PDI抑制剂对小鼠体内移植静脉粥样硬化狭窄的抑制作用及机制研究

Coronary atherosclerosis is an important cause of death of heart infarction, and autologous vein transplantation is an important method to treat the disease. However, 50% of the transplanted vessels in the 5-10 years after surgery were narrowed and blocked, which hindered the promotion of the operation. Whether or not to inhibit the postoperative atherosclerosis stenosis is the key problem to be solved in this study. Recent studies had shown that mouse veins were grafted into carotid arteries of non-diabetic and diabetic mice for 8 weeks, the vein graft arterializations in non-diabetic mice or atherosclerosis in diabetic mice were observed, respectively. Histological analysis indicated that simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMC) in the vein grafts were the main causes of venous atherosclerosis (VS). Mechanically, research results showed that weak expression of protein disulfide isomerase (PDI) in VSMC triggered cell proliferation, while high PDI expression contributed to cell apoptosis. In vitro study represented that inhibition of PDI activity could simultaneously inhibit the increases in proliferation and apoptosis of VSMCs. But there are no reports to show whether in vivo inhibition of PDI can inhibit the simultaneous increases of proliferation and apoptosis of VSMCs in the grafted veins. Thus, our academic hypothesis is that inhibition of PDI results in simultaneous inhibition of VSMC proliferation and apoptosis, and ultimately inhibits vein graft atherosclerosis formation. On the basis of established arterial-venous bridge model, PDI inhibitors will be used, and cellular/molecular biology, biochemistry, signal transduction and epigenetic inheritance techniques will be adopted to observe the effect of PDI inhibitors on vascular remodeling. The results would provide important experimental data and new treatment strategies, new drug targets and new molecular mechanisms for the prevention and treatment of vein graft atherosclerosis.

冠脉粥样硬化是心梗致死重要原因，自体静脉移植是治疗该病的重要手段，但术后5-10年50%的移植血管狭窄堵塞，阻碍了该手术推广，能否抑制静脉移植术后狭窄是本研究要解决的关键问题。最新研究显示，静脉移植到正常小鼠颈总动脉8周，移植静脉动脉化，而移植到高血糖鼠则粥样硬化。组织学发现移植静脉平滑肌细胞（SMC）增生和凋亡同时增加是静脉粥样硬化（VS）发生主要原因。机制探讨显示，SMC中低水平表达PDI促进细胞增生，而高水平表达PDI则促进凋亡。体外抑制PDI活性，可同时抑制细胞增生和凋亡增加，但体内能否同时抑制增生和凋亡迄今无报道。学术假设：抑制PDI同时抑制细胞增生和凋亡，最终抑制体内移植VS形成。拟在已建立的动-静脉桥模型上，施加PDI抑制剂，用细胞/分子生物学、生物化学、信号转导及表观遗传等方法，观察PDI抑制剂对血管重构影响，为防治移植VS提供重要的实验资料和新策略、新靶点、新机制。

为了研究蛋白质二硫键异构酶（PDI）抑制剂对高血压机械力对动脉粥样硬化（AS）的影响机制，本研究使用饲喂高脂的ApoE基因敲除小鼠建立AS模型，小檗碱（黄连素，BBR）灌胃4、8周，收集动-脉静脉血液及血管，测定血脂与血糖。血管做石蜡切片、HE染色，观察病理改变，自身静脉作对照。体外，分离的血管平滑肌细胞（VSMC）静息培养，给予机械牵张力（SS）刺激不同时间后收集细胞，观察细胞增殖、凋亡及相关信号变化；并探讨VSMC对相同胞外刺激而呈现不同反应的异质性机制。结果显示：高血脂可诱导AS发生，但静脉结构无变化。高血脂组的动脉血与静脉血的血脂、血糖浓度均比对照组高，但小鼠自身的动脉、静脉血脂、血糖浓度均无明显差异。BBR可抑制AS发生发展，但对血脂、血糖浓度的影响不明显。SS可引起VSMC迁移增加、增殖与凋亡同时增加，许多相关信号被激活，如内质网应激通路、PKC-δ、-alpha通路、MAPK通路等；BBR可抑制VSMC机械力诱导的上述效应与信号。结论：血压升高产生的机械力对血管病变起决定作用，高血脂起协同促进作用。BBR可抑制机械力诱导的VSMC迁移、增殖与凋亡同时增加，并抑制相关信号分子激活，实现抑制高血脂引起的AS发生发展。本研究可为高血压血管重构与疾病发生的防治及机制研究提供重要资料，探讨BBR防治心血管病的机制并拓展老药新用途。此外，为了深入揭示PDI在介导机械力信号调控细胞氧化应激、内质网应激以及激酶激活等通路起其至关紧要的作用，本研究构建了可诱导型小鼠VSMC-PDI基因敲除小鼠，目前已经获得PDI基因敲除纯合子小鼠，并正在构建与筛查ApoE基因与PDI基因双敲除小鼠纯合子，已经取得重要进展。该模型鼠将引入高脂饮食诱导高血脂、STZ诱导高血糖以及动脉缩窄诱导高血压等模型，用于后续的PDI介导高血压机械力、高血脂、高血糖单独或联合对血管重构的作用与机制研究，为防治“三高”相关的血管疾病的致病作用与机制研究提供重要工具。