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
Effects of lipoproteins and insulin on phospholipid transfer protein content and mRNA expression in human hepatoblastoma (HepG2) cells
中文摘要
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。
英文摘要
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.
