OVEREXPRESSION OF HUMAN LECITHIN CHOLESTERYL ACYLTRANSFERASE IN TRANSGENIC MICE

为了评估LCAT在高密度脂蛋白代谢中的作用，6.2kb 利用人类LCAT(HLCAT)基因片段开发了三个 分离血浆水平高表达hLCAT的转基因小鼠系 比对照组(1微克/毫升)高出10、14和100倍。LCAT活动在 45只杂合子和纯合子小鼠的范围从582+/-92到 3695q340nmol/ml/h(NL=31q4nmol/ml/h)。Northern杂交 分析表明，hLCAT在小鼠肝脏中具有组织特异性表达。 与24个年龄和性别匹配的兄弟姐妹相比，转基因小鼠的比例更高 血浆TC(NL的133-237%)、CE(NL的141-267%)和高密度脂蛋白胆固醇(123-209% NL)，但含PL、B的血浆甘油三酯水平相似 脂蛋白、载脂蛋白A-I和载脂蛋白A-II。HLCAT-TG血浆的FPLC分析 发现了富含CE和PL的更大尺寸的高密度脂蛋白颗粒。 年龄/性别匹配的转基因动物(M=7，F=8)和对照(M=14，F=13)动物 然后给予高胆固醇(HF)饮食21d，以调查 LCAT在调节饮食反应中的潜在作用。餐前血脂 对照组TC=97加/减11，高密度脂蛋白=72q10，In 转基因为TC=121加/减16，高密度脂蛋白胆固醇=84加/减17。节食后 对照组血脂水平为TC=290正/负55，高密度脂蛋白=85正/负15， 转基因为TC=313+/-83，高密度脂蛋白=115+/-27。因此， 在HF饲料上转基因小鼠显著高于对照组(P<0.05)。 与对照组相比，高密度脂蛋白-C以及降低的总胆固醇/高密度脂蛋白比率没有差异 在TG、PL、CE、LCAT质量和活性中。转基因小鼠的FPLC分析 血浆中高密度脂蛋白胆固醇、CE和PL显著升高 降低IDL/LDL-C、CE和PL。 小鼠125I-apoA-I和131I-apoA-II的动力学研究 研究LCAT过度表达导致的潜在机制 以提高高密度脂蛋白水平。载脂蛋白A-I和载脂蛋白A-II分解代谢延迟 转基因(N=5)(FCR=1.9和3.4d-1)与对照(FCR=2.6)相比 和4.2d-1)小鼠。因此，转基因小鼠的高脂蛋白血症 HLCAT的过表达是高密度脂蛋白分解代谢延迟的结果。

In order to evaluate the role of LCAT in HDL metabolism, a 6.2 kb fragment of the human LCAT (hLCAT) gene was utilized to develop three separate transgenic mouse lines overexpressing hLCAT at plasma levels 10,14 and 100 fold higher than control (1 ug/ml) mice. LCAT activity in 45 heterozygous and homozygous mice ranged from 582plus/minus 92 to 3695q340 nmol/ml/h (NL=31q4 nmol/ml/h). Northern blot hybridization analysis demonstrated tissue specific expression of hLCAT in mouse liver. Compared to 24 age and sex-matched siblings, transgenic mice had elevated plasma TC (133-237% of NL), CE (141-267% of NL) and HDL-C (123-209% of NL) but similar plasma levels of triglycerides, PL, B-containing lipoproteins, apoA-I and apoA-II. FPLC analysis of hLCAT TG plasma revealed larger sized HDL particles enriched in CE and PL. Age/sex matched transgenic (M=7, F=8) and control (M=14, F=13) animals were then placed on a high chol-fat (HF) diet for 21 d to investigate the potential role of LCAT in modulating dietary responses. Pre-diet lipid values (mg/dl) in controls were TC=97plus/minus11, HDL=72q10 and in transgenics were TC=121 plus/minus16, HDL-C=84 plus/minus17. Post-diet lipid values in controls were TC=290plus/minus55, HDL= 85plus/minus15, and in transgenics were TC=313plus/minus83, HDL=115plus/minus27. Thus, on the HF diet transgenic mice had significantly higher (pless than0.05) HDL-C as well as reduced TC/HDL ratios than controls, without differences in Tg, PL, CE, LCAT mass and activity. FPLC analysis of transgenic mouse plasma revealed significant increases in HDL-C, CE and PL with reciprocal decreases in IDL/LDL-C, CE and PL. Mouse 125I-apoA-I and 131I-apoA-II kinetic studies were performed to investigate the underlying mechanisms by which LCAT overexpression leads to increased HDL levels. ApoA-I and apoA-II catabolism was delayed in transgenic (N=5) (FCR=1.9 and 3.4d-1) when compared to control (FCR=2.6 and 4.2d-1) mice. Thus, hyperalphalipoproteinemia in transgenic mice overexpressing hLCAT results from delayed catabolism of HDL.