OVEREXPRESSION OF HUMAN LECITHIN CHOLESTERYL ACYLTRANSFERASE IN TRANSGENIC MICE

转基因小鼠中人卵磷脂胆固醇酰基转移酶的过度表达

• 批准号: 3757645
• 负责人: S SANTAMARINA-FOJO
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3757645
• 关键词: apolipoproteins; blood lipid; blood lipoprotein metabolism; cholesterol; dietary lipid; enzyme activity; gene expression; genetically modified animals; high density lipoproteins; high performance liquid chromatography; human genetic material tag; hyperlipoproteinemia; laboratory mouse; northern blottings; nutrition related tag; pathogenic diet; phosphatidylcholine sterol acyltransferase; protein purification

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.

为了评估 LCAT 在 HDL 代谢中的作用，一个 6.2 kb 利用人类 LCAT (hLCAT) 基因片段开发了三种 分离在血浆水平过表达 hLCAT 的转基因小鼠品系 比对照 (1 ug/ml) 小鼠高 10,14 和 100 倍。 LCAT 活性 45 只杂合子和纯合子小鼠的范围从 582plus/minus 92 到 3695q340 nmol/ml/h (NL=31q4 nmol/ml/h)。 Northern印迹杂交 分析表明 hLCAT 在小鼠肝脏中的组织特异性表达。 与 24 名年龄和性别匹配的兄弟姐妹相比，转基因小鼠的 血浆 TC（NL 的 133-237%）、CE（NL 的 141-267%）和 HDL-C（NL 的 123-209%） NL) 但甘油三酯、PL、B 的血浆水平相似 脂蛋白、apoA-I 和 apoA-II。 hLCAT TG 血浆的 FPLC 分析 揭示了富含 CE 和 PL 的较大尺寸 HDL 颗粒。 年龄/性别匹配的转基因（M=7，F=8）和对照（M=14，F=13）动物 然后进行 21 天的高胆脂 (HF) 饮食，以研究 LCAT 在调节饮食反应中的潜在作用。餐前脂质 对照中的值（mg/dl）为 TC=97plus/minus11，HDL=72q10，在 转基因为TC=121加/减16，HDL-C=84加/减17。节食后 对照组的血脂值为 TC=290plus/minus55，HDL= 85plus/minus15， 在转基因中，TC=313plus/minus83，HDL=115plus/minus27。因此， HF饮食转基因小鼠显着高于（p小于0.05） HDL-C 以及 TC/HDL 比值低于对照，无差异 Tg、PL、CE、LCAT 质量和活性。转基因小鼠的FPLC分析 血浆显示 HDL-C、CE 和 PL 显着增加，且呈倒数 IDL/LDL-C、CE 和 PL 降低。 进行小鼠 125I-apoA-I 和 131I-apoA-II 动力学研究 研究 LCAT 过度表达导致的潜在机制 提高 HDL 水平。 ApoA-I 和 apoA-II 分解代谢延迟 与对照 (FCR=2.6) 相比，转基因 (N=5) (FCR=1.9 和 3.4d-1) 和4.2d-1)小鼠。因此，转基因小鼠的高α脂蛋白血症 hLCAT 过度表达是 HDL 分解代谢延迟的结果。