以转录因子AP-1为靶点的decoy ODNs对大鼠心肌成纤维细胞增殖及胶原合成的抑制作用

Effect of AP-1 decoy oligodeoxynucleotides on neonatal rat cardiac fibroblast proliferation and collagen synthesis

目的探讨AP-1decoy ODNs对大鼠心肌成纤维细胞(CFs)增殖及细胞胶原合成的抑制作用,为高血压心肌纤维化的防治提供理论依据。方法通过2.5%胰酶分离培养新生SD大鼠心脏成纤维细胞,采用四氮唑盐(MTT)比色法测定细胞数目,羟脯氨酸比色法测定培养细胞上清胶原合成,流式细胞分析技术(FCM)检测细胞周期,分别观察不同浓度的AP-1decoy ODNs对AngⅡ诱导的CFs增殖,胶原的合成及细胞周期的影响。结果CFsMTT比色法显示10-7mmol/LAngⅡOD490值明显高于空白对照组(0.451±0.014和0.342±0.096,n=9,P<0.05);100nmol/L和200nmol/L组的OD490值分别为0.329±0.04和0.214±0.25,均较AngⅡOD490值显著降低(P<0.05);AngⅡ作用24h后能够显著增加CFs胶原合成,decoy ODNs可以抑制这种作用,其中100nmol/L和200nmol/L均有显著性抑制作用。FCM细胞周期分析表明,AngⅡ作用24h后能够明显增加S期细胞百分率(21.93±0.71%和10.93±0.2%,n=6,P<0.01),随着decoy ODNs浓度增加,S期细胞百分率明显降低,其中100nmol/L和200nmol/L作用具有显著性意义(P<0.01)。但突变的decoy ODNs对CFs的增殖及胶原合成均没有明显的影响。结论AP-1decoy ODNs可以抑制AngⅡ诱导的CFs增殖和胶原的合成,其作用机制可能与影响CFs细胞周期有关。

Objective To investigate the inhibitory effects of AP-1 decoy oligodeoxynucleotides （ODNs） on angiotensin II （AngⅡ）-induced proliferation and collagen synthesis in neonatal rat cardiac fibroblasts （CFs）. Methods The CFs of neonatal SD rats were cultured in serum-free medium for 24 h and stimulated with 10.7 mol/L AngⅡ in the presence of AP-1 decoy ODNs or mutational AP-1 decoy ODNs at varied concentrations. MTT assay was employed for quantitative evaluation of the CF proliferation. Collagen synthesis in the CFs was assessed with hydroxyproline, and the cell cycle distribution determined with flow cytometry （FCM）. Results With the increase of the concentration of AP-1 decoy ODNs, the absorbance at 490 nrn （OD490） of the CFs decreased gradually as shown by MTT assay. Treatment with 100 or 200 nmoVL AP-1 decoy ODNs resulted in Significantly lowered OD490 of the CFs as compared with that of AngⅡ group. The concentration of hydroxyproline increased significantly after treatment with 10.7 mol/L AngⅡ in comparison with the control group （P〈0.05）. Hydroxyproline concentration in cells treated with 100 or 200 nmol/L AP-1 decoy ODNs was significantly lower than that in the 104 mol/L AngⅡ-treated cells. AP-1 decoy ODNs decreased the cell percentage in S phase and increased hydroxyproline concentration, but increased the percentage of cells in Go/G1 phase. AP-1 decoy ODNs at 100 and 200 nmol/L did not obviously affect AngⅡ-induced CF prolifersation and collagen synthesis （P〈0.01）. Conclusion AP-1 decoy can inhibit AngⅡ-induced rat CF proliferation and collagen synthesis possibly by affecting the cell cycle distribution.