放射性同位素标记法检测脂蛋白脂肪酶活性及其应用

Determination of Lipoprotein Lipase Activity Using Radioactive Substrate Emulsion and Its Application

脂蛋白脂肪酶活性测定国内常用滴定法和比色法 ,但这两种方法都不够灵敏和可靠。为准确检测脂蛋白脂肪酶的活性 ,我们在国内首次建立了放射性同位素标记法 ,并用此方法检测了基因体外表达的脂蛋白脂肪酶 ,以及摄食条件下C5 7小鼠肝素后血浆中及心肌、脂肪和肝脏组织中的脂蛋白脂肪酶的活性。测定使用含3H标记三油酸甘油酯的甘油乳剂为底物。标本与底物 37℃孵育反应后通过抽提将甘油酯与游离脂肪酸分开。脂解产生的游离脂肪酸的放射活性与标本中脂肪酶的活性成正比。表达人脂蛋白脂肪酶的重组腺病毒感染培养的 2 93A细胞后 ,肝素后培养液中表达很高的脂蛋白脂肪酶活性。 5只小鼠肝素后血浆及心肌、脂肪组织中脂蛋白脂肪酶活性检测结果均值分别为 314 .3U L以及 349和 6 1mU g。结果表明 ,放射性同位素标记法用于脂蛋白脂肪酶基因表达的研究灵敏、准确和稳定。小鼠血浆和组织标本脂蛋白脂肪酶活性检测为今后利用小鼠进一步研究脂蛋白脂肪酶提供了一些资料。

Aim To study lipoprotein lipase (LPL) gene espression in vitro and observe the mouse LPL activity in post-heparin plasma and various tissue samples we adopted a standard method using radioactive emultion as substrate. Methods 3H-labeled triolein emulsion was prepared and used for subsequent incubation with different enzyme sources. Results There was high LPL activity expression in cultured 293A cells infected with recombinant adenovirus encoding human LPL. The LPL activity of post-heparin plasma,cardiac muscle,and adipose tissue of mice were 314.3 U/L, 349 mU/g and 61 mU/g respectively. Conclusions The method of determination for LPL activity was sensitive,accurate and stable. The results of LPL activity of post-heparin plasma and various tissue samples of mice provided helpful data for further research.