Role of apo (a) in atherogenesis. A study using transgenic rabbits expressing human apo (a)

表达人载脂蛋白(a)的转基因家兔制备转基因家兔：我们使用肝脏特异性启动子小鼠转铁蛋白启动子和人类载脂蛋白(a) cDNA，其中包含17个kringle 4拷贝。共2836个受精卵进行显微注射，得到96只幼崽，其中11只幼崽基因组中有人类载子(a)转基因。经过多次育种，我们得到了3种血浆中可检测到载脂蛋白(a)的转基因家兔。转基因家兔的特性：经Northern blot分析证实，人载脂蛋白(a)在肝脏中表达。SDS-PAGE测定人载脂蛋白(a)大小约为500kD。在转基因兔血浆中，人载脂蛋白(a)与兔载脂蛋白ob结合形成Lp (a)复合物，但脂蛋白谱不受载脂蛋白(a)表达的影响。在鼠粮中，转基因家兔不发生任何动脉粥样硬化病变。载脂蛋白(a)转基因家兔饲喂胆固醇饲料16周。当食用高胆固醇食物时，转基因家兔比非转基因家兔更容易发生动脉粥样硬化。转基因家兔所有动脉树病变均增加，包括主动脉、髂动脉、颈动脉和冠状动脉。免疫组织化学染色研究表明载脂蛋白(a)可能通过调节SMC表型改变来促进病变的发展。WHHL转基因兔：将WHHL与转基因兔杂交，获得WHHL转基因兔，研究LDL受体是否参与Lp (a)分解代谢。我们发现，在LDL受体缺乏的情况下，Lp (a)在血浆中明显积累，表明Lp (a)的清除在一定程度上受LDL受体的控制。

Transgenic rabbits expressing human apoprotein (a)1. Production of transgenic rabbits :We used liver-specific promoter mouse transferrin promoter and human apo (a) cDNA containing 17 copies of kringle 4. After microinjection of zygotes (2836 in total), we got 96 pups and found that 11 pups had human apo (a) transgene in their genome. After several breeding, we got 3 lines of transgenic rabbits that have detectable apo (a) in the plasma.2. Characterization of transgenic rabbits :Human apo (a) was expressed in the liver as confirmed by Northern blot analysis. Human apo (a) size was about 500kD as determined by SDS-PAGE.In transgenic rabbit plasma, human apo (a) was associated with rabbit apoB to form Lp (a) complex but lipoprotein profile was not effected by apo (a) expression. On a chow diet, transgenic rabbits do not develop any atherosclerotic lesions.3. Characterization of apo (a) transgenic rabbits fed a cholesterol diet for 16 weeks. When on a cholesterol diet, transgenic rabbits developed more atherosclerosis than did nontransgenic control. Increased lesions were found in all arterial trees in transgenic rabbits, including aorta, iliac and carotid arteries, and coronary artery. Immunohistochemical staining study showed that apo (a) may enhance the lesion development by modulating SMC phenotype changes.4. WHHL transgenic rabbits :After cross-breeding between WHHL and transgenic rabbits, we got WHHL transgenic rabbits to investigate whether LDL receptor is involved in Lp (a) catabolism. We found that in the setting of LDL receptor deficiency, Lp (a) was markedly accumulated in the plasma suggesting that Lp (a) clearance is controlled in part, by LDL receptor.