| ObjectiveCigarette smoking was a major atherosclerosis risk factors, which was more harmful to cardiovascular system than to smoking-induced lung cancer. Tobacco consumption in China was the world's largest.In China 60%of men and 3%of women smoke.there were about 350 million smokers. The dangers of smoking on human health were enormous.In 2000, the world's 169 million people died from smoking-induced cardiovascular disease, account for 11%of cardiovascular disease deaths. In 2002, the death of 500 million people was caused by smoking, while China had 120 million people died from smoking each year.Basic fibroblast growth factor (basic Fibroblast Growth Factor, bFGF) was one of heparin-binding growth factor family, which was widely distributed in the body. Promoting cell proliferation was a major one of bFGF1 biological characteristics. The VSMC (vascular smooth muscle cells) proliferation was the pathological basis of atherosclerosis, restenosis and pulmonary hypertension and other cardiovascular diseases.In this study, through cigarette smoke extract stimulated rat VSMC, we observed its effect on the proliferation of VSMC and explored bFGF'mechanism in CSE stimulated VSMC proliferation by bFGF antibodies neutralized biological function of bFGF.Methods1. VSMC cultureThe cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), pH7.4, containing 10%fetal bovine serum (FBS), at 378 in a humidified atmosphere of 5% CO2. The medium was changed once every two days.when Cells were passaged by washing once in phosphate buffered solution (PBS) followed by trypsinization. Subcultured strains were cultured in DMEM, pH7.4, containing 10%FBS. Subcultured strains were used between passages 3 and 8. Cell were used in trial, when growed 70%-80%.2.experimental grouping(1)The rat aortic smooth muscle cells were treated with various concentrations of cigarette smoke extract( 0%,2.5%,5.0%,10.0%,20.0%), the VSMC treated without CSE were control group, then MTT assay was performed to observe the changes of the proliferation of VSMC. The expression of proliferating cell nuclear antigen (PCNA) and Fibroblast Growth Factor 2(bFGF) were detected by immunocytochemistry. In addition, reverse transcription polymerase chain reaction (RT-PCR) was applied to detect the mRNA expression of bFGF.(2)Treated with 5%CSE(the optimal concentration)at 0h(control group),4h,8h, 12h,24h and observed the expression of bFGF mRNA,bFGF and PCNA protein.(3)Treated with bFGF antibody and 5.0%CSE at 24h,in addition to observed MTT A value, bFGF and PCNA protein.3.Experiment methodsThe effect of CSE on proliferation of VSMC was observed by 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)metabolism measuring. The protein expression of bFGF and PCNA was measured by immunocytochemistry.RT-PCR was done to detect the mRNA level of bFGF.Results(1)CSE can stimulate increasment of MTT absorbance(A)value. the absorbance(A) value of 2.5%(P<0.05),5.0%CSE(P<0.01) group were significantly increased compared to the control group. The absorbance(A)value of 10%,20%CSE group were not significantly increased compared to the control group(P>0.05).There was the maximal increase in 5.0%CSE group. The VSMC in control group expressed bFGF mRNA, bFGF and PCNA protein at low levels. Increased bFGF mRNA, bFGF and PCNA protein expression were evident after 2.5%CSE treatment. Maximal increase in bFGF mRNA, bFGF and PCNA protein expression occurred after 5.0%CSE treatment. Then expression were still elevated than control group's after 10%,20%CSE treatment. (2)The VSMC in control group expressed bFGF mRNA, bFGF and PCNA protein at low levels. Increased bFGF mRNA, bFGF and PCNA protein expression were evident at 4h after CSE treatment. Maximal increase in bFGF mRNA occurred at 8h,while peak expression of bFGF and PCNA protein was at 12h.(3)Treatment with bFGF antibody nearly inhibited CSE-induced MTT A value,bFGF and PCNA protein expression.ConclusionCSE maybe increased rat VSMC proliferation by high expression of bFGF. |
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