与启动子序列相同的单链寡聚脱氧核苷酸拮抗胰岛素对人α1(I)型胶原启动子的激活

Sense Single-stranded Phosphorothioate Oligodeoxynucleotide Promoter Competitors Inhibits Insulin Activated of Collagen gene Expression

目的探讨正义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。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NIH3T3细胞,加入不同剂量的胰岛素,测CAT值;(2)合成全硫代的ssPTODN,其序列为COL1A1基因-129～-105的25个碱基。稳定转染的细胞克隆内加入不同剂量的ssPTODN共孵育24h,加入2.5μmol/mL的胰岛素,测定CAT值。结果(1)胰岛素浓度(μmol/mL)在0,0.25,2.5,10时的CAT值(pg/mL)分别是920±94,1021±99,1595±81,1711±121。2.5μmol/mL及10μmol/mL的胰岛素显著增加了COL1A1基因启动子活性(P<0.05);二者的作用差异无显著性(P>0.05)。(2)胰岛素剂量为2.5μmol/mL,ssPTODN浓度(μg/mL)分别为0,20,40,80时,各组的CAT值(pg/mL)相应为1660±104,1625±64,1396±76,1040±135;ssPTODN浓度为80μg/mL时,受胰岛素刺激所增加的CAT值得到显著抑制(P<0.05),回复到基础水平附近。结论ssPTODN能够抑制胰岛素对人COL1A1基因启动子的激活。

Objective To investigate the effect of sense single-strand phosphorothioate oligodeoxynucleotide（ssPTODN） on promoter activity of human COL1A1 gene induced by insulin. Methods （1）The construct of pCOL1A1 2.5 containing -2 483 to ± 42 bp of the promoter and chloromycetin acetyl transferase （CAT） gene as a reporter was stably transfected into NIH 3T3 cells . The cells were treated with co-insulin of various dosage. （2）Various dosage＇s ssPTODN coinciding with -129 to -105 bp of COL1A1 gene were incubated with the transfected cells, then treated with insulin of 2.5μmol/mL. Results （1）The CAT value（pg/ mL）of insulin in 0, 0.25, 2.5, 10μmol/mL were 920 ± 94, 1 021 ± 99, 1 595 ± 81, 1 711 ± 121 respectively. （2）The CAT values（pg/mL）of ssPTODN of 0, 20, 40, 80μg/mL were 1 660 ±104, 1 625 ± 64, 1 396 ± 76, 1 040 ± 135 respectively, 80μg/mL ssPTODN decreased the stimulating effect of insulin . Conclusion ssPTODN can inhibit the promoter activity of human α（I） collagen gene induced by insulin.