细胞导向药物TGFα-SAP涂层支架改善PCI术后血管重构及其内在机制的研究

Current drug-eluting stent has reduced the rate of restenosis but also brought the problem of thrombosis, which is related to the endothelial injury caused by the non-selective cytotoxicity of drug coating. Therefore, selective inhibition of the proliferation of smooth muscle cells (SMC) becomes an urgent problem facing current interventional treatment. While the mechanism of restenosis is related to the phenotypic transition, migration, and proliferation of SMC, vascular progenitor cells are also involved in the reconstruction process after arterial intima injury. After the transition of SMC, EGFR(Epidermal Growth Factor Receptor) is significantly upregulated, while its conformation and binding capacity change too. Upon binding with its embryonic ligand TGFα, semi-toxin saporin selectively inhibits the proliferation and migration of SMC via EGFR pathway, while the dissociated TGFα post endocytosis also has the ability to chemoattract ECs（endothelial cell） and EPCs (endothelial progenitor cells EPCs) migration and promote endothelial repair through a paracrine mechanism. Therefore, the present project synthesizes the cell-targeting agent TGFα-SAP by SPDP chemical conjugation method and further investigates its role and mechanism in inhibiting intimal hyperplasia and promoting endothelial repair, by using the coated stent delivery system and the miniature pig model of combined atherosclerosis and coronary damage. Moreover, human SMC, EC, smooth muscle progenitor cells and endothelial progenitor cells are also cultured to explore the mechanism of receptor mediating cell proliferation,differentiation and apoptosis;to explore the regulation mechanisms of receptor polymerization;to explore the mechanisms of intervention of progenitor cells involving in vascular reconstruction, through methods such as cytotoxicity, chemotaxis, ligand binding, co-immunoprecipitation, and gene silencing, to provide experimental basis for its application in preventing the restenosis and thrombosis clinically.

现有药物支架降低了再狭窄发生率，但也带来了血栓形成的问题，这与涂层药物非选择性细胞毒作用导致的内皮损伤有关，选择性抑制SMC增殖是目前介入治疗研究的难题。再狭窄机制与SMC表型转化及迁移增殖有关，血管祖细胞也参与重构。SMC表型转化后EGFR明显上调且结合力改变，其胚胎期配体TGFα与半毒素Saporin联结后更易经EGFR靶向抑制SMC的迁移增殖，而TGFα内吞后可再进入旁分泌循环趋化EC或EPC迁移，促进内皮修复。本课题以SPDP化学联接法合成细胞导向药物TGFα-SAP，通过动脉粥样硬化合并冠脉损伤的小型猪模型和涂层支架给药系统，探讨其抑制内膜增生和促进内皮修复的作用和机制，并培养人SMC、EC、SMPC和EPC，通过细胞毒性、趋化、配体结合实验和免疫沉淀、RNA干扰等方法探讨其受体介导和多聚化调控机制以及干预祖细胞参与血管重建的作用和机制，为其预防再狭窄和血栓形成的应用提供实验依据

现有药物支架降低了再狭窄发生率，但也带来了血栓形成的问题，这与涂层药物非选择性细胞毒作用导致的内皮损伤及内皮化不全有关，选择性抑制平滑肌细胞增殖并促进内皮修复是目前介入治疗研究的难题。再狭窄机制与SMC 表型转化及迁移增殖有关，内膜增生时平滑肌细胞发生表型转化并伴随EGFR明显上调且结合力改变，其胚胎期配体 TGFα 与半毒素 Saporin 联结后更易经 EGFR 靶向抑制 SMC 的迁移增殖，而 TGFα内吞后可再进入旁分泌循环趋化 EC或EPC迁移，促进内皮修复。我们发现以其胚胎期的主要配体TGFα作为导向因子联结半毒素蛋白Saporin经EGFR可有效抑制表型转化后平滑肌细胞的增殖，并促进内皮修复。本课题组在国内外首次以SPDP方法联接合成细胞导向药物TGFα-SAP，并通过培养的人平滑肌细胞和内皮细胞，在体外实验中证实TGFα-SAP通过上调的EGFR受体介导可有效抑制增殖平滑肌细胞的增殖，同时减少对内皮细胞的细胞毒性作用。而与bFGF-SAP相比，TGFα-SAP对增殖平滑肌细胞具有更特异的选择性细胞毒性作用。而选择性EGFR酪氨酸激酶抑制剂Gefitinib也可选择性抑制平滑肌细胞增殖，其机制促进平滑肌细胞凋亡以及抑制AKT通路有关。同时课题组通过检测EGFR的表达变化，发现增生内膜和增殖平滑肌细胞的EGFR的表达均有明显上调，同时EGFR表型分布也有明显变化，而通过EGFR的介导，TGFα-SAP和Gefitinib均对增殖平滑肌细胞显示出较内皮细胞明显增强的选择性细胞毒性作用。同时课题组首次构建了TGFα-SAP涂层支架和涂层球囊给药系统，以及Gefitinib涂层球囊，通过建立的大鼠颈动脉球囊拉伤模型和动脉粥样硬化合并冠脉损伤的小型猪模型，通过涂层支架和涂层球囊给药系统从在体实验观察证实TGFα-SAP其抑制内膜增生和改善再狭窄的作用，同时减少细胞毒性药物对内皮修复的抑制作用，减少PCI植入术后支架内血栓形成。而Gefitinib涂层球囊同样也可通过抑制AKT/PI3K通路抑制球囊损伤后大鼠颈总动脉的内膜过度增生，结果提示EGFR信号转导系统在血管损伤后平滑肌细胞表型转变和内膜过度增生过程起着重要作用，该结果为EGFR靶向药物及其药物输送系统在预防再狭窄和血栓形成的临床应用提供依据。