核因子-κB在凝血酶促自发性高血压大鼠血管平滑肌细胞增殖中的作用

目的：研究凝血酶诱导培养的自发性高血压大鼠（SHR）血管平滑肌细胞（VSMCs）的增殖作用，探讨其引起VSMCs增殖与NF—κB途径的关系及可能机制。方法：以0．01、0．1、0．2、0．5、1．0、2．0U／mL的凝血酶与培养的VSMCs共孵育24h和用0．5U／mL凝血酶与VSMCs分别孵育1、2、6、12、24、48h：采用WST-1代谢活性和^3H—TdR掺入率测定VSMCs细胞增殖率；免疫荧光法和蛋白质印迹技术检测基础状态下和0．5U／mL凝血酶干预的VSMCs中NF—κB的细胞内定位及其核中NF—κB的蛋白含量；以已知的AngⅡ和PDGF对VSMCs的增殖作用为阳性对照，研究NF-κB途径与凝血酶诱导的VSMCs增殖关系,100μmol／LPDTC（NF—κB特异性阻断剂）预处理后检测凝血酶引起VSMCs的增殖率。结果：浓度为0．2～2．0U／mL的凝血酶对VSMCs分别有明显的促增殖作用（P〈0．05和P〈0．01）；但浓度为0．5～2．0U／mL的凝血酶作用24h对VSMCs的促增殖作用差异无统计学意义（P〉0．05）。0．5U／mL凝血酶促VSMCs的增殖呈双峰样改变．1h和24h分别达峰值。基础状态下VSMCs的NF-κB主要分布于细胞浆，凝血酶干预后VSMCs的NF—κB主要分布于细胞核。PDTC预处理明显抑制凝血酶促VSMCs增殖的作用（P〈0．01）。结论：浓度0．2～0．5U／mL范围内的凝血酶呈浓度依赖性方式促进培养SHR的VSMCs增殖：而且在时间上呈双峰样改变，提示凝血酶的促增殖作用存在延迟现象。凝血酶能够激活VSMCs的NF—κB，使其从细胞浆转位至细胞核。凝血酶引起VSMCs增殖与NF-κB途径有关联，NF—κB可能是VSMC增殖的细胞内信号转导的共同通路。

Effect of nuclear factor-κB on proliferation of vascular smooth muscle cells promoted by thrombin in spontaneously hypertensive rats

AIM：To study the dose-effect and time-effect on proliferation of vascular smooth muscle cells（VSMCs） in spontaneously hypertensive rats（SHR） promoted by hrombin and to explore the relationship between VSMCs proliferation and NF-κB path as well as their possible mechanism.METHODS：VSMCs were incubated with thrombin at the following concentrations of 0.01,0.1,0.2,0.5,1.0,2.0 U/mL for 24 h and with 0.5 U/mL thrombin for 1,2,6,12,24,48 h respectively;The proliferation rate of VSMCs was determined with both WST-1 metabolic activity and 3H-TdR incorporation efficiency;The expression of NF-κB in VSMCs was tested with both immumofluorescence and western blot in basic condition and with 0.5 U/mL thrombin intervention.AngⅡ and PDGF on proliferation of VSMCs were used as positive control group.In order to research the relationship of NF-κB path and VSMCs proliferation induced by thrombin,after pretreatment with 100 μmol/L PDTC（NF-κB special inhibitor）,VSMCs proliferation induced by thrombin was also investigated.RESULTS： Thrombin concentrations of 0.2-2.0 U/mL significantly promoted VSMCs proliferation respectively（P〈0.05 or P〈0.01）.But VSMCs proliferation was not different in the thrombin at the concentrations of 0.5-2.0 U/mL at 24 h（P〉0.05）.VSMCs proliferation with 0.5 U/mL thrombin exhibited double hump when compared with the Control Group.The peak values at 1 h and 24 h showed that thrombin promotion VSMCs proliferation was not only rapid and brief but also delayed and retarded.The expression of NF-κB of VSMCs in basic condition was mainly distributed in cytoplasm while that of VSMCs interfered by thrombin was in nucleus.The PDTC pre-treatment could obviously inhibit VSMCs proliferation induced by thrombin（P〈0.01）.CONCLUSION： Thrombin promotes VSMCs proliferation in the manner of dose dependent at the concentrations of 0.2-0.5 U/mL.The proliferation curve induced by thrombin exhibits double hump which suggests that thrombin promotion VSMCs proliferation is delayed.Thrombin