problem. At present, replacement therapies of MSCs are carried out basically in two ways-by injecting MSCs into local vasculature and into damaged tissue, and the former is more prevalent. MSCs migrate across the capillary endothelium, as the first step of a tissue regenerating process. Previous findings have characterized the capability of MSCs of homing to ischemic or injured tissue, in the process of which adhesion molecules and chemotatic factors were supposed to play a central part. In experiments performed here, we studied the expression of adhesion molecules of rat MSCs and the mechanism of their adhesion to vascular endothelial cells which had not been clearly understood.Objective: To study the influence of inflammatory cytokine TNF-αon the expression of adhesion molecules of rat MSCs, to study the optimal stimulation of MSCs with inflammatory factors in inducing adhesion molecule expression, and to discuss a feasible way of MSC transplantation.Methods: The primary MSCs were isolated from rat bone marrow and cultured in vitro and cells at passage and later were used in the following experiments: (1)MSCs were exposed to TNF-αof different concentration for 24h (2 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml respectively); analyses of expression of VCAM-1, ICAM-1, L-selectin, VLA-4 on cell surface were performed using flow cytometry . (2) Aortic endothelial cells were isolated from rat and cultured with the TNF-αstimulated MSCs for 12h and the adhesion rate was calculated by observation; in comparison to the result obtained from flow cytometry in the first step, an optimal concentration of TNF-αwas yielded. (3) Changes in the specific antigen of MSCs was identified by flow cytometry. (4) Ischemia injury models were made in the hindlimbs of the rats; 1, 1-Dioctadecyl-3, 3, 3, 3-tetramethylindocarbocyanine Iodade (DiI) labeled MSCs, previously treated with TNF-α, were injected intravenously 24h after operation; the animals were executed another 24h later, and muscle samples in the ischemic area were obtained and made into frozen section; the number of MSCs was counted under a fluorescent microscope.Results: (1) In physiological state, rat MSCs express high level of ICAM-1, while the expression of VCAM-1, L-selectin and VLA-4 was relatively low; Stimulated with TNF-α, the expression of VCAM-1 increased concentration-dependently, while the expression of L-selectin, ICAM-1 and VLA-4 did not change markedly; (2) Exposed to TNF-αof 10 ng/ml and 20 ng/ml, the adhesion ability of MSCs to the endothelial cells obviously increased; (3) The specific antigen of MSCs was unchanged, when the stem cells pretreated with 10 ng/ml TNF-α; (4) More MSCs rested in damaged tissue of the rat hindlimbs, compared to the control groups, when the stem cells pretreated with 10 ng/ml TNF-α.Conclusion: (1) TNF-αcan increase the expression of VCAM-1 and can not effect the expression of ICAM-1, L-selectin and VLA-4 on rat MSCs. (2) TNF-αcan promote MSCs adhere to endothelial cells, promote MSCs migrate to the damaged tissue. The mechanism may is concerned with increase of VCAM-1. (3) Low dose TNF-αhas no distinct influence to the character of MSCs. (4) The intima tunica of rat aorta was exposed outside and cultured can obtain pure endothelial cells. (5) Thrombus microparticles can induce ideal ischemia injury models in the hindlimbs of the rats;... |
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