| Objective: Through investigating the effects on the abilities of anti-thrombus, synthesis of vasoactive substances and expression of adhesion molecules of the endothelial cells induced from rat bone marrow mesenchymal stem cells (MSCs) after flow loading. And approaching possibility to construct seeded cells which can well satisfied with the needs for ischemic heart disease treatment and cardiovascular tissue engineering by connecting flow shear stress and the process of endothelial differentiation of MSCs.Methods: (1). S-D rats bone marrow MSCs could be separated and purified in vitro by the Percoll density gradient separation,. Morphology characteristics, cell surface markers and the potency of multi-directional differentiation were applied to identify the cells. The purified cells were induced to endothelial linage in special media contain VEGF and bFGF for 2 weeks, the changes of cell morphology were observed under an inverted phase contrast microscope, the expression of CD31 was measured by immunofluorescence strain(IF), immunohisto- chemistry (IH) strain were used to investigate the factorâ…§related antigen, a transmission electron microscope (TEM) was applied to observe the cells ultramicro-structure.. (2) the purified MSCs were divided into control group, 5 dyn/cm~2 group and 5 dyn/cm~2 group according the intensity of shear stress loading. As soon as the 3 hours stress loading finished , all groups were induced to endothelial linage for 14 days, the mRNA and protein expression levels of t-PA, eNOS, ET-1, ICAM-1, VCAM-1 were measured by real-time RT-PCR and flow cytometry. The shear loading groups cells were cultured for more 3 days and then passaged to next generation, and all mentioned factors were measured again. (3). At the same time, the amount of NO synthesis, total anti-oxidation competence(T-AOC), and the mNRA expression level of gp91phox, a subunit of NADPH oxidase, were all detected.Results: (1). After induced for 2 weeks ,the cells acquired endothelial cell morphology, arrangement and ultramicro structure, and expressed the endothelial special antigen-CD31 andâ…§factor. (2). after exposing to physiological shear stress, the mRNA expression levels of t-PA, eNOS, ET-1, ICAM-1, VCAM-1 of P1 group had a obviously increase in an intensity depend manner. When the cells were passaged ,the mRNA expression levels of ICAM-1 kept on increasing,and that of the rest decreased .among them , mRNA expression of eNOS and ET-1 decreased significantly. (3). after exposing to physiological shear stress, the change of the protein expression levels of eNOS,ET-1,ICAM-1,VCAM-1 of P1 group differed with shear stress intensity, the expression levels of eNOS decreased in 5 dyn/cm~2 and increased in 10 dyn/cm~2. the other factors level increased regardless of shear intensity. When cells passaged to next generation, eNOS remained lower in 5 dyn/cm~2 and decreased to control level in 10dyn/cm~2. (4). after exposing to physiological shear stress for 3 hours, the yield of NO and T-AOC of endothelial cells induced from MSCs increased, and the mRNA level of gp91phox decreased in an intensity-dependent manner.Conclusion: (1). Adult SD rat bone marrow MSCs can be induced to endothelial cells in vitro,it would be an ideal seeded cell source for cardiovascular disease treatment and cardiovascular tissue engineering. (2). Physiological fluid shear stress could refined regulate the anti-thrombus ability, the anti-oxidation ability, the formation of vasoactive substances and the expression levels of cell adhesion molecules. The effects and intensity of regulation differed with different factors, which may due to different factors reacted to shear stress in respectively special molecule mechanism. (3). It would be a efficient method for preconditioning the seeded cells by exposing physiological fluid shear stress to MSCs, it could make the cells acquire strong proliferation and differentiation ability, superordinary anti-thrombokinesis, anti-inflammation and anti atherosclerotic character, and it would helpful to the development of IHD treatment and cardiovascular tissue engineering. |
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