【摘要】：目的:帶核定位信號的維甲酸受體(Nuclear localization signal retinoic acid receptor alpha:NLS-RARα)在急性早幼粒細胞白血病(Acute promyelocytic leukemia:APL)的發(fā)生發(fā)展中扮演著重要的角色,它是由中性粒細胞彈性蛋白酶(Neutrophil elastase:NE)切割早幼粒細胞白血病-維甲酸受體(Promyelocytic leukemia-retinoic acid receptor alpha:PML-RARα)融合蛋白形成的。然而,NLS-RARα對APL的作用機制尚未明確,本研究論文探討NLS-RARα對APL細胞株NB4細胞的作用及其機制。方法:免疫印跡實驗和CCK-8增殖實驗分別檢測全反式維甲酸(All-trans retinoic acid:ATRA)處理后的NB4細胞分化標志物C/EBPβ、CD11b,絲裂原活化蛋白激酶p38α(Mitogen-activated protein kinase p38α:p38αMAPK)表達水平和NB4細胞增殖水平;分子克隆技術(shù)構(gòu)建p38αMAPK真核表達質(zhì)粒,雙熒光素酶實驗檢測p38αMAPK的轉(zhuǎn)錄激活活性;利用慢病毒介導的NLS-RARα基因過表達,進一步用免疫印跡實驗驗證過表達效率并檢測NLS-RARα對NB4細胞分化標志物C/EBPβ、CD11b和p38αMAPK表達水平的影響,同時,CCK-8增殖實驗檢測NLS-RARα對NB4細胞增殖水平的影響;間接免疫熒光實驗分析nls-rarα與p38αmapk的空間共定位,免疫共沉淀實驗分析nls-rarα與p38αmapk的相互作用;應用p38αmapk活性抑制劑pd169316研究atra激活p38αmapk后再募集nls-rarα還是atra募集已與p38αmapk結(jié)合的nls-rarα后激活p38αmapk。結(jié)果:全反式維甲酸(all-transretinoicacid:atra)處理組nb4細胞分化指標c/ebpβ、cd11b,磷酸化的絲裂原活化蛋白激酶p38α(phosphorylated-mitogen-activatedproteinkinase:p-p38αmapk)的量增加(p0.05),細胞增殖水平下降(p0.05),p38αmapk表達水平無差異(p0.05);成功構(gòu)建p38αmapk真核表達質(zhì)粒,p38αmapk真核表達質(zhì)粒轉(zhuǎn)染組的熒光素酶活性增加(p0.05);成功在nb4細胞中過表達nls-rarα,當未用atra處理細胞時,過表達nls-rarα組nb4分化指標c/ebpβ和cd11b的表達水平下降(p0.05),p38αmapk和p-p38αmapk的量無改變(p0.05),當用atra處理細胞時,過表達nls-rarα組nb4分化指標c/ebpβ、cd11b和p-p38αmapk的量減少(p0.05),p38αmapk表達水平無差異(p0.05),細胞增殖水平上升(p0.05);免疫熒光實驗和免疫共沉淀實驗證實nls-rarα與p38αmapk直接相互作用;p38αmapk抑制劑pd169316的在lv-nls-rarα-nb4細胞中的應用發(fā)現(xiàn),與atra處理組相比,atra+pd169316處理組lv-nls-rarα-nb4細胞中的分化指標c/ebpβ、cd11b和p-p38αmapk的量減少(p0.05),nls-rarα和p38αmapk表達水平無差異(p0.05),細胞增殖水平上升(p0.05),pd169316在293t細胞中的應用發(fā)現(xiàn)抑制劑處理組和未處理組中p38αMAPK和NLS-RARα都能發(fā)生相互作用。結(jié)論:ATRA募集已與p38αMAPK結(jié)合的NLS-RARα后,能通過激活p38αMAPK促進APL細胞株NB4細胞分化而抑制其增殖,然而在此過程中,NLS-RARα能夠通過下調(diào)p-p38αMAPK而抑制ATRA對NB4細胞的效應。
[Abstract]:Objective: retinoic acid receptor (Nuclear localization signal retinoic acid receptor alpha:NLS-RAR 偽) with nuclear localization signal plays an important role in the pathogenesis and development of acute promyelocytic leukemia (Acute promyelocytic leukemia:APL). It is formed by neutrophil elastase (Neutrophil elastase:NE) cleavage of promyelocytic leukemia-retinoic acid receptor (Promyelocytic leukemia-retinoic acid receptor alpha:PML-RAR 偽) fusion protein. However, the mechanism of NLS-RAR 偽 on APL is not clear. In this study, the effect of NLS-RAR 偽 on APL cell line NB4 cells and its mechanism are discussed. Methods: Western blot assay and CCK-8 proliferation assay were used to detect the differentiation markers C/EBP 尾 and CD11b, of NB4 cells treated with all trans retinoic acid (All-trans retinoic acid:ATRA). The expression of mitogen-activated protein kinase p38 偽 (Mitogen-activated protein kinase p38 偽: p38 偽 MAPK) and the proliferation of NB4 cells were observed. The eukaryotic expression plasmid p38 偽 MAPK was constructed by molecular cloning, and the transcriptional activation activity of p38 偽 MAPK was detected by double luciferase assay. Using lentivirus-mediated overexpression of NLS-RAR 偽 gene, the overexpression efficiency of NLS-RAR 偽 and the expression of C/EBP 尾, CD11b and p38 偽 MAPK in NB4 cells were examined by Western blotting assay. At the same time, the expression levels of C/EBP 尾, CD11b and p38 偽 MAPK were detected. CCK-8 proliferation assay was used to detect the effect of NLS-RAR 偽 on the proliferation of NB4 cells. The spatial co-localization of nls-rar 偽 and p38 偽 mapk was analyzed by indirect immunofluorescence assay, and the interaction between nls-rar 偽 and p38 偽 mapk was analyzed by immunoprecipitation assay. Using p38 偽 mapk activity inhibitor pd169316 to study whether atra activates p38 偽 mapk before recruiting nls-rar 偽 or atra recruitment with nls-rar 偽 combined with p38 偽 mapk to activate p38 偽 mapk. Results: in all trans retinoic acid (all-transretinoicacid:atra) treated group, c/ebp 尾, cd11b, phosphorylated mitogen-activated protein kinase p38 偽 (phosphorylated-mitogen-activatedproteinkinase:p-p38 偽 mapk) increased (p0.05). The level of cell proliferation was decreased (p0. 05), but the expression of p38 偽 mapk was not different (p0. 05). The eukaryotic expression plasmid p38 偽 mapk was constructed successfully. The luciferase activity of p38 偽 mapk eukaryotic expression plasmid transfected group was increased (p0. 05). Nls-rar 偽 was successfully expressed in nb4 cells. When the cells were not treated with atra, the expression levels of c/ebp 尾 and cd11b in nb4 differentiation index were decreased (p0.05), and the levels of p38 偽 mapk and p-p38 偽 mapk were not changed (p0.05). When the cells were treated with atra, the nb4 differentiation indexes c/ebp 尾, cd11b and p-p38 偽 mapk decreased (p0.05), the expression of p38 偽 mapk was not different (p0.05), and the cell proliferation was increased (p0.05). The direct interaction between nls-rar 偽 and p38 偽 mapk was confirmed by immunofluorescence and co-immunoprecipitation. The application of p38 偽 mapk inhibitor pd169316 in lv-nls-rar 偽-nb4 cells showed that the quantity of c/ebp 尾, cd11b and p-p38 偽 mapk in lv-nls-rar 偽-nb4 cells treated with atra pd169316 was lower than that in atra treatment group (p0. 05). There was no difference in the expression of nls-rar 偽 and p38 偽 mapk (p0.05), but the level of cell proliferation increased (p0.05). The application of pd169316 in 293t cells showed that p38 偽 MAPK and NLS-RAR 偽 could interact with each other in both the inhibitor group and the untreated group. Conclusion: ATRA recruitment combined with p38 偽 MAPK NLS-RAR 偽 can inhibit the proliferation of NB4 cells by activating p38 偽 MAPK. However, NLS-RAR 偽 can inhibit the effect of ATRA on NB4 cells by down-regulating p-p38 偽 MAPK.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.7

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