フローサイトメトリーによるDiI-LDLを用いたLDLレセプター活性測定法

Abstract

A simple, specific flow cytometric method for the diagnosis of familial hypercholesterolemia (FH), caused by mutation of the LDL receptor gene, has been developed. This method involving measurement of fluorescent-labeled LDL (DiI-LDL) uptake by lymphocytes has been evaluated. Lymphocytes isolated from blood were incubated in lipoproteinfree medium for 72h to upregulate the LDL receptor.After incubation, cells were collected and then suspended in PBS containing 1% BSA. Each cell suspension (1×105 cells/100μl) was incubated at 37°C for 2h with 1μg of DiI-LDL. After being washed, the fluorescent intensity uptaken by cells was measured by FACScan flow cytometry. In contrast to freshly isolated lymphocytes from subjects with normolipidemia, which showed very little uptake of DiI-LDL, lymphocytes after upregulation of the LDL receptor showed nearly a 10-fold increase. Uptake was inhibited by excess unlabeled LDL, 0.2mg/ml heparin and 2μM EGTA, but not 100μg/ml acetylated LDL, indicating that uptake of DiI-LDL by lymphocytes was mediated through a specific LDL receptor. Comparative studies demonstrated a correlation between the uptake of DiI-LDL and degradation of 125I-LDL. To determine if this method could be used for the diagnosis of FH patients with a defective LDL receptor, the uptake of DiI-LDL by lymphocytes from thirty-two patients with clinically diagnosed FH was compared to that of fifty-six control subjects with a normal lipid profile. In 30 of 32 FH patients, LDL receptor activity was 23 to 76%, whereas more than 80% of activity in control subjects. Normal receptor activities were observed in 20 of 21 hyperlipidemias without FH. These findings suggest that this simple flow cytometric method can be useful in the diagnosis of FH patients with a defective LDL receptor. This procedure is less complicated than techniques currently available for the measurement of functional LDL receptors, and has the reproducibility with intea- and inter-assay variations of less than 10% and 15%, respectively.