葛根素对血管平滑肌细胞增殖及Bcl-2蛋白和凝血酶受体mRNA表达的影响

Effects of Puerarin on Proliferation of Vascular Smooth Muscle Cells and Expression of Bcl-2 Protein and Thrombin Receptor mRNA

目的观察葛根素对凝血酶诱导的血管平滑肌细胞增殖及Bcl-2蛋白和凝血酶受体mRNA表达的影响，旨在认识葛根素作用的分子机制。方法以细胞计数法和流式细胞仪DNA含量测定，细胞周期分析法观察凝血酶及葛根素对血管平滑肌细胞增殖和DNA合成的影响。凝血酶及葛根素等各处理因素作用24h后，用免疫印迹法检测Bcl-2蛋白表达．以半定量逆转录聚合酶链反应检测凝血酶受体mRNA的表达。结果凝血酶对血管平滑肌细胞有明显促增殖作用，促增殖效应在24h末达峰值，且凝血酶浓度在0．1～1．0u／L之间有剂量依赖关系；葛根素呈剂量依赖性地抑制凝血酶诱导的细胞增殖、DNA合成及血管平滑肌细胞Bcl-2蛋白的表达；高浓度（1．5×10^3mol／L）葛根素可显著抑制凝血酶诱导的凝血酶受体nRNA上调。结论葛根素能抑制凝血酶诱导的血管平滑肌细胞增殖，这可能与其抑制Bcl-2蛋白有关，并部分与其抑制凝血酶受体mRNA表达有关。

Aim To observe the role of puerarin on the proliferation of vascular smooth muscle cells （ VSMC ） induced by thrombin and the effect of puerarin on the Bcl-2 protein and thrombin receptor （TR） mRNA expression. Methods Cell number and cell cycle analysis using riow cytometry were adopted as two different indicators of effects on proliferation of VSMC. Western blot was used to indicate the changes of Bcl-2 protein, and reverse transcription-polymerase chain reaction （RT-PCR） was used to evaluate TR mRNA expression, with treatment of thrombin and puerarin after 24 h. Results Thrombin can significandy increase the cell numbers of VSMC, the peak of proliferation curve is at 24 h, and the effect of T is in a dose dependent manner （0.1 - 1.0 u/L）. 1.5 × 10^-5 - 1.5 × 10^-3 mol/L puerarin can signifieandy suppress dais stimulation of VSMC proliferation and DNA synthesis. Western blot demonstrated that treated with thtrombin and puerarin after 24 h, thrombin can signifieandy increase Bcl-2 protein. 1.5 × 10^-5 - 1.5 × 10^-3 mol/L puerarin can significantly suppress tiffs increase. RT-PCR demonstrated that thrombin can increase TR mRNA expression signifieandy, and 1.5 × 10.3 mol/L puerarin can suppress dais increase. Conclusions Puerafin can suppress the profiferation and DNA synthesis of VSMC promoted by thrombin. Tiffs inhibitory effects of puerarin are closely related with the suppression of Bcl-2 protein, and paitly related with the suppression on TR mRNA.