| Objective:To establish the model of smooth muscle cell differentiated from bone mesenchymal stem cell (BMSC-SMC) in vitro, compare the foam effects of oxidized low density lipoprotein (ox-LDL) on BMSC-SMC and VSMC, investigate the roles of BMSC- SMC in the occurrence and development of artherosclerosis(As).Methods: BMSC was isolated from the femoral bone of SD rats by adherent method, and VSMC from thoracic aorta. BMSC-SMC was differentiated from BMSC by special condition medium. The specific markers of BMSC and smooth muscle cell differentiated from bone mesenchymal stem cell (BMSC-SMC) was identified by immunofluorescence (IF) staining. After treated with 80mg/Lox-LDL for 0,12,24,48,72h and with0,20,40 and 80 mg/Lox-LDL for 72h, Oil Red O staining was adopted to observe the lipid droplet and high performance liquid chromatography(HPLC) to detect the contents of total cholesterol and cholesterol ester in BVSMC and VSMC. After treated by ox-LDL with 80mg/L for 72h, Western blotting was performed to detect the different expression of scavenger receptor CD36 in BMSC-SMC and VSMC.Results:1,The isolated BMSC present a slim-spindle appearance. Immunofluorescence (IF) staining showed that the cells expressed CD44,which was the specific markers of BMSC, but not CD34.the cells(BMSC-SMC) induced by condition medium containing b-FGF and TGF-βfor 15day present slim-spindle smooth muscle-like cells. Immuno-fluorescence (IF) staining showed that the cells expressed smooth muscleα-actin and smooth muscle Myosin heavy chain I,which were the specific markers of SMC. the cells are named smooth muscle cell differentiated from bone mesenchymal stem cell, (BMSC- SMC)。2,After treated by ox-LDL with 80mg/L for0,12,24,48,72h, the contents of total cholesterol and cholesterol ester in BMSC-SMC increased gradually with time prolonged, and the contents of total cholesterol reached 304.3±26.4μg/mg in 72h, compared with untreated cells 28.8±3.6μg/mg. which raised 9.5times(P〈0.05);the ratio of cholesterol ester to total cholesterol was 70.1% and raised 2.2times(P〈0.05),which correspond to the characteristic of foam cells.compared with that of VSMC treated with the same method. After treated by ox-LDL with 80mg/L for 72h,the contents of total cholesterol reached 215.7.3±18.9μg/mg,the ratio of cholesterol ester to total cholesterol in VSMC was 60.1%. compared with BMSC-SMC, variance is significance(P<0.05)After treated by ox-LDL with 0,20,40,80mg/L for 72h, the contents of total cholesterol in BMSC-SMC reached29.4±2.2,137.3±7.3,171.3±11.7,295.9±20.3μg/mg; the ratio of cholesterol ester to total cholesterol was 23.6%,38.4%,48.8%,68.5%,suggest increased gradually with concentration of ox-LDL , Cellular totol cholesteroland and Ester cholesterol also increased gradually.After treated by ox-LDL with 80mg/L for 72h, Oil Red O staining showed that the volume of BMSC-SMC was augmentation and a lot of lipid droplet appeared in cells, which was the typical form of foam cells. Lipid droplet in cells was significantly increased compared with that of VSMC.3,Western blotting analysis showed that the expression of CD36 was low in BSMC-MSC. After treated by ox-LDL with 80mg/L for 72h, the expression of CD36 was significantly increased,the same treated in VSMC the expression of CD36 was increased too, but the extent of increasion compared with that of VSMC is weak(P〈0.05).Conclusion 1,Compared with VSMC that isolated from Aortic tunica media, BMSC-SMC can accumulate a lot of lipid droplet and form foam cells after treated by ox-LDL, suggested that BMSC-SMC maybe participate and facilitate the occurrence and development of artherosclerosis(As)2,The accumulation of cholesterol ester and foam in BVSMC after treated by ox-LDL is relate with the up-regulation of scavenger receptor CD36 expression. |
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