本文關(guān)鍵詞：含啟動子序列的單鏈寡核苷酸拮抗胰島素對人α1（Ⅰ）型膠原啟動子的激活　出處：《福建醫(yī)科大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 寡脫氧核糖核苷酸 胰島素 膠原 啟動子 轉(zhuǎn)錄 共轉(zhuǎn)染

【摘要】：目的:1.探討含啟動子序列的單鏈寡脫氧核糖核苷酸(ssPTODN)抑制胰島素對人 α1(I)型膠原(COL1A1)基因啟動子的激活。2.進(jìn)一步確定胰島素在人成 纖維細(xì)胞COL1A1 基因上的反應(yīng)元件 方法:1. 將含有氯霉素乙�；D(zhuǎn)移酶(CAT) 基因與人COL1A1 基因-2483～ +42bp 片段的重組質(zhì)粒(pCOLH12.5)穩(wěn)定轉(zhuǎn)染至人胚肺成纖維細(xì)胞 WI-38 中, 建立穩(wěn)定轉(zhuǎn)染的WI-38 細(xì)胞株,于該細(xì)胞株中加入不同劑量 的胰島素,測CAT 值;2.合成全硫代的不同序列ssPTODN,其中 ssPTODN1 的序列為COL1A1 基因-129 至-105 間的25 個堿基。瞬時轉(zhuǎn)染 不同劑量的ssPTODN1 于克隆的細(xì)胞株中,靜息后,分別加入2.5μmol/ml 的胰島素,測CAT 值。 結(jié)果:1.成功建立了整合有pCOLH12.5 的WI-38 細(xì)胞株P(guān)N1。2.胰島素濃度在 0、0.25、2.5、10μmol/ml 時的CAT 值(pg/ml)分別是920±94、1021 ±98、1595±101、1711±117。2.5μmol/ml 及10μmol/ml 的胰島素顯著 增加了COL1A1 基因啟動子活性(P0.01),二者的作用無顯著性差異 (P0.05)。3.胰島素劑量為2.5μmol/ml,ssPTODN1 濃度分別為0、 40nM、80nM 、120nM、160nM時,細(xì)胞株P(guān)N1的各組校正后CAT值(pg/ml) 相應(yīng)為:1593±87、1535±49、1370±54、1147±93、899±85;單獨(dú)胰 島素作用組的CAT 校正值為1601±23(pg/ml);ssPTODN1 濃度為120nM 和160nM 時,受胰島素刺激所增加的CAT 值得到顯著抑制(P0.01), 但160nM 劑量時所抑制的水平低于啟動子的基礎(chǔ)CAT 值。 結(jié)論: 1. ssPTODN1 能夠抑制胰島素對人成纖維細(xì)胞COL1A1 基因啟動子的激 活。2.胰島素在人成纖維細(xì)胞COL1A1 基因上的反應(yīng)元件被定位于該 基因啟動子近端-129 至-105 區(qū)域內(nèi)。
[Abstract]:Objective: 1. To investigate the inhibitory effect of insulin on human 偽 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 鹵94 鹵94, 1021 鹵98, 595 鹵101, respectively. Insulin at 1711 鹵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 鹵87 / ml, 1535 鹵49 / 1370 鹵54, respectively. 1147 鹵93,899 鹵85; The corrected CAT value of islet alone group was 1601 鹵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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R346

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