本文選題：多效生長因子 + 基因沉默��； 參考：《蘭州大學》2008年碩士論文

【摘要】： 目的:在多效生長因子(Pleiotrophin,Ptn)基因穩(wěn)定沉默的小鼠胚胎成纖維細胞Ptn-siRNA B/MEF241中,研究Pleiotrophin對Schlafen2(Slfn2)基因表達的影響,以及在此調(diào)控過程中可能涉及的細胞因子;探討Ptn沉默誘導Slfn2基因表達可能涉及的信號通路。 方法:前期研究發(fā)現(xiàn),多效生長因子(Pleiotrophin,Ptn)基因穩(wěn)定沉默的小鼠胚胎成纖維細胞Ptn-siRNA B/MEF241中Schlafen2(Slfn2)基因高表達。在本實驗中,(1)為了驗證Ptn是否可能通過調(diào)控某種分泌型因子來抑制Slfn2表達,用Ptn穩(wěn)定沉默細胞的培養(yǎng)液培養(yǎng)對照NC細胞,通過Northern雜交法檢測培養(yǎng)不同時間后NC細胞中Slfn2表達水平變化,同時應用RT-PCR檢測Ptn沉默細胞內(nèi)IL-1a和IL-1f6表達水平,以及外源性PTN因子(終濃度50ng/μl)對Ptn沉默細胞內(nèi)IL-1表達水平的影響;分別使用重組人白細胞介素-1受體拮抗劑(rhIL-lra)和IL-1a中和抗體阻斷IL-1的作用通路,檢測Ptn穩(wěn)定沉默細胞中Slfn2基因表達變化。(2)為了探討Ptn沉默誘導Slfn2基因表達可能涉及的信號通路,應用Western印跡檢測Ptn沉默細胞及NC細胞JNK磷酸化基礎水平,以及外源性PTN因子(終濃度50 ng/μl)對Ptn沉默細胞JNK磷酸化水平的影響;用SP600125和SB203580特異性阻斷JNK和p38通路,Northern印跡分析Ptn沉默細胞Slfn2基因轉(zhuǎn)錄水平的變化。(3)為了進一步驗證Ptn沉默細胞內(nèi)表達上調(diào)的IL-1是否也對JNK通路具有調(diào)控作用,分別使用重組人白細胞介素-1受體拮抗劑(rhIL-lra)和IL-1a中和抗體阻斷IL-1的作用通路,應用Western印跡檢測Ptn穩(wěn)定沉默細胞中JNK磷酸化水平的變化。 結(jié)果:(1)Ptn沉默細胞中Slfn2表達顯著上調(diào),Ptn沉默細胞培養(yǎng)液可以誘導對照細胞Slfn2表達;Ptn沉默后細胞內(nèi)IL-1a和IL-1f6表達顯著上調(diào),外源性PTN能抑制沉默細胞中IL-1a和IL-1f6的表達;IL-1受體拮抗劑(rhIL-lra)和IL-1a中和抗體可抑制Ptn沉默細胞中Slfn2表達。(2)Ptn沉默誘導細胞內(nèi)磷酸化JNK蛋白高表達,此誘導效應可被外源性PTN抑制;阻斷JNK通路呈時間依賴性抑制Ptn沉默細胞中Slfn2基因表達;阻斷p38通路對Ptn沉默細胞中Slfn2表達水平?jīng)]有明顯影響。(3)IL-1受體拮抗劑(rhIL-1ra)和IL-1a中和抗體可抑制Ptn沉默細胞中JNK磷酸化水平。 結(jié)論:(1)Ptn對Slfn2基因表達具有負性調(diào)控作用,在此調(diào)控過程中IL-1家族是其中一種非常重要的調(diào)控因子。(2)Ptn可能通過抑制其下游JNK/MAPK通路來負調(diào)控Slfn2的表達。(3)Ptn沉默細胞內(nèi)表達上調(diào)的IL-1也對JNK/MAPK通路具有調(diào)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.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前1條

1 霍艷英;胡迎春;周喬丹;楊柳;張博;李剛;吳德昌;;多效生長因子(Pleiotrophin)基因沉默后的基因表達譜初步分析[J];中國生物化學與分子生物學報;2007年05期

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本文編號：1959413