脂蛋白及胰島素對人肝真瘤 (HepG2) 細胞內磷脂質轉移蛋白含量與 mRNA 表 現的影響

1. 脂蛋白及胰島素對人肝真瘤(HepG2)細胞內磷脂質轉移蛋白含量與mRNA表 現的影響 • 人體血漿中的磷脂質轉移蛋白(phospholipid transfer protein,PLTP)已 • 證實 會促進磷脂質由脂質體(liposomes)、極低密度脂蛋白(VLDL)或低 • 密度脂蛋白 (LDL)轉移至高密度脂蛋白(HDL)，以及在促進HDL之轉換作 • 用上扮演極重要角 色，但其在體內的生理功能仍鮮為人知。本研究之 • 目的乃是以人類肝真瘤(HepG2細胞作為實驗模型，來探討HepG2細胞中 • PLTP含量和基因之轉錄作用是否會 受到不同脂蛋白及胰島素的調節 • 影響。實驗乃先將HepG2細胞以90% MEM (minimum essential • medium)、10 % 胎牛血清和1 mM丙酮酸鈉為生長培養基， 其中添加 50 • U/mL 盤尼西林和 50 ug/mL 鏈黴素，於 37℃ 和 5% CO2 環境下 培育生 • 長。當細胞生長至 90% 滿時，則轉換至不含血清及抗生素的基本培養基 • 培養 24 小時，之後添加 HDL(50 ug/mL)、LDL(50 ug/mL) 或VLDL (50 • ug/mL) 等不同脂蛋白或胰島素(1 ug/mL) 於培養液中 12-24 小時，而 • 以沒有添加任何脂蛋白及胰島素者作為控制組。收集 12 和 24 小時的培 • 養液進行蛋白質含量分析，並且收集 HepG2 細胞分析 PLTP 含量和基因 • 表現，其中 PLTP 含量是利用 電泳法 (10 % SDS-PAGE) 及Western • blotting 分析，而基因表現則是以 slot blotting 來分析 PLTP mRNA • 含量，並且以影像分析系統定量之。結果顯示， 不論 12 或 24 小時之 • 培養液中，添加 LDL、VLDL 和胰島素組之蛋白質含量顯 著低於控制組 • (p<0.05)，且添加不同脂蛋白和胰島素組之 24 小時培養液中蛋 白質含 • 量較 12 小時有顯著地增加 (p<0.05)。HepG2 細胞中 PLTP 含量 (MW • 44 kDa)，於添加不同脂蛋白、胰島素組與控制組間無顯著性差異，而且 • PLTP mRNA 分析結果顯示：添加不同脂蛋白、胰島素組與控制組間亦無 • 顯著性差異。 所以，不同脂蛋白及胰島素對 HepG2 細胞中 PLTP 含量與 • 基因轉錄並無明顯調 節作用。

2. Effects of lipoproteins and insulin on phospholipid transfer protein content and mRNA expression in human hepatoblastoma (HepG2) cells • Human plasma phospholipid transfer protein (PLTP) was • identified to promote phospholipid transfer from liposomes, • very low density lipoprotein (VLDL) or low density • lipoprotein (LDL) to high density lipoprotein (HDL). PLTP • also plays an important role to facilitate HDL conversion. • However, its physiological function is less known. The • purpose of this study was to investigate the effects of • lipoproteins and insulin on PLTP content and gene • transcription in human hepatoblastoma (HepG2) cells. HepG2 cells • were grown in 90 % MEM (minimum essential medium) • supplemented with 10 % of fetal bovine serum, 1 mM • sodium pyruvate, 50 U/mL penicillin, and 50 ug/ml • streptomycin at 37℃ and in 5 % CO2 atmosphere. Upon 90 % • confluency, the cells were switched to serum-free media • without antibiotics for 24 hours. After 24-hour serum-free • incubation, HDL (50 ug/mL), LDL (50 ug/mL), VLDL (50 ug/ • mL) or insulin (1 ug/mL) was added to the media for 12-24 hours. • The control group was without any addition of lipoproteins or • insulin. The conditioned media were collected at 12 and • 24 hours for protein analysis. Cells were collected for the • measurements of PLTP content using 10 % SDS-PAGE and • Western blotting, and PLTP mRNA expression using slot blotting. • The results showed that protein secretion into the conditioned • media in LDL, VLDL and insulin treatment groups was • significantly lower than the control group at both 12 and • 24 hours (p<0.05). In addition, protein secretion was • significantly increased at 24h than at 12h in all groups • (p<0.05). The cellular PLTP exhibited one major band • with molecular weight of 44.0 kilodaltons (kDa) in • HepG2 cells. The cellular PLTP was not significantly • different among the five groups. The level of PLTP mRNA was • similar among the five groups. In conclusion, lipoproteins • and insulin did not significantly affect PLTP content and gene • transcription in HepG2 cells.