本文選題：高遷移率蛋白1 + 聚ADP-核糖聚合酶1��； 參考：《華中科技大學(xué)》2012年碩士論文

【摘要】：目的：高遷移率族蛋白1(HMGB1)作為重要的晚期炎癥介質(zhì)在炎癥(包括SAP)的發(fā)生發(fā)展中起到重要作用，HMGB1在翻譯后修飾包括乙�；⒘姿峄�、甲基化和ADP-核糖基化。HMGB1主動分泌過程中，乙�；揎棸缪萘撕苤匾慕巧龠M(jìn)HMGB1向胞漿移位和細(xì)胞外分泌。近年的研究發(fā)現(xiàn)PARP-1參與了炎癥過程的HMGB1核內(nèi)定位和向細(xì)胞外分泌的過程，可能與HMGB1的ADP-核糖基化修飾有關(guān)。本研究旨在研究HMGB1的聚ADP-核糖基化修飾及其對乙酰化修飾的影響，探討PARP-1調(diào)控HMGB1細(xì)胞內(nèi)定位和細(xì)胞外分泌的可能機(jī)制，為PARP-1抑制劑在臨床上用于抗HMGB1相關(guān)炎癥的治療提供更多的理論依據(jù)。 方法：以LPS(100ng/ml)、LPS+3-AB(10mmol/l)刺激RAW264.7細(xì)胞2h和4h，用RIPA裂解法提取出細(xì)胞全蛋白；用HMGB1抗體進(jìn)行免疫沉淀富集HMGB1；用PAR抗體、乙�；�-賴氨酸抗體進(jìn)行Western-blot檢測HMGB1的聚ADP-核糖基化和乙酰化水平。體外構(gòu)PARP-1以6-生物素-17-NAD為底物對HMGB1進(jìn)行聚ADP-核糖基化，用HRP標(biāo)記的鏈霉親和素抗體進(jìn)行免疫印跡檢測生物素化-ADP核糖；體外CBP以乙酰輔酶A為底物對HMGB1進(jìn)行進(jìn)一步的的乙�；靡阴；�-賴氨酸抗體進(jìn)行Western-blot檢測蛋白的繼續(xù)乙酰化水平。 結(jié)果：單用LPS刺激RAW264.7細(xì)胞活化PARP-1后，HMGB1的聚ADP-核糖基化水平明顯高于對照組（0h），乙酰化水平也明顯高于對照組；同時以(PARP-1活性抑制劑)3-AB刺激以后，HMGB1的聚ADP-核糖基化水平明顯低于LPS組，乙酰化水平也明顯低于LPS組,差異具有統(tǒng)計學(xué)意義(P0.05)。體外HMGB1在PARP-1作用下進(jìn)行聚ADP-核糖基反應(yīng)后，結(jié)果顯示PARR-1組HMGB1的聚ADP-核糖基化水平明顯高于control組（PARR-1熱滅活對照組），，繼續(xù)乙�；揭裁黠@高于control組，差異具有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論：HMGB1的聚ADP-核糖基化修飾促進(jìn)其乙酰化修飾。
[Abstract]:Aim: high mobility group protein 1 (HMGB1) plays an important role in the occurrence and development of inflammation (including SAP1) as an important late inflammatory mediator. HMGB1 plays an important role in posttranslational modification, including acetylation, phosphorylation, methylation and ADP- ribonylation. HMGB1 is active in the secretion of HMGB1. Acetylation plays an important role in promoting the translocation and exocrine secretion of HMGB1 to the cytoplasm. In recent years, it has been found that PARP-1 is involved in the localization and exocrine of HMGB1 in the process of inflammation, which may be related to the ADP- ribosylation modification of HMGB1. The aim of this study was to investigate the effects of HMGB1 polyADP- ribonylation modification and its effect on acetylation modification, and to explore the possible mechanism of PARP-1 regulating intracellular localization and exocrine secretion of HMGB1 cells. To provide more theoretical basis for the clinical use of PARP-1 inhibitors in the treatment of HMGB1-related inflammation. Methods: RAW264.7 cells were stimulated with LPSN 100ng / ml LPS-3-ABN 10mmol / L for 2h and 4h, the whole cell proteins were extracted by RIPA lysis method, the HMGB1 was enriched by immunoprecipitation with HMGB1 antibody, and the levels of polyADP- ribose glycosylation and acetylation of HMGB1 were detected by Western-blot with PAR antibody and acetyllysine antibody. In vitro PARP-1 was used as the substrate of 6-biotin-17-NAD to carry out polyADPribylation of HMGB1, and HRP labeled streptavidin antibody was used to detect biotinylated ADP ribose by Western blotting, and in vitro CBP used acetyl coenzyme A as substrate for further acetylation of HMGB1. The level of continuous acetylation of protein was detected by Western-blot with acetylated-lysine antibody. Results: the level of polyADP- ribonylation of HMGB1 in RAW264.7 cells activated by LPS alone was significantly higher than that in control group, and the acetylation level was significantly higher than that in control group. At the same time, the level of polyADP- ribonylation of HMGB1 was significantly lower than that of LPS group, and the level of acetylation was significantly lower than that of LPS group after stimulation with PARP-1 activity inhibitor 3-AB. The difference was statistically significant (P 0.05). The results showed that the level of polyADP- ribosylation of HMGB1 in PARR-1 group was significantly higher than that of control group, and the level of continued acetylation was significantly higher than that of control group. The results showed that the level of HMGB1 in PARR-1 group was significantly higher than that in control group, and the level of continued acetylation in control group was significantly higher than that in control group (P 0.05). Conclusion the polyADP- ribonucleylation modification of HMGB1 promotes the acetylation modification of HMGB1.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R3411

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊智勇,王春友,熊炯p

本文編號：1862016