丹蒌片促进兔髂动脉粥样硬化药物涂层支架置入后再内皮化的调控机制研究

Main cause of MACE after coronary stent implantation is in-stent thrombosis, while as the later is intimately associated with delayed endothelialisation. In-stent thrombosis is a severe complication due to its high mortality. Rapid reendothelialization of implanted coronary artery stent is a way for preventing in-stent thrombosis.It has been revealed that endothelial progenitor cells(EPCs) and endothelial cells(ECs) are involved in the process of re-endothelialization after DES implanting. PI3K/Akt signal transduction pathway plays an important role in the proliferation,migration and(or) mobilization,differentiation of EPCs and ECs. Management of removing phlegm and activating blood circulation,a kind of therapy of the traditional Chinese medicine,has been proved to improve the prognosis of coronary patients underwent DES implanting.As a represent drug used in the management of removing phlegm and activating blood circulation,Danlou tablet is a Chinese patent drug. Researches have proved that Danlou effects the progress of re-endothelialization by means of acting on EPCs and ECs. Further research needed to investigate the ditailed mechanism of how Danlou effects the progress of re-endothelialization after stent implanting. .Experiments is planed to perform in Vivo and vitro to prove the hypothesis of Danlou promoting endothelialization by means of PI3K/Akt signal transduction pathway. In vivo,we make rabbit model of atherosclerosis and DES implanting in iliac artery and then to observe and study proliferation, migration and differentiation of EPCs and ECs by organ-cell-molecular level.In vitro,we culture rabbit EPCs and human umbilical vein ECs(HUVECs) with Danlou, then examine the proliferation, migration and differentiation of EPCs and ECs,and changes of related ion channel protein to explor the detailed mechanism of how Danlou effecting the reendothelialization after stent implanting.

冠心病药物涂层支架(DES)置入后严重心脏事件与支架内血栓形成关系密切，内皮化不良是支架内血栓形成的重要原因。研究显示内皮祖细胞（EPCs）和内皮细胞（ECs）参与支架后再内皮化，PI3K/Akt通路对EPCs和ECs增殖、迁移和/或动员、分化起着重要作用。化痰活血法预防冠心病支架内血栓，促进内皮细胞增殖，但作用机制并不清楚。我们推测化痰活血法代表药丹蒌片能通过PI3K/Akt信号通路，促进内皮祖细胞和内皮细胞的增殖、迁移和/或动员、分化，从而促进再内皮化，预防支架内血栓形成。为证实该假说，通过制作兔髂动脉粥样硬化DES置入模型及细胞培养,从器官-细胞-分子水平，以PI3K/Akt信号通路为主线，研究丹蒌片对EPCs、ECs的影响，阐明其对DES置入后再内皮化的作用机制，为化痰活血法促进再内皮化、预防支架内血栓、改善DES置入后临床预后提供理论而和实验依据。

药物涂层支架（DES）的治疗减少了冠心病支架内再狭窄的发生率，但因损伤内皮，延迟支架后再内皮化，增加了晚期支架内血栓的发生率。丹蒌片是化痰活血法治疗冠心病的代表药物，具有降脂、改善内皮功能、抗动脉粥样硬化等作用。本研究首先通过整体动物实验观察丹蒌片在DES置入后不同阶段的内皮化的作用及PI3K/Akt信号通路蛋白的影响；通过细胞实验研究丹蒌片通过PI3K信号通路对雷帕霉素作用后内皮细胞、内皮祖细胞、平滑肌细胞增殖、迁移、凋亡的影响。为丹蒌片改善DES置入后内皮化，防治支架内血栓提供理论基础。在兔腹主动脉粥样硬化DES置入模型中，研究发现DES置入后初期支架处内皮爬行延迟，后期支架处尽管内膜覆盖，但内皮化不良，易于血小板的黏附、血栓的形成；同时激活炎症反应、抑制PI3K/Akt信号通路的激活。丹蒌片在DES置入后的早期即能有效的促进内皮的爬行和内皮化，而在晚期联合瑞舒伐他汀的治疗在促进支架再内皮化的同时，抑制新生内膜的增加，减少支架内再狭窄的发生；调节PI3K/Akt信号通路的表达。通过雷帕霉素干预内皮细胞、内皮祖细胞的离体实验，研究发现雷帕霉素通过PI3K信号通路抑制内皮细胞、内皮祖细胞的增殖、迁移，促进凋亡的作用；通过平滑肌细胞的培养，证实炎症因子同型半胱氨酸促进平滑肌细胞增殖和迁移。而丹蒌片能通过PI3K信号通路改善雷帕霉素对内皮细胞和内皮祖细胞的作用，联合瑞舒伐他汀治疗抑制平滑肌增殖和迁移、促进凋亡。进一步说明了在DES置入后的早期，丹蒌片通过上调PI3K/Akt信号通路内皮细胞和内皮祖细胞功能的增加，促进早期内皮化的完成；而在DES置入后期通过PI3K/Akt抑制平滑肌细胞细胞的增殖，达到DES置入后血管的稳态愈合。综上所述，DES置入后内皮细胞、内皮祖细胞功能受损，平滑肌细胞过度激活引起支架内皮化不良，早期易于发生支架内血栓，晚期发生支架内再狭窄；同时伴随PI3K/Akt信号通路蛋白表达的改变。丹蒌片具有促进早期支架再内皮化，联合瑞舒伐他汀治疗抑制晚期支架内再狭窄的作用，PI3K/Akt信号通路可能是其作用机制。