补体旁路激活致血管平滑肌细胞增殖活化及干预研究

Proliferation and activation of vascular smooth muscle cells induced by activated complement alternative pathway and intervention study

目的研究补体旁路途径激活诱导人血管平滑肌细胞(VSMC)增殖活化及其干预作用。方法采用CVF特异激活血浆补体旁路途经。将VSMC与补体旁路激活产物共同孵育,采用倒置相差显微镜和MTT法分别检测细胞形态变化和增殖活性,ELISA法检测培养上清中E-selectin、ICAM-1和VCAM-1含量,Western blot检测p-NF-κB p65、IKK和NF-κB p65蛋白表达水平,双荧光素酶报告基因检测试剂盒检测NF-κB p65转录活性,采用PDTC对上述指标的变化进行干预。结果补体旁路激活导致VSMC增殖活化,上调表达E-selectin、ICAM-1和VCAM-1,其中ICAM-1和VCAM-1含量在6 h达到高峰,E-selectin在12 h出现明显上调;VSMC经补体旁路激活产物刺激后,NF-κB p65磷酸化明显增加,NF-κB p65核内转录活性升高,IKK和NF-κB p65蛋白表达上调,PDTC对上述相关指标的变化有明确的干预作用。结论补体旁路激活可导致VSMC增殖活化,其机制与NF-κB信号通路活化有密切关系,且NF-κB抑制剂PDTC对其增殖活化具有明显的干预作用。

Aim To study the effect of proliferation and activation of vascular smooth muscle cells(VSMCs)induced by activated the complement alternative pathway and intervention.Methods Normal human plasma was specifically activated the complement alternative pathway by incubated with cobra venom factor(CVF).Exposed VSMCs to the activated complement products,the change of cell morphology and the cell viability were assayed by inverted phase contrast microscope and MTT method,respectively.The supernatant was assayed for expression of E-selectin,ICAM-1 and VCAM-1 by using ELISA reagent kits.The protein expression levels of p-NF-κB p65,NF-κB p65 and IKK were assayed by Western blot.The nucleus transcrip-tional activity of NF-κB p65 was tested by the dual luciferase reporter assay system.Pyrrolidine dithiocarbamate(PDTC)was used to intervene the proliferation and activation of VSMCs.Results VSMCs were activated and induced to proliferation after exposed to the products of activated complement alternative pathway.The expressions of E-selectin,VCAM-1 and ICAM-1 were up-regulated.The contents of ICAM-1 and VCAM-1 reached the peak at 6 h and the E-selectin increased significantly at 12 h.Meanwhile,the phosphorylation level of NF-κB p65,nucleus transcriptional activity of NF-κB p65 and the protein expression of IKK and NF-κB p65 increased.The above mentioned changes were clearly inhibited by PDTC.Conclusions The activated complement alternative pathway can initiate proliferation and activation of VSMCs,and its mechanism goes hand in hand with activation of NF-κB signaling pathway.PDTC,a specific inhibitor of NF-κB,can effectively inhibit the proliferation and activation of VSMCs.