利用基因敲除家兔研究salusins影响动脉粥样硬化发生发展的机制

Salusin-α和β是同一基因(TOR2A)的选择性剪接产物。研究两者分别与动脉粥样硬化(AS)的关系，需TOR2A敲除模型,以排除内源性salusins干扰。家兔脂代谢特征与人相似，适合AS研究。本研究拟用TALEN技术制备TOR2A敲除家兔，在其皮下埋植微渗透泵，分别输注salusin-α、β或生理盐水，输注生理盐水的野生型家兔为空白对照组。所有家兔饲喂高脂饲料，诱导AS，观察家兔血脂、脂蛋白、载脂蛋白及相关酶水平，分析salusins对脂代谢的影响。比较四组家兔主动脉AS病变大小、免疫组化检测AS斑块组成成分，并结合组织学方法分析斑块稳定性。用细胞实验研究salusins对胆固醇代谢和炎症过程的影响，结合基因芯片技术筛选出可能的信号通路。用信号关键分子抑制剂干预细胞，研究信号转导机制。综合体内外研究，分析salusins影响AS发生的机制，为心血管疾病的防治提供新的理论和实验依据。

Salusin-α and salusin-β are considered to be processed from preprosalusin, an alternative-splicing product of the torsion dystonia-related gene(TOR2A), after frameshift reading and digestion at dibasic amino acids. Knock out animal model are needed to reveal the effects of salusins on the progression of atherosclerosis. Rabbits have been extensively utilized as an ideal model for atherosclerosis because of its size, easy manipulation, and extraordinary response to dietary cholesterol. In this project, TOR2A knock out rabbits,which will be created by transcription activator-like effector nucleases (TALEN) techniques, will be performed the subcutaneous continuous infusion of salusin-α,salusin-β or saline,respectively through osmotic mini-pumps in three groups knock out rabbits. Wide type rabbits infused of saline will be used as control group. All rabbits will be fed high cholesterol diet to induce atherosclerosis. We also plan to analyze how salusins influences lipid metabolism by comparing the plasma lipid level,lipoprotein profile and lipid related enzymes. The characteristics of atherosclerotic lesion will be compared in four groups by immunohistochemisty staining, Western Blot and real time PCR. In vitro study will be performed on cultured primary macrophages to analyze the cholesterol metobolism. Human umbilical vein endothelial cells will be used in transcriptomics study and the following signal pathway study. The perspective results of this study are expected to be useful for the identification of an novel drug target for atherosclerosis.