灯盏花素抑制血管平滑肌细胞增殖的分子基础研究

Molecular basis study on inhibition of erigeron breviscapus element on the proliferation of vascu- lar smooth muscle cell

目的研究反义凝血酶受体寡核苷酸（antisense thrombin receptor oligo—deoxy—nucleotides，Antisense TR ODNs）及灯盏花素在凝血酶（Thrombin，Ⅱa）诱导的血管平滑肌细胞增殖中的作用，比较二者对血管平滑肌细胞凝血酶受体基因、蛋白表达功能及肌醇代谢的影响，以期在分子水平探讨高血压脑出血预防用药的作用机制以及药物筛选方法。方法应用细胞转染、反转录聚合酶链式反应（reverse transcription polymerase chain reaction，RT—PCR）、免疫印迹（Western blot）、酶联免疫吸附测定（ELISA）、氚-胸腺嘧啶核苷（^3H—TdR）及。H肌醇掺入实验等对体外培养的第4～6代SD大鼠血管平滑肌细胞的凝血酶受体基因、蛋白表达功能、核酸及肌醇代谢进行测定。探索灯盏花素和反义凝血酶受体对血管平滑肌细胞增殖的影响。结果灯盏花素和反义凝血酶受体寡核苷酸均明显抑制凝血酶诱导的大鼠血管平滑肌细胞的增殖，与对照组、正义组相比差异有统计学意义（P〈0．05），RT—PCR、Western blot及ELISA试验结果显示灯盏花素和反义凝血酶受体显著抑制了大鼠血管平滑肌细胞凝血酶受体mRNA和蛋白的表达；且明显抑制了血管平滑肌细胞磷酸肌醇代谢。灯盏花素和反义凝血酶受体对平滑肌细胞增殖的抑制效应差异无统计学意义。结论灯盏花素和凝血酶受体反义序列明显抑制血管平滑肌细胞增殖，其作用机制为通过抑制凝血酶受体基因的表达（mRNA和蛋白的表达），抑制细胞内信号传递来完成。灯盏花素在预防治疗高血压脑出血中的作用机制之一是通过阻断凝血酶受体表达抑制血管平滑肌细胞增殖而实现的。

Objective Applying cell transfection, RT-PCR Western blot, ELISA, ^3H -TDR and ^3H inositol unincorporation etc, to assay the thrombin receptor gene and protein expression, which was cultured by the fourth - sixth generation Sprague - Dawley rat （ SD ） ＇s vascular smooth muscle cell （VSMC） in vitro. To explore the influence of Erigeron breviscapus element and antisense thrombin receptor oligo - deoxy - nucleotides on the proliferation of VSMC. Results Erigeron breviscapus element and antisense thrombin can inhibit the proliferation of SD＇s VSMC, which is induced by thrombin. Comparing the obverse with control group, the statistic difference still exists （ P 〈 0. 05 ） . The experimental result of RT - PCR, Western blot and ELISA indicates that Erigeron breviscapus element and antisense thrombin restrains the expression of mRNA and protein in SD＇s VSMC distinctly, and inhibits phosphoric inositol metabolism of Vascular Smooth Muscle Cell distinctly as well. There is no difference in inhabitation effects between Erigeron breviscapus element and atisense thrombin receptor. Methods Applying cell transfection, RT - PCR Western blot, ELISA, ^3 H - TDR and 3 H inositol unincorporation etc, to assay the thrombin receptor gene and protein expression, which was cultured by the fourth - sixth generation Sprague - Dawley rat （ SD ） ＇s VSMC in vitro. To explore the influence of Erigeron breviscapus element and antisense thrombin receptor oligo - deoxy - nucleotides on the proliferation of VSMC. Results Erigeron breviscapus element and antisense thrombin can inhibit the proliferation of SD ＇s VSMC, which is induced by thrombin. Comparing the obverse with control group, the statistic difference still exists （ P 〈 0. 05 ）. The experimental result of RT - PCR, Western blot and ELISA indicates that Erigeron breviscapus element and antisense thrombin restrains the expression of mRNA and protein in SD＇s VSMC distinctly, and inhibits phosphoric inositol metabolism of VSMC distinctly as well. There is no difference in inhabitation effects between Erigeron breviscapus element and atisense thrombin receptor. Conclusion Erigeron breviscapus element and antisense sequence of thrombin can inhibit the proliferation of SD＇s VSMC. The mechanism of action is fulfilled through inhibiting gene expression of thrombin receptor in order to suppress intracellular signal transmission. One of the action mechanisms of Erigeron breviscapus element on hypertensive cerebral hemorrhage is to suppress the proliferation of VSMC through blocking the expression of thrombin receptor which provide the theoretic and experimental basis to screen the drug action situs in cardio- cerebral disease with genetic technique.