Influence Mechanism Of C-myc In Proliferation Of Vein Smooth Muscle Cell And Restenosis After Coronary Artery Bypass Grafting

Part one Study on role of c-myc in Restenosis after Coronary Artery Bypass GraftingObjective:To study the actions of c-myc protein in restenosis after coronary artery bypass grafting (CABG).Methods:A total of 25 healthy pure breed New Zealand white rabbits were randomly divided into 5 groups according to weight,5 in each group. The external jugular vein is placed at ipsilateral common carotid artery and sampling at 6h,2d,7d,14d and 28d. The expression of c-myc positive cell population was observed in different time using immunohistochemistry and morphological analysis. The thickness and ratio of luminal intima and media were measured by computer image analytical method.Results:The luminal intima and media thickness at day 7 is significantly thickening (P<0.01) from 6h while it has not changed obviously (P>0.05) at day 14 and 28 compered to day 7. c-myc proteins are gradually increased from 6h to day 7, reached a peak (P<0.01) at day 7; started declining from day 14-28. The difference has statistical significance (P<0.01) compared to day 7.Conclusion:c-myc positive cell population has reached a peak after transplantation, which is identical with the peak of fast intimal proliferation. It indicates that c-myc protein expression is closely associated to intimal proliferation. It can act as an indicator for intimal proliferation after vascular injuries in the early stage of reactions.Part two the influence of ET-1 in endothelial cells conditioned medium on the proliferation and the expression of c-myc of vascular smooth muscle cellsObjective:To study the influence of ET-1 in endothelial cells conditioned medium on the proliferation and the expression of c-myc of vascular smooth muscle cells, and the machanism between them.Methods:First, TNF-a was used to stimulate human saphenous vein endothelial cells and then ET-1 ELISA kits was used to measure the expression of ET-1 in the cell culture supernatant, and we determine the best dose and time of TNF-a stimulation. At last, we confirm that the best dose and time of TNF-a stimulation which leads to endothelial cells scereting ET-1 is lOng/ml and 24h. Then a final concentration of 10 ng/ml and 0 ng/ml of TNF-a was used to stimulate human saphenous vein endothelial cells and collected the cell culture supernatant. The supernatant fluid which was treated with 10 ng/ml TNF-a for 24h called fluid A, The supernatant fluid which was treated with 0 ng/ml TNF-a for 24h called fluid B, and then continue the subsequent cell experiments. The human saphenous vein smooth muscle cells were seeded in cell culture plates and divided into four groups: ? stimulation group:fluid A and the DMEM cell culture medium was added at the ratio of 1:1, ? antagonism group: fluid A and the DMEM cell culture medium was added at the ratio of 1:1, and added the anti-ET-1 monoclonal antibody (50ng/ml), ? control group:fluid B and the DMEM cell culture medium was added at the ratio of 1:1, ? blank group:only serum-free DMEM medium. After grouping, cultured smooth muscle cells were collected 24h later, and then MTT assay were used to detect the proliferation of smooth muscle cells, RT-PCR and weatern blot was used to detect the expression of c-myc mRNA and c-myc protein in smooth muscle cells.Results:1. TNF-a stimulate human saphenous vein endothelial cell and leads to its release of ET-1 at dose-and time-dependently manner, ? dose effect:the secretion level of ET-1 was increasing as the increase of the stimulation dose of TNF-a, and when the stimulation dose were Ong/ml?5ng/ml?10ng/ml?20ng/ml?40ng/ml and time was 24h, the secretion levels of ET-1 were 23.41±1.03?53.98±0.97?71.78±1.09? 76.77±1.00?69.66±1.04 (pg/ml).? time effect:when the stimulation dose of TNF-a was lOng/ml, the secretion level of ET-1 was increasing as the increase of the stimulation time of TNF-a, and the the secretion levels of ET-1 were 12.29±0.91? 45.42±1.33?55.98±0.91?71.78±1.09?67.45± 1.24 (pg/ml) when the stumilation time were 0h?6h?12h?24h?48h, respectively; 2. ET-1 in endothelial cell conditioned medium promote the expression of c-myc mRNA in human saphenous vein smooth muscle cells, and relative expression of c-myc mRNA of the four groups of cells 9.435±0.625,6.285±0.585,3.165±0.195,1, the relative expression level of c-myc mRNA in stimulation group was significantly higher than the antagonism group, control group and blank group (p<0.05); 3. ET-1 in endothelial cell conditioned medium promote the expression of c-myc protein in human saphenous vein smooth muscle cells, and relative expression of c-myc protein of the three groups of cells 1.002±0.0695,0.789±0.0240,0.292±0.0225,0.112±0.0115, the relative expression level of c-myc protein in stimulation group was significantly higher than the antagonism group, control group and blank group (p<0.05); 4. ET-1 in endothelial cell conditioned medium promote the proliferation of human saphenous vein smooth muscle cells, and the OD490 value of the three groups of cells 1.455±0.077, 0.964±0.067,0.618±0.043, the OD490 value of stimulation group was significantly higher than the antagonism group, control group and blank group (p<0.05)Conclusion:TNF-a stimulates human saphenous vein endothelial cells releasing ET-1, and ET-1 promotes smooth muscle cells expressing c-myc mRNA and the secretion of c-myc protein, and thereby promote cell proliferation of vascular smooth muscle cells.Part three The influence of RNA interfering to c-myc on the biological activity of human saphenous vein smooth muscle cellObjective:We have found that overexpression of c-myc is associated with restenosis after coronary artery bypass surgery of coronary heart disease (CHD) in part 1, it maybe an indicator of intimal proliferation, and the main reason of intimal proliferation is that smooth muscle cells waver into the intima and proliferate. The present study was aimed to investigate the influence of RNA interference to c-myc gene on proliferation and apoptosis of human saphenous vein smooth muscle cell, to provide a theoretical basis and methods for the prevention of restenosis after coronary artery bypass.Methods:The design tools on the Invitrogen company website was used to design and synthesize the siRNA sequence of c-myc, and also synthesize irrelevant siRNA sequence of c-myc as the negative control. And then Lipofectamine 2000 liposome was used to transfect the siRNA into human saphenous vein smooth muscle cells, and transfection efficiency was detected by inverted fluorescent microscope, MTT assay and colony formation was used to detect the influence of c-myc siRNA on the proliferation of smooth muscle cell, RT-PCR and western blot was used to detect the expression level of c-myc mRNA and protein of smooth muscle cells after transfected with c-myc siRNA, and finally flow cytometry was used to detect apoptosis of smooth muscle cells after transfected with c-myc siRNA.Results:1. the transfection efficiency of c-myc siRNA was 88%±2.43%, which shows that the transfection is perfect; 2. c-myc siRNA significantly inhibited proliferation of human smooth muscle endothelial cell with time-dependent after transfection, and the growth inhibition rates were 25.92±3.373,53.66±1.822,50.83±0.236 after transfection 24h,48h and 72h, which significantly higher than the negative control group and normal control group (p<0.05); 3. c-myc siRNA significantly inhibited colony formation of human smooth muscle endothelial cell after transfection, and colony formation rates of the normal control group, negative control group and c-myc siRNA transfection group were 15.00 ± 1.323,14.33 ± 1.364,2.0 ± 0.289, the colony formation rates of c-myc siRNA transfection group was significantly lower than the negative control group and normal control group (p<0.05); 4. c-myc siRNA significantly inhibited the expression of c-myc mRNA of human smooth muscle endothelial cell after transfection, and the relative expression level of c-myc mRNA of the normal control group, negative control group and c-myc siRNA transfection group after 24h?48h and 72h were 1,0.9633 ± 0.0151,0.8580 ± 0.0104,0.5863± 0.0109,0.2007±0.0084, the results show that the mRNA expression levels of c-myc siRNA transfection group were significantly lower than the negative control group and normal control group (p<0.05); 5. c-myc siRNA significantly inhibited the expression of c-myc protein of human smooth muscle endothelial cell after transfection, and the relative expression level of c-myc protein of the normal control group, negative control group and c-myc siRNA transfection group after 24h?48h and 72h wee 0.7105±0.0215?0.6570±0.0150?0.4155±0.0095?0.2225±0.0095? 0.1050±0.0070, the results show that the mRNA expression levels of c-myc siRNA transfection group were significantly lower than the negative control group and normal control group (p<0.05); 6. c-myc siRNA significantly promote cell apoptosis of human smooth muscle endothelial cell after transfection, and cell apoptosis of the normal control group, negative control group and c-myc siRNA transfection group were 13.30±0.875,14.46±1.200,42.54±1.354, the cell apoptosis of c-myc siRNA transfection group was significantly higher than the negative control group and normal control group (p<0.05).Conclusion:1. c-myc siRNA can significantly inhibit cell proliferation and colony formation of smooth muscle cell; 2. c-myc siRNA can significantly inhibit the expression of c-myc mRNA and c-myc protein in smooth muscle cells and with a time-dependent manner; 3. c-myc c siRNA can induce apoptosis of smooth muscle cells...