| Objective: Through establishing in vitro vascular calcification model of bovine aortic smooth muscle cell (BASMC) induced by p-glycerophosphate, to observe the expression of non-collagenous bone associated proteins (NCPs) and the status of cell proliferation, apoptosis or death during calcification, as well as to discuss their possible mechanisms; Furthermore, to determine the effect of Fluvastatin on vascular calcification in vitro and its possible mechanism. Methods:1. Primary BASMCs were obtained by the explant method. The cells up to passage 8 were incubated in calcification medium (DMEM containing 15% fortified bovine calf serum, 10 mmol/L p-glycerophosphate) for 10 days to induce calcification. Calcification was measured in cultures by staining with alizarin red s and Von Kossa's method or by quantification of calcium deposition. Meanwhile, alkaline phosphatase (ALP) activity of cell layer was measured by ALP assay kit, and osteocalcin (OC) in culture supernatant was quantified by radioimunoassay.2. Thiazolyl blue (MTT) colorimetry assay and Trypan blue exclusion assay were adopted to evaluate BASMCs proliferation cultured in serial concentrations of P-glycerophosphate. Flow cytometry was used to detect apoptosis and cell cycle during calcification.3. During vascular calcification in vitro, fluvastatin at concentrations of 10-6,10-7 or 10-8 mol/L was added to calcification medium respectively, the effect of fluvastatin on calcification was assessed by quantification of calcium deposition in cell layer, ALP activity, OC in culture supernatant. RT-PCR and Western blot analysis were used to observe the expression ofosteopontin (OPN) and osteonectin (ON) in control cultured SMCs, calcification group as well as fluvastatin-treated group. Results:1. After BASMCs were cultured in calcification medium for 10 days, extensive calcium deposition were seen in cell layer by inverted optical microscope and transmission electron microscopy, which represented 28.44 times compared to the control and increased by 293 times compared to the beginning of the culture (P<0.01). Von Kossa and alizarin red s calcification staining were positive. As a result, in vitro vascular calcification model was successfully established.2. As -glycerophosphate increased calcium deposition in a time-dependent manner, ALP activity of cell layer or OC concentration in culture supernatant was also increased over time, and all those were higher than uncalcified controls at various time points (P<0.01). A higher level of the OPN mRNA and protein expression were detected by RT-PCR and Western blot analysis in calcification group, while a lower level of ON expression compared with uncalcified control group.3. BASMC proliferation was inhibited by -glycerophosphate in a dose/time-dependent manner. The optical density (OD) value and the average viable cell number in calcification group had been significantly increased since the 4th day compared with uncalcified control (P<0.01).4. In calcification group, apoptotic indices took on an increasing tendency over time, and were also correlated with calcium deposition of cell layer (r =0.9, P<0.05).5. In calcification group, cell cycle analysis revealed that the cell percentage for G1 phase was slightly increased, for S phase was decreased, while for G2-M phase waschanged little, with a decreased proliferative index (PrI).6. Fluvastatin treatment resulted in significantly decreased calcium deposition of the cultures in a dose-dependent manner, with the most obvious result at concentration of 10-6mol/L to decrease calcium deposition by 27% (P<0.05).7. In fluvastatin-treated group, ALP activity of cell layer did not fluctuate significantly, but OC concentration in culture supernatant was obviously decreased (P<0.05); meanwhile, RT-PCR or Western blot analysis demonstrated decrease expression of OPN and increase expression of ON respectively at gene...
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