Effect of oxidative low-density lipoprotein on the proliferation of bone marrow stem cell- derived smooth muscle cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish the model of bone mesenchymal stem cell-derived smooth muscle cells (BMSC-SMCs) and investigate the role of BMSC-SMCs in the development and progression of artherosclerosis.</p><p><b>METHODS</b>BMSCs were isolated from the femoral bone of SD rats by adherent tissue culture method, and vascular smooth muscle cells (VSMCs) were obtained from the thoracic aorta. The differentiation of BMSCs into BMSC-SMCs was induced in the conditioned medium. The specific markers of BMSCs and BMSC-SMCs were identified by immunofluorescence (IF) staining. After treatment with 80 mg/L oxidative low-density lipoprotein (ox-LDL) for 72 h, the growth characteristics of BMSC-SMCs and VSMCs were observed. Flow cytometry was applied to analyze the cell cycle of BMSC-SMCs and VSMCs.</p><p><b>RESULTS</b>BMCS-SMCs transformed into foam cells after treatment with ox-LDL, which was more obvious in comparison with VSMCs. The growth curve of BMSC-SMCs and VSMCs presented with an S-shape pattern with the cell doubling time of 20 and 32 h, which was reduced to 15 and 28 h after treatment with 80 mg/L ox-LDL, respectively. Flow cytometry showed that exposure to 80 mg/L ox-LDL significantly increased G(0)/G(1) and decreased S and G(2)/M phase cells in both BMSC-SMCs (P<0.01, n=3) and VSMCs (P<0.05, n=3) in comparison with the control cells.</p><p><b>CONCLUSION</b>BMSC-SMC might be involved in the formation of fatty core and accelerate the development of atherosclerosis.</p>

Mechanisms of foam cell formation in smooth muscle cell derived from bone marrow stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a model of smooth muscle cells differentiated from bone mesenchymal stem cells (BMSC-SMCs) in vitro and explore the relationship between scavenger receptors A (SR-A) and caveolin-1.</p><p><b>METHODS</b>BMSCs were isolated from the femoral bone of SD rats by adherent culture. After treatment of the BMSC-SMCs with 80 mg/L ox-LDL for 72 h, Western blotting was performed to detect the expression of scavenger receptor SR-A, cell cholesterol transport protein ATP-binding cassette transporter Al (ABCA1) and caveolin-1.</p><p><b>RESULTS</b>BMCS-SMCs became foam cells after treatment with ox-LDL. BMSC-SMC gave rise to more foam cell formation than VSMCs did. Western blotting showed that treatment with 80 mg/L ox-LDL for 72 h resulted in significantly increased expression of SR-A and significantly decreased expressions of ABCA1 and caveolin-1.</p><p><b>CONCLUSIONS</b>Treatment of BMCS-SMCs with ox-LDL results in cholesterol ester accumulation in the cells to result in foam cells, the mechanism of which involves up-regulation of scavenger receptor SR-A expression and down-regulation of the reverse cholesterol transport protein ABCA1 and caveolin-1 expression.</p>

Preparation and identification of monoclonal antibody against human thrombomodulin / 生理学报

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.