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

目的 探讨AP-1 decoy 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-1 decoyODNs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);100 mnol/L和200 mmol/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24 h后能够显著增加CFs胶原合成,decoy ODNs可以抑制这种作用,其中100 nmol/L和200 nmol/L均有显著性抑制作用.FCM细胞周期分析表明,AngⅡ作用24 h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decoyODNs对CFs的增殖及胶原合成均没有明显的影响.结论 AP-1 decoy 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.

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

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 nm (OD490) of the CFs decreased gradually as shown by MTT assay. Treatment with 100 or 200 nmol/L 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 10(-7) 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 G0/G1 phase. AP-1 decoy ODNs at 100 and 200 nmol/L did not obviously affect AngII-induced CF proliferation 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.