摘要
目的:1.探讨含启动子序列的单链寡脱氧核糖核苷酸(ssPTODN)抑制胰岛素对人 α1(I)型胶原(COL1A1)基因启动子的激活。2.进一步确定胰岛素在人成 纤维细胞COL1A1 基因上的反应元件
方法:1. 将含有氯霉素乙酰基转移酶(CAT) 基因与人COL1A1 基因-2483~ +42bp 片段的重组质粒(pCOLH12.5)稳定转染至人胚肺成纤维细胞 WI-38 中, 建立稳定转染的WI-38 细胞株,于该细胞株中加入不同剂量 的胰岛素,测CAT 值;2.合成全硫代的不同序列ssPTODN,其中 ssPTODN1 的序列为COL1A1 基因-129 至-105 间的25 个碱基。瞬时转染 不同剂量的ssPTODN1 于克隆的细胞株中,静息后,分别加入2.5μmol/ml 的胰岛素,测CAT 值。
结果:1.成功建立了整合有pCOLH12.5 的WI-38 细胞株PN1。2.胰岛素浓度在 0、0.25、2.5、10μmol/ml 时的CAT 值(pg/ml)分别是920±94、1021 ±98、1595±101、1711±117。2.5μmol/ml 及10μmol/ml 的胰岛素显著 增加了COL1A1 基因启动子活性(P<0.01),二者的作用无显著性差异 (P>0.05)。3.胰岛素剂量为2.5μmol/ml,ssPTODN1 浓度分别为0、 40nM、80nM 、120nM、160nM时,细胞株PN1的各组校正后CAT值(pg/ml) 相应为:1593±87、1535±49、1370±54、1147±93、899±85;单独胰 岛素作用组的CAT 校正值为1601±23(pg/ml);ssPTODN1 浓度为120nM 和160nM 时,受胰岛素刺激所增加的CAT 值得到显著抑制(P<0.01), 但160nM 剂量时所抑制的水平低于启动子的基础CAT 值。
结论: 1. ssPTODN1 能够抑制胰岛素对人成纤维细胞COL1A1 基因启动子的激 活。2.胰岛素在人成纤维细胞COL1A1 基因上的反应元件被定位于该 基因启动子近端-129 至-105 区域内。
Objective:(1)To investigate the effect of sense single-strandphosphorothioate oligodeoxynucleotide(ssPTODN) conciding with promotergenes on promoter activity of human COL1A1 gene induced by insulin.(2)Tolocate the response element of insulin on human COL1A1 gene moreprecisely.
Methods: (1) The construct of pCOLH_12.5 containing –2483 to +42 bp ofthe promoter and CAT gene as a reporter was stably transfected into WI-38cells.The cell clone integrated with pCOLH_12.5 were treated with insulinof various dosages.(2) Four different ssPTODNs were synthesized. Variousdosage’s ssPTODN1 coinciding with position -129 to -105 of COL1A1 genewere transitently transfected into the cell clone, consequently treatedwith insulin of 2.5μmol/ml.
Results: (1) the WI-38 cell clone named PN1 containing pCOLH12.5 wasSuccessfully constructed. (2) At 0、0.25、2.5、10μmol/ml insulin,the CATvalues(pg/ml)were 920±94、1021±98、1595±101(P<0.01) and 1711±117a(P<0.01) respectively. But it showed no significance between 2.5 and10μmol/ml insulin (P>0.05).(3) At 0、40、80 、120、160nM ssPTODN1, theCAT values(pg/ml)were 1593±87、1535±49、1370±54、1147±93(P<0.01)and 899±85(P<0.01) respectively in the presence of 2.5μmol/ml insulin.At 120nM ssPTODN1, insulin-induced increase in CAT value was abrogated
to basal level.Conclusions: (1)The ssPTODN coinciding with position -129 to -105 ofCOL1A1 gene can inhibit the promoter activity of human α1(I) collagen geneinduced by insulin.(2)The location of insulin response element on humanCOL1A1 gene is suggested between -129 and -105 in the promoter.
引文
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