碱性成纤维细胞生长因子在烟草烟雾诱导大鼠血管平滑肌细胞增殖中的作用

The Cigarette Smoke Extract Induced VSMC Proliferation Via the Basic Fibroblast Growth Factor

目的探讨烟草烟雾提取物对大鼠血管平滑肌细胞增殖的影响及碱性成纤维细胞生长因子在其中的作用。方法按不同浓度烟草烟雾提取物(0、2.5%、5%、10%和20%)分为对照组、低浓度、中等浓度、高浓度和过高浓度烟草烟雾提取物组刺激血管平滑肌细胞,采用MTT法观察细胞增殖变化,免疫细胞化学法测定碱性成纤维细胞生长因子和增殖细胞核抗原蛋白的表达,同时用逆转录-聚合酶链反应法检测碱性成纤维细胞生长因子mRNA表达。用筛选出的最适烟草烟雾提取物浓度处理大鼠血管平滑肌细胞不同时间(0、4、8、12 h和24 h)后,检测碱性成纤维细胞生长因子mRNA及碱性成纤维细胞生长因子和增殖细胞核抗原蛋白的变化。用碱性成纤维细胞生长因子抗体和最适浓度烟草烟雾提取物干预血管平滑肌细胞24 h后检测细胞增殖及碱性成纤维细胞生长因子和增殖细胞核抗原蛋白表达的变化。结果 (1)与对照组相比,低浓度烟草烟雾提取物组(P<0.05)和中浓度组血管平滑肌细胞增加明显(P<0.01),而高浓度组和过高浓度组与对照组比较差异无显著性(P>0.05)。碱性成纤维细胞生长因子mRNA、蛋白和增殖细胞核抗原蛋白在对照组中有少量表达,低浓度烟草烟雾提取物组表达增加(P<0.01),中浓度烟草烟雾提取物组达到高峰,高浓度和过高浓度烟草烟雾提取物组仍高于对照组(P<0.01)。(2)对照组(不加烟草烟雾提取物组即0 h组)血管平滑肌细胞中有少量碱性成纤维细胞生长因子mRNA、蛋白和增殖细胞核抗原蛋白表达。低浓度烟草烟雾提取物刺激4 h后细胞内碱性成纤维细胞生长因子mRNA、蛋白和增殖细胞核抗原蛋白表达增加(P<0.01),碱性成纤维细胞生长因子mRNA于8 h达高峰;碱性成纤维细胞生长因子和增殖细胞核抗原蛋白于12 h达高峰。(3)碱性成纤维细胞生长因子抗体可显著抑制5%烟草烟雾提取物诱导的血管平滑肌细胞增殖和碱性成纤维细胞生长因子、增殖细胞核抗原蛋白表达增加。结论低浓度和中浓度烟草烟雾提取物对大鼠血管平滑肌细胞的促增殖作用逐渐增加;高浓度和过高浓度组时促增殖作用反而减弱。烟草烟雾提取物可能是通过增加碱性成纤维细胞生长因子的表达促进大鼠血管平滑肌细胞的增殖。

Aim To investigate the effect of cigarette smoke extract（CSE） on the proliferation of vascular smooth muscle cells（VSMC） and the relevant roles of basic fibroblast growth factor（bFGF）. Methods（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 0 h（control group）,4 h,8 h,12 h,24 h,the expression of bFGF mRNA,bFGF and PCNA protein were observed.（3）Treated with bFGF antibody and 5.0%CSE at 24 h,MTT A value,bFGF and PCNA protein were observed. Results（1）CSE can stimulate the increase of MTT absorbance（A）value.The absorbance（A）value of 2.5%（P0.05）,5.0%CSE（P0.01） group were significantly increased compared to the control group.The absorbance（A）value of 10%,20%CSEgroup were not significantly increased compared to the control group（P0.05）.There was the maximal increase in5.0% CSE group.The VSMC in control group expressed bFGF mRNA,bFGF and PCNA protein at low levels.In-creased bFGF mRNA,bFGF and PCNA protein expression were evident after 2.5%CSE treatment.Maximal increase inbFGF mRNA,bFGF and PCNA protein expression occurred after5.0%CSE treatment.Then expression were still elevat-ed compared with control group’s after 10%,20%CSE treatment.（2）The VSMC in control group expressed bFGF mR-NA,bFGF and PCNA protein at lowlevels.Increased bFGF mRNA,bFGF and PCNA protein expression were evident at4h after CSE treatment.Maximal increase in bFGF mRNAoccurred at8 h,while peak expression of bFGF and PCNApro-tein was at12 h.（3）Treatment with bFGF antibody nearly inhibited CSE-induced MTTA value,bFGF and PCNA proteinexpression.Conclusion The proliferation of VSMC increased gradually in low and middle concentrations（2.5%,5.0%CSE group）,while decreased in high and higher concentrations（10%,20%CSE group）on the contrary.CSE maybe in-creased rat VSMC proliferation by high expression of bFGF.