含启动子序列的单链寡核苷酸拮抗胰岛素对人α1（Ⅰ）型胶原启动子的激活

发布时间：2017-12-31 14:17

本文关键词：含启动子序列的单链寡核苷酸拮抗胰岛素对人α1（Ⅰ）型胶原启动子的激活　出处：《福建医科大学》2005年硕士论文　论文类型：学位论文

【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(P0.01),二者的作用无显著性差异 (P0.05)。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(P0.01), 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。
[Abstract]:Objective: 1. To investigate the inhibitory effect of insulin on human 伪 1 I) collagen type 1 by single strand oligodeoxyribonucleotide (ssPTODN) containing promoter sequence. Activation of gene promoter. 2. Further identification of insulin response elements in COL1A1 gene of human fibroblasts Methods: 1.Recombinant plasmid pCOLH12.5 containing chloramphenicol acetyltransferase (COL1A1) gene and human COL1A1 gene (-2483-42 BP) was constructed. Stable transfection into WI-38 of human embryonic lung fibroblasts. The stable transfected WI-38 cell line was established, and different doses of insulin were added to the cell line. The CAT value was measured. 2. Synthesis of different sequences of ssPTODN. The sequence of ssPTODN1 was 25 bases of COL1A1 gene from -129 to -105. Transient transfection of different doses of ssPTODN1 was carried out. In cloned cell lines. After resting, 2. 5 渭 mol/ml insulin was added to measure CAT value. Results: 1.The WI-38 cell line PN1.2 integrated with pCOLH12.5 was successfully established. The insulin concentration was 0.252.5. The CAT values at 10 渭 mol/ml were 920 卤94 卤94, 1021 卤98, 595 卤101, respectively. Insulin at 1711 卤117.2.5 渭 mol/ml and 10 渭 mol/ml significantly increased the promoter activity of COL1A1 gene (P0.01). There was no significant difference in the effect between the two groups. The insulin dose was 2.5 渭 mol / ml ssPTODN1, and the concentration of insulin was 0, 40nM, 80nM, respectively. At 120nM 160nM, the corrected CAT value (PG / ml) of PN1 was 1 1593 卤87 / ml, 1535 卤49 / 1370 卤54, respectively. 1147 卤93,899 卤85; The corrected CAT value of islet alone group was 1601 卤23g / ml. When the concentration of ssPTODN1 was 120nM and 160nM, the increased CAT value stimulated by insulin was significantly inhibited (P 0.01). But the inhibitory level at 160 nm dose was lower than the basic CAT value of promoter. Conclusion: 1. SsPTODN1 can inhibit the activation of COL1A1 gene promoter of human fibroblasts by insulin. 2. Insulin in human fibroblast COL1A1. The response elements of the gene were located in the proximal region of the gene promoter-129 to-105.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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