| BackgroundAtherosclerosis (AS) is one of the most important reason that induced cardiovascular diseases. More and more proof show that AS is overaction inflammatory to injury of blood vessel. TNF-α(Tumor necrosis factor-α, TNF-α) is one of glycosidoprotein mainly created by mononuclear macrophage. TNF-αis also one of criticality regulatory factors in inflammatory reaction that created by various inflammatory cells and playing a important role in the development of AS. Syndecan-4 is a transmembrane heparan sulfate proteoglycan belonging to the syndecans family. Syndecan-4 is the one of a major class of heparin sulphate proteoglycans in the vasculature. Current research show that Syndecan-4 is a kind of co-receptor linking with growth factor regulates many cell biological effect, as an important part in the cell expansion, recognition, adhesion, migration, multiplication regulation at equal pace mediates inflammatory reaction. Consequently, it is fairly necessary to find a new path of atherosclerotic treatment by researching the a series of potential functional capacity of syndecan-4 in the development of AS.ObjectiveTo cultivate human umbilical vein endothelial-like Cell cells and rat vascular smooth muscle cells in vitro, and make use of MTS/PES,Western blotting to observe the proliferation of human umbilical vein endothelial-like Cell cells and rat vascular smooth muscle cells and the effection to syndecan-4 protein expression, finally to explain potential functional capacity of syndecan-4 in the development of AS at cell and molecular level.Methods1. The detection of cell's proliferation:Exp one: Human umbilical vein endothelial-like cells were passed on 96 well assay in vitro and stimulated by different concentrations of TNF-α(1, 10, 20, 100ng/ml) for 24 hours and 36hours in vitro respectively, each concentration and each time have two arraies hole, in total 16 holes. To repeat the experiment 6 times at the same condition, we get 96 data. Take the same number holes that have no TNF-αas control group, we also have 96 control data to observe if there have a change between TNF-αpost-stimulatory cell and non-irritant cell. The ratio of cell proliferation was determined by non-radioactive MTS/PMS assay.Exp two: Rat aortic vascular smooth muscle cells were passed on 96 well assay in vitro and stimulated by different concentrations of TNF-α(1, 10, 20, 100ng/ml) for 24 hours in vitro, each concentration have two arraies hole, in total 16 holes. To repeat the experiment 6 times at the same condition, we get 96 data. Take the same number holes that have no TNF-αas control group, we also have 96 control datas to observe if there have a change between TNF-αpost-stimulatory cell and non-irritant cell. The ratio of cell proliferation was determined by non-radioactive MTS/PMS assay.2. The detection of syndecan-4's expressionExp one. Human umbilical vein endothelial-like cells were cultured in vitro, and treated with different level of TNF-α(1, 10, 20, 100ng/ml) for 24 hours and 36hours respectively, then the cell were lysed and the concentration of protein was measured using Coomassie brilliant blue G-250. The expression of syndecan-4 protein in human umbilical vein endothelial-like cells was evaluated by immunoblotting technique using anti-syndecan-4 antibody. Each concentration and time repeat 4 times, we get 4 data in all, and set up TNF-αnon-irritant as control each time, in order to calculate gray scale.Exp two. Rat aortic vascular smooth muscle cells were cultured in vitro, and treated with various concentrations of TNF-α(1, 10, 20, 100ng/ml) for 24 hours, then cell were lysed and the concentration of protein was measured using Coomassie brilliant blue G-250. The expression of syndecan-4 protein in rat vascular smooth muslce cells was evaluated by the immunoblotting technique using anti-syndecan-4 antibody. Each concentration repeat 4 times, we get 4 data in all, and set up TNF-αnon-irritant as control each time, in order to calculate gray scale.Results1,Effects of TNF-αon proliferation of human umbilical vein endothelial-like CellThe ratio of human umbilical vein endothelial-like cells proliferation in 24 huors was 1.956±0.214 in the control group, and was 2.154±0.250, 2.26±0.151, 2.118±0.205 and 2.106±0.136 in TNF-αgroups corresponding to TNF-αconcentrations of 100, 20, 10, 1ng/ml respectively. It was shown that TNF-αcan significantly stimulated the proliferation of human umbilical vein endothelial-like cells when the concentration uses was above 1ng/L (F=29.522, P＜0.001). The 20ng/ml is the most significant(F=29.522, P＜0.001). The ratio of human umbilical vein endothelial-like cells proliferation in 36 hours was 1.956±0.214 in the control group, and was 2.067±0.328, 2.207±0.150, 2.052±0.126 and 2.051±0.178 in TNF-αgroups corresponding to the TNF-αconcentrations of 100, 20, 10, 1ng/ml respectively. It was shown that TNF-αcan significantly increased the ratio of proliferation of the cell when the concentration uses was above 1ng/ml (F=21.719, P＜0.001). The 20ng/ml is the most significant(F=21.719, P＜0.001). But compared with 24 hours TNF-αgroups, the ratio of human umbilical vein endothelial-like cells proliferation after 36 hours was partly decreased, respectively.2,Effects of TNF-αon expression of syndecan-4 protein in human umbilical vein endothelial-like cellsTake control group's OD as 1, the expression of syndecan-4 in human umbilical vein endothelial-like cells after 24 hours by TNF-αare as follows 2.429±0.765, 3.284±0.582, 6.780±0.658 and 4.026±0.445 in TNF-αgroups corresponding to the TNF-αconcentrations of 1, 10, 20, 100ng/ml respectively. The 20ng/ml is the most significant (F=36.846, P＜0.001), 100ng/ml is higher than 1ng/ml (F=36.846, P=0.004). The expression of syndecan-4 in human umbilical vein endothelial-like cells after 36 hours by TNF-αare as follows 1.322±0.114, 2.239±0.079, 2.573±0.660 and 2.155±0.108 in TNF-αgroups corresponding to the TNF-αconcentrations of 1, 10, 20, 100ng/L respectively, The expression of syndecan-4 is the highest in treated with 20ng/ml TNF-α(F=129.214, P＜0.001). It is suggested that TNF-αcan significantly enhanced the expression of syndecan-4 in human umbilical vein endothelial-like cells, but the expression of syndecan-4 was reduced after 36 hours by TNF-α.3. Effects of TNF-αon proliferation of rat aortic vascular smooth muscle cellsThe ratio of proliferation of the vascular smooth muscle cells were 1.001±0.064 in the control group, and 1.215±0.231, 1.566±0.060, 1.125±0.198, and 1.082±0.272 in TNF-α-treated groups corresponding to TNF-αconcentrations of 100, 20, 10 and 1ng/ml, respectively. It was shown that TNF-αsignificantly stimulated the vascular smooth muscle cells proliferation at the concentration above 1ng/ml as compared with the control group (F=132.899, P=0.044). The 20ng/ml is the most significant (F=132.899, P＜0.001).4. Effects of TNF-αon expression of syndecan-4 protein in rat aortic vascular smooth muscle cellsTake control group's OD as 1, the expression of syndecan-4 in rat aortic vascular smooth muscle cells after 24 hours by TNF-αare as follows 1.24±0.126, 1.639±0.345, 4.937±0.598 and 3.708±0.37 in TNF-αgroups corresponding to the TNF-αconcentrations of 1, 10, 20, 100ng/ml respectively. It is shown that TNF-αcan significantly enhanced the expression of syndecan-4 in rat vascular smooth muscle cells (F=77.502, P＜0.001). The 100ng/ml is higher than 1ng/ml.ConclusionsOur study suggests that TNF-αcan significantly stimulated the proliferation of human umbilical vein endothelial-like cells and rat aortic vascular smooth muscle cells, respectively. TNF-αcan also significantly enhanced expression of syndecan-4 protein in human umbilical vein endothelial-like cells and rat aortic vascular smooth muscle cells, respectively. This founding may represent an additional component of the pro-inflammatory, growth-stimulating pathways that are. activated in response to changes in vascular wall.
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