Inhibitory Effects Of XPD On HUVSMC Proliferation Induced By PDGF-BB And Its Signal Mechanism

OBJECTIVE:To investigate the effects of XPD on vascular smooth muscle cell(VSMC)proliferation induced by platelet-derived growth factors-BB(PDGF-BB), and the role of Glycogen synthase kinase-3β(GSK3β) in the Inhibitory Effects of XPD on HUVSMC Proliferation induced by PDGF-BB. METHODS:1. Human Umbilical Vein Smooth muscle Cell were cultured; Establish the model of PDGF-BB inducing vascular smooth muscle cell proliferation,and cultivate it under various conditions.2. Optimize the PDGF-BB concentration and the incubation time. Harvest cells for 24 hours with final PDGF-BB concentration in the medium at 0ng/ml,10 ng/ml,30 ng/ml,50 ng/ml,80 ng/ml. Cell was detected by MTT and the best concentration of PDGF-BB was chosen.Cells were incubated with PDGF-BB at the concentration selected for 0,12,24,36,and 48 hours, followed by MTT detection.3. Incubate cells with PDGF-BB at the concentration selectedfor 0,1,8,16, 24,32,40 and 48 hours, then harvest cells and extract cellular proteins. The expression of XPD protein was detected by Western Blotting.4. Extract the recombinant plasmid p EGFP-N2 / XPD and vacant vector plasmid. The expression of XPD protein was detected by Western Blotting.5. Incubate transfected recombinant plasmid p EGFP-N2/XPD and transfected p EGFP-N2 VSMC cells with PDGF-BB. The experiments were divided into five groups:(1) Blank control group;(2)p EGFP-N2 group;(3)p EGFP-N2/XPD group;(4)PDGF-BB group;(5)PDGF-BB+p EGFP-N2 group;(6)PDGF-BB+ p EGFP- N2/ XPD group. By means of Western Blotting, the expressions of XPD, GSK3β, p-GSK3β, CDK4,cyclin D1 protein were detected. By means of Flow Cytometry, cell cycle was detected.6. HUVSMC were transfected with recombinant plasmid p EGFP-N2/XPD, and cultured 24 hours with 0.25μM GSK3β inhibitor. The experiments were divided into four groups:(1)Blank control;(2)DMSO group;(3)p EGFP-N2/XPD group;(4)p EGFP-N2/XPD+ SB216763 group. Via Western Blotting, the expressions of CDK4,cyclin D1 protein was detected.7. Statistical method: analyzed by SPSS19.0 statistical software, experimental data was expressed as mean±SD. P <0.05 was considered statistically significant. RESULTS:1. Optimization of the concentration of PDGF-BB and incubation time:MTT results showed that:OD492nm was ascending in dose-dependent manners within the concentration of PDGF-BB at 10 ng/ml,30 ng/ml,80 ng/ml; OD492 nm was ascending in time-dependent manners within 0,2,24 hours for incubation time.The concentration of PDGF-BB at 30 ng/ml via an additional 24 hours’ incubation, was chosen for the subsequent experiments.2. Incubate cells with PDGF-BB at the concentration 30 ng/ml for 0,1,8,16,24,32, 40 and 48 hours. Western Blotting results showed that the expression of XPD protein was descending in time-dependent manners within 0,1,8,16 hours for incubation time(P<0.01), while time-dependent manners within 24,32,40,48 hours(P<0.05).3. After transfected with recombinant plasmid p EGFP-N2 / XPD,Western Blotting results showed the expression levels of XPD higher than blank control( 0.91±0.34 vs 0.40±0.34,P<0.01), which indicated the recombinant plasmid p EGFP-N2/XPD was transfected successfully.4. Western Blotting results showed: XPD overexpression made the expression levels of p-GSK3β(0.11±0.02 vs 0.33±0.02,P<0.01),CDK4 protein dropped by(0.20±0.01 vs 0.54±0.03, P<0.01) and cyclin D1(0.22±0.02 vs 0.29±0.01, P<0.01) protein descend.And the transfection could inhibited the effect of PDGF-BB,accelerate expressions of p-GSK3β(0.17±0.005 vs 0.62±0.01,P<0.01),CDK4(0.46±0.02 vs 0.80±0.03,P<0.01),cyclin D1(0.23±0.01 vs 0.51±0.02,P<0.01) protein in VSMC.5. Flow cytometry results showed: overexpression of XPD caused cell cycle G0 /G1 increasing(P<0.01),cell cycle S decreasing(P<0.05),and inhibited PDGF-BB promoting cell cycle G0/G1 of VSMC to decrease(P<0.01),cell cycle S of VSMC to increase(P≤0.01).6. Western Blotting results showed:the expressions of CDK4(0.26±0.03 vs 0.12±0.02,P<0.01),cyclin D1(0.25±0.02 vs 0.11±0.01,P<0.01)protein in p EGFP-N2/XPD+ SB216763 group elevated, comparing with p EGFP-N2/XPD group. CONCLUSION:1. Overexpression of XPD can inhibit VSMC proliferation which induced by PDGF-BB;2. GSK3β may mediate XPD to inhibit PDGF-BB inducing VSMC proliferation;3. GSK3β may mediate XPD to inhibit VSMC proliferation through CDK4 and cyclin D1.