组织因子途径抑制物对apoE-/-小鼠动脉粥样硬化斑块稳定性的影响及分子机制研究

Atherosclerosis(AS) is a serious disease threatening human health. It has been shown that monocyte chemoattractant protein (MCP) -3, a proinflammatory cytokine, might be closely related to the instability of AS plaque. We have found the inhibitory effect of tissue factor pathway inhibitor(TFPI) on intimal hyperplasia and restenosis in which inflammatory response was involved. The preliminary study demonstrated that TFPI could inhibit the expression of MCP-3 in vascular smooth muscle cell. In this study, the apoE-/- mice as well as vascular smooth muscle cell(VSMC) and macrophages cultured in vitro were used as experimental subjects. Based on above considerations, we have made some researches listed as follows. ①We established AS plaque by casing technology in carotid artery of apoE-/- mice. ② We constructed Ad-MCP-3-shRNA by RNA interference technology, which were then transfected into the plaque and cells cultured in vitro in order to observe the expression and location of MCP-3 and CCR2(the receptor of MCP-3) and the stability of AS plaque. ③ We transfected Ad-TFPI into the AS plaque of carotid artery as well as cells cultured in vitro so as to observe the expression and location of MCP-3 and CCR2 and the effect of the overexpression of TFPI on the stability of AS plaque. ④ We also detected the effect of MCP-3 gene silence and overexpression of TFPI on the expression of MAPK pathway. This study will confirm the effect of TFPI on the stability of AS plaque and the mechanisms. This will provide a new target for the treatment on the stability of AS plaque.

动脉粥样硬化(AS)是严重威胁人类健康的疾病，已知促炎因子单核细胞趋化蛋白(MCP)-3与AS斑块不稳定性密切相关。我们发现组织因子途径抑制物(TFPI)对内膜增生、再狭窄等炎症反应参与的疾病均有抑制作用，预实验发现TFPI能够抑制平滑肌细胞中MCP-3表达。本实验以apoE-/-小鼠以及培养的血管平滑肌细胞和巨噬细胞为实验对象，进行以下研究:①颈动脉套管技术建立颈动脉AS斑块模型；②用RNA干扰技术合成MCP-3-shRNA重组腺病毒载体，转染至斑块及细胞内，观察MCP-3及其受体CCR2在斑块和细胞内表达、定位以及斑块稳定性的变化；③ 通过腺病毒转染使TFPI基因在斑块和体外细胞中过表达，观察其对MCP-3和CCR2表达、定位以及斑块稳定性的影响；④ 检测MCP-3沉默和TFPI过表达对MAPK通路表达的影响。本研究将确定TFPI在AS斑块稳定性中的作用及机制，为AS的治疗提供新靶点。

动脉粥样硬化（AS）所导致的冠心病、心肌梗塞等心血管疾病是危害人类健康的重大疾患，趋化因子是斑块不稳定的一个重要介质。在前期工作中我们发现组织因子途径抑制物(TFPI)能够制血管平滑肌细胞中单核细胞趋化蛋白(MCP)-3表达，推测其可能继而抑制血管内膜增生。本实验以apoE-/-小鼠以及培养的血管平滑肌细胞和巨噬细胞为实验对象，用RNA干扰技术选择性的沉默MCP-3，系统的研究MCP-3及其受体CCR2对AS发生、发展以及斑块稳定性的影响，同时检测TFPI过表达对MCP-3及其受体CCR2表达的影响以及对斑块稳定性的作用，取得成果如下：① 成功构建颈动脉AS不稳定斑块模型；②用RNA干扰技术合成MCP-3-shRNA重组腺病毒载体，转染至颈动脉斑块，在体内实验中证明MCP-3沉默能够有效抑制颈动脉局部斑块面积，并通过增加局部胶原含量、减少脂质的含量、减少局部斑块内巨噬细胞的含量以及抑制斑块内MCP-3和CCR2的表达从而增加局部斑块稳定性；③ 将MCP-3-shRNA重组腺病毒载体转染至体外培养的血管平滑肌细胞和巨噬细胞中，发现MCP-3沉默通过抑制细胞中MCP-3和CCR2的表达以及抑制MAPK及PI3K通路磷酸化激活从而抑制细胞增殖；④ 通过腺病毒载体将TFPI基因转染至颈动脉斑块中，证明能够有效抑制颈动脉局部斑块面积，并通过增加局部胶原含量、减少脂质含量、减少局部斑块内巨噬细胞含量以及抑制斑块内MCP-3和CCR2的表达从而增加局部斑块稳定性；⑤ 过腺病毒载体将TFPI基因转染至体外培养的血管平滑肌细胞和巨噬细胞中，发现TFPI通过抑制细胞中MCP-3和CCR2的表达以及抑制MAPK及PI3K通路磷酸化激活从而抑制细胞增殖。本研究证明了MCP-3在动脉粥样硬化斑块稳定性中的发挥的作用，为AS的治疗提供新靶点，同时确定了TFPI促进AS斑块稳定性中的作用及机制，为AS的治疗提供了新的方法和思路。