| Nowadays, tissue engineered blood vessels (TEBVs) is one of the most hot spot in life science study. But it is still the major problem limited TEBVs study, that the adhesion and vasoreactivity of seeded cells are not good enough. Human bone mesencymal stem cells (hBMSCs) are one kind of the idealest seed cells, but there is no study on multiplication and apoptosis of hBMSCs under shear stress. In this study, hBMSCs were exposed to different intensity/duration of shear stress, to elucidate the effect of shear stress on multiplication and apoptosis of hBMSCs. And this is the first proteomic application to discover the changed key proteins of hBMSCs under shear stress, which would be critical targets for enhancing function of seed cells at genetic/protein level.Aims1. To investigate the effect of different intensity/duration of shear stress on multiplication and apoptosis of hBMSCs.2. To discover the changed key proteins of hBMSCs under shear stress, which would be critical targets for enhancing function of seed cells at genetic/ protein level.Methods1. Density gradient centrifugation method was applied to isolate hBMSCs, and cultured hBMSCs were then identified by specific surface CD molecule flow cytometry. And hBMSCs were induced to differentiate into adipocytes and osteoplasts, which were assessed separately by oil red-O dyeing and Osteocalcin immunohistochemistry.2. The third generation of hBMSCs were exposed to different intensity (1dyne/cm2, 10 dyne/cm2, or 20 dyne/cm2) for different duration (2h, 6h, or 24h), and flow cytometry was applied to assess the multiplication and apoptosis of hBMSCs.3. Extracted proteins of hBMSCs, which were exposed to shear stress (at 1dyne/cm2 for 6h) or not, were separated by two-dimensional gel electrophoresis. MALDI-TOF-MS was applied to identify the varied proteins and acquire the specific information. Besides, western blot and immunofluorescence provided the selected key proteins with further proof.Results1. Flow cytometry of isolated cells demonstrated that CD29 positive, CD34 negative, and CD45 negative, which supposed to be characteristics of hBMSCs. And cultured cells could be induced into adipocytes (oil-red O positive), or osteoplasts (osteocalcin positive).2. Under shear stress of 1dyne/cm2, a high raise of multiplication (cells at S+G2 period) was observed, compared to their counterparts at 10dyne/cm2 and 20dyne/cm2. And after the duration of 6h under different intensity of shear stress, a visible increase of apoptosis and necrosis was also assessed.3. 2-DE map of hBMSCs exposed to shear stress (at 1dyne/cm2 for 6h) displayed about 760±57 spots, while its counterpart displayed 750±53 spots. 32 specific proteins were identified. Among them, 10 proteins were up-regulated, 3 proteins were down-regulated, and 19 proteins kept unchanged. Shear stress especially induced sustained increases in the expression of AnnexinA2 and GAPDH, which was also proved by western blot and immunofluorescence.Conclutions1. hBMSCs isolated from human bone marrow are cultured and multiplied well. Variety of identification confirmed that they match the characteristics of hBMSCs. After passaged and expanded twice in vitro, the quantity of cells can reach the amount of 5×107, which can meet the need of proteomic study.2. It is an original discovery that exposure to 1dyne/cm2 shear stress could improve multiplication of hBMSCs, while after 6 hour exposure to shear stress, apoptosis and necrosis both increase dramatically.3. By proteomic study, we first demonstrate that shear stress exposure (at 1 dyne/cm2 for 6h) can up-regulate significantly the expression of AnnexinA2 and GAPDH of hBMSCs, which could be the key point to improve the function of seed cells. And it suggests the critical targets for seed cells of TEBVs at protein level.
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