多效生长因子Pleiotrophin对Schlafen2基因表达的影响及其调控机制的研究

发布时间：2018-05-31 11:10

  本文选题：多效生长因子 + 基因沉默　； 参考：《兰州大学》2008年硕士论文

【摘要】： 目的:在多效生长因子(Pleiotrophin,Ptn)基因稳定沉默的小鼠胚胎成纤维细胞Ptn-siRNA B/MEF241中,研究Pleiotrophin对Schlafen2(Slfn2)基因表达的影响,以及在此调控过程中可能涉及的细胞因子;探讨Ptn沉默诱导Slfn2基因表达可能涉及的信号通路。 方法:前期研究发现,多效生长因子(Pleiotrophin,Ptn)基因稳定沉默的小鼠胚胎成纤维细胞Ptn-siRNA B/MEF241中Schlafen2(Slfn2)基因高表达。在本实验中,(1)为了验证Ptn是否可能通过调控某种分泌型因子来抑制Slfn2表达,用Ptn稳定沉默细胞的培养液培养对照NC细胞,通过Northern杂交法检测培养不同时间后NC细胞中Slfn2表达水平变化,同时应用RT-PCR检测Ptn沉默细胞内IL-1a和IL-1f6表达水平,以及外源性PTN因子(终浓度50ng/μl)对Ptn沉默细胞内IL-1表达水平的影响;分别使用重组人白细胞介素-1受体拮抗剂(rhIL-lra)和IL-1a中和抗体阻断IL-1的作用通路,检测Ptn稳定沉默细胞中Slfn2基因表达变化。(2)为了探讨Ptn沉默诱导Slfn2基因表达可能涉及的信号通路,应用Western印迹检测Ptn沉默细胞及NC细胞JNK磷酸化基础水平,以及外源性PTN因子(终浓度50 ng/μl)对Ptn沉默细胞JNK磷酸化水平的影响;用SP600125和SB203580特异性阻断JNK和p38通路,Northern印迹分析Ptn沉默细胞Slfn2基因转录水平的变化。(3)为了进一步验证Ptn沉默细胞内表达上调的IL-1是否也对JNK通路具有调控作用,分别使用重组人白细胞介素-1受体拮抗剂(rhIL-lra)和IL-1a中和抗体阻断IL-1的作用通路,应用Western印迹检测Ptn稳定沉默细胞中JNK磷酸化水平的变化。 结果:(1)Ptn沉默细胞中Slfn2表达显著上调,Ptn沉默细胞培养液可以诱导对照细胞Slfn2表达;Ptn沉默后细胞内IL-1a和IL-1f6表达显著上调,外源性PTN能抑制沉默细胞中IL-1a和IL-1f6的表达;IL-1受体拮抗剂(rhIL-lra)和IL-1a中和抗体可抑制Ptn沉默细胞中Slfn2表达。(2)Ptn沉默诱导细胞内磷酸化JNK蛋白高表达,此诱导效应可被外源性PTN抑制;阻断JNK通路呈时间依赖性抑制Ptn沉默细胞中Slfn2基因表达;阻断p38通路对Ptn沉默细胞中Slfn2表达水平没有明显影响。(3)IL-1受体拮抗剂(rhIL-1ra)和IL-1a中和抗体可抑制Ptn沉默细胞中JNK磷酸化水平。 结论:(1)Ptn对Slfn2基因表达具有负性调控作用,在此调控过程中IL-1家族是其中一种非常重要的调控因子。(2)Ptn可能通过抑制其下游JNK/MAPK通路来负调控Slfn2的表达。(3)Ptn沉默细胞内表达上调的IL-1也对JNK/MAPK通路具有调控作用
[Abstract]:Objective: To study the effect of Pleiotrophin on the expression of Schlafen2 (Slfn2) gene in mouse embryonic fibroblasts (Ptn-siRNA B/MEF241), which is stable and silent in Pleiotrophin (Ptn) gene, and to explore the possible cytokines in this regulation process, and to explore the possible signaling pathway involved in the expression of Slfn2 gene expression induced by Ptn silencing.
Methods: the previous study found that the Schlafen2 (Slfn2) gene was highly expressed in Ptn-siRNA B/MEF241 of mouse embryonic fibroblasts with Pleiotrophin (Ptn) gene stably and silenced. (1) in this experiment, (1) to verify that Ptn may inhibit Slfn2 expression by regulating a certain secretory factor, and the culture fluid of the cell is stabilized with Ptn. The expression level of Slfn2 in NC cells was detected by Northern hybridization, and the expression level of IL-1a and IL-1f6 in Ptn silenced cells and the effect of exogenous PTN factor (50ng/ Mu L) on the expression level in the silent cells were detected by RT-PCR, and the recombinant human leukocyte was used respectively by RT-PCR. -1 receptor antagonist (rhIL-lra) and IL-1a neutralization antibody blocking the IL-1 pathway to detect the change of Slfn2 gene expression in Ptn stable silencing cells. (2) in order to explore the possible signaling pathways involved in Ptn silence induced Slfn2 gene expression, Western blotting was used to detect the basal level of Ptn silencing cells and NC cell JNK phosphorylation, as well as exogenous proteins. The effect of factor (final concentration 50 ng/ Mu L) on the level of JNK phosphorylation in Ptn silencing cells; the specific blocking of JNK and p38 pathways with SP600125 and SB203580 and the change in the Slfn2 gene transcriptional level of Ptn silenced cells by Northern blotting. (3) to further verify whether the up regulation of the expression in the silent cells also has a regulatory effect on the pathway. Do not use recombinant human interleukin -1 receptor antagonist (rhIL-lra) and IL-1a neutralizing antibody blocking the IL-1 pathway. Western blot was used to detect the changes in the level of JNK phosphorylation in Ptn stable silent cells.
Results: (1) the expression of Slfn2 in Ptn silencing cells was significantly up-regulated, and Ptn silencing cell culture fluid could induce the expression of Slfn2 in the control cells, and the expression of IL-1a and IL-1f6 in the cells was significantly up-regulated after Ptn silencing, and exogenous PTN could inhibit the expression of IL-1a and IL-1f6 in the silenced cells. Medium Slfn2 expression. (2) Ptn silencing induced high expression of phosphorylated JNK protein in cells, which can be inhibited by exogenous PTN; blocking JNK pathway is time dependent inhibition of Slfn2 gene expression in Ptn silencing cells; blocking p38 pathway has no significant influence on Slfn2 expression level in Ptn silencing cells. (3) IL-1 receptor antagonist (rhIL-1ra) and Neutralization antibody can inhibit the level of JNK phosphorylation in Ptn silencing cells.
Conclusion: (1) Ptn has a negative regulatory effect on Slfn2 gene expression, and IL-1 family is one of the most important regulatory factors in this regulation. (2) Ptn may negatively regulate the expression of Slfn2 by inhibiting its downstream JNK/MAPK pathway. (3) the up regulation of IL-1 in Ptn silencing cells also regulates the JNK/MAPK pathway.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

【参考文献】

相关期刊论文 前1条

1 霍艳英;胡迎春;周乔丹;杨柳;张博;李刚;吴德昌;;多效生长因子(Pleiotrophin)基因沉默后的基因表达谱初步分析[J];中国生物化学与分子生物学报;2007年05期

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本文编号：1959413