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目的观察葛根素对凝血酶诱导的血管平滑肌细胞(VSMC)增殖的影响,对原癌基因c-fos和bc l-2蛋白质以及凝血酶受体(TR)mRNA表达的作用。方法以细胞计数法,流式细胞仪测定DNA含量,细胞周期分析法观察凝血酶及葛根素对VSMC增殖和DNA合成的影响。在凝血酶及葛根素作用24 h后,用W estern印迹法检测c-fos和bc l-2蛋白表达,以半定量逆转录聚合酶链式反应(RT-PCR)检测TR mRNA的表达。结果凝血酶对VSMC有明显促增殖作用,促增殖效应在24 h末达峰值(细胞计数凝血酶组8.64×104/m l±0.12×104/m l,对照组4.20×104/m l±0.11×104/m l,P<0.05),且凝血酶浓度在0.1~1.0 U/L之间呈剂量依赖关系;葛根素呈剂量依赖性地抑制凝血酶诱导的细胞增殖、DNA合成以及VSMC c-fos和bc l-2蛋白的表达(葛根素浓度为1.5×10-3mol/L时,抑制率分别为42.6%±5.2%、58.2%±7.9%、44.5%±7.5%和39.6%±6.4%,均P<0.05);高浓度(1.5×10-3mol/L)的葛根素可显著抑制凝血酶诱导的TR mRNA上调(抑制率为17.6%±1.7%,P<0.05)。结论葛根素能抑制凝血酶诱导的VSMC增殖,这可能与其抑制c-fos和bc l-2蛋白有关,并部分与其抑制TR mRNA表达有关。
Objective To observe the effects of puerarin on the proliferation of vascular smooth muscle cells (VSMC) induced by thrombin, and the effects of pro-oncogene c-fos and bcl-2 protein and thrombin receptor (TR) mRNA expression. Methods The DNA content was measured by flow cytometry using cell counting method. Cell cycle analysis was used to observe the effects of thrombin and puerarin on proliferation and DNA synthesis of VSMC. After 24 h of thrombin and puerarin treatment, the expression of c-fos and bcl-2 protein was detected by Western blotting, and the expression of TR mRNA was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results Thrombin promoted proliferation of VSMC significantly, and the pro-proliferative effect peaked at the end of 24 h (cell count thrombin group 8.64×104/ml±0.12×104/ml, control group 4.20×104/ml±0.11×104/ Ml, P<0.05), and thrombin concentration was dose-dependent in a range of 0.1-1.0 U/L; puerarin dose-dependently inhibited thrombin-induced cell proliferation, DNA synthesis, and VSMC c-fos and bcl -2 protein expression (puerarin concentration 1.5 × 10-3mol / L, the inhibition rate was 42.6% ± 5.2%, 58.2% ± 7.9%, 44.5% ± 7.5% and 39.6% ± 6.4%, respectively, P < 0.05); high concentrations (1.5×10-3mol/L) of puerarin can significantly inhibit thrombin-induced TR mRNA upregulation (inhibition rate was 17.6% ± 1.7%, P <0.05). Conclusion Puerarin can inhibit the proliferation of VSMC induced by thrombin, which may be related to its inhibition of c-fos and bcl-2 proteins, and partly related to its inhibition of TR mRNA expression.