利用基因敲入家兔研究载脂蛋白A-II在动脉粥样硬化发生中的作用

Plasma levels of high density lipoproteins (HDL) are inversely associated with risk of atherosclerosis. HDL protects vascular functions by its anti-atherogenic actions. HDL particles contain two major apolipoproteins (apo): apoA-I and apoA-II. Compared with apoA-I, pathophysiological roles of apoA-II in vivo is not fully understood. This is mainly due to lack of appropriate animal models because mouse metabolic system along with mouse apoA-II structures is quite different from those of humans. Rabbits are one of the best animal models for the study of lipid metabolism and atherosclerosis because they are sensitive to a cholesterol diet. In addition, rabbits are naturally deficient in apoA-II gene therefore, rabbit HDLs contain only apoA-I without apoA-II. The purpose of this project is to elucidate the functional roles of apoA-II on lipid metabolism and atherosclerosis. For this undertaking, we will first (1)generate apoA-II gene knock-in (KI) rabbits in which human apoA-II gene is introduced into rabbit apoA-I gene locus using TALEN technology;(2)then, we will characterize the lipid and lipoprotein metabolism in apoA-II KI rabbits compared with wild-type rabbits on a chow diet;(3)we will examine these apoA-II KI rabbits in terms of their susceptibility to cholesterol diet-induced atherosclerosis; and(4)coronary plaque stability and myocardial infarction in the setting of LDL receptor deficiency using WHHL rabbits, (5)we will also investigate the molecular mechanisms such as cholesterol efflux, anti-inflammatory properties, anti-oxidization using isolated HDLs from apoA-II KI and wild-type rabbits. This is the first study to examine apoA-II pathophysiological functions in lipoprotein metabolism using apoA-II KI rabbit models and our results will not only provide a novel insight into understanding apoA-II molecular mechanisms in atherosclerosis but also explore the possibility of using apoA-II as a therapeutic strategy for treatment of atherosclerosis.

血清高密度脂蛋白(HDL)与动脉硬化疾病发生呈负相关,HDL具有抗动脉硬化保护血管的功能。HDL有两种主要载脂蛋白即apoA-I和apoA-II,和apoA-I相比,apoA-II的生理功能以及在脂质代谢异常和动脉硬化的发生中的作用目前还没有阐明。本项研究的目的是(1)利用TALEN靶向基因敲除敲入技术，首先开发研制apoA-II基因敲入家兔模型;(2)研究apoA-II基因敲入家兔在正常饮食条件下的脂质代谢特点;(3)利用体外细胞培养和分子生物学手段研究含有不同apoA-I和apoA-II的HDL功能特性;(4)研究高脂饮食诱导情况下apoA-II基因敲入家兔的动脉硬化的特性;(5)利用LDL受体缺失WHHL家兔,研究apoA-II对冠状动脉斑块的形成及心肌梗死的发生的影响。以此揭示HDL的抗动脉硬化分子机制,为将来开发以apoA-II为靶点的治疗动脉硬化新方法提供理论依据。

载脂蛋白AII (apoAII) 是高密度脂蛋白(HDL)的主要蛋白成分之一，但是与apoAI相比，apoAII在脂质代谢的生理功能以及在高脂血症和动脉硬化发生中的作用并不清楚。由于野生型家兔先天缺失apoAII基因，他们的HDL的蛋白成分里只有apoAI没有apoAII。我们在本项研究的主要目的是：TALEN靶向基因敲除敲入技术，独自开发建立apoA-II基因敲入（knock-in，KI）同时敲除内源性apoA-I基因家兔模型，研究在没有apoA-I背景下，apoA-II在脂质代谢中的生理学功能，及apoA-II在动脉硬化发生中的作用。.重要结果：（1） apoA-II-家兔的肝脏和血液中只表达apoAII而没有apoAI。在喂养正常饲料的情况下，与野生型家兔相比，apoA-II-KI家兔出现明显总胆固醇升高和甘油三酯的降低。通过超速离心密度梯度方法分析脂蛋白的变化发现，和野生型家兔相比，apoA-II-KI的VLDL和IDL水平减少而HDL1和HDL2增加，但是HDL3没有变化。利用琼脂胶电泳分析脂蛋白发现，apoA-II-KI的HDL明显高于野生型家兔而且出现VLDL和IDL的减低。我们也对apoA-II-KI的脂蛋白进行了液相色谱分析，结果表明，通过导入apo-AII取代apo-AI使HDL颗粒变小，明显出现以颗粒小的HDL2占优势，在液相色谱图像上出现HDL峰值向前移动。 Western blot分析及蛋白组学分析载脂蛋的变化发现，apoA-II-KI家兔的HDL与野生家兔相比出现一下几种显著变化，第一是颗粒小的LDL中apoB100明显增加，此外，apoE的含量在HDL颗粒中增加15倍，apoAIV和apoAV也明显增加。KI家兔和野生型家兔喂养高胆固醇饲料16周，ApoAII-KI家兔对胆固醇饲料明显要拮抗性，以此与对照组相比，出现较低的血脂水平，主动脉和冠状动脉粥样硬化的病变明显减少。我们从野生型和KI家兔里提取HDL进行体外细胞培养，发现apoAII的HDL具有与apoAI一样的抗炎症和促进胆固醇从巨噬细胞里流出的功能。.本项研究的科学意义在于，第一首次建立了新型apoAII动物模型，第二提一次阐明apoAII具有抗高脂血症及动脉硬化的作用，为今后开发以apoAII为靶点的抗动脉硬化治疗药物开辟了新的道路。