本文選題：高遷移率族蛋白1 + 多聚(ADP-核糖)聚合酶1��； 參考：《華中科技大學》2012年博士論文

【摘要】：第一部分PARP-1對LPS誘導的HMGBl核漿移位和釋放的影響 目的：研究LPS激活RAW264.7細胞PARP-1活性變化的時間規(guī)律,以及PARP-1對RAW264.7細胞內HMGBl分泌的影響,探討PARP-1在炎癥反應中的作用。 方法：通過cell ELISA法測定LPS刺激后RAW264.7細胞內PARP-1活性變化的時間規(guī)律；用LPS(100ng/m1)+DMSO.LPS+3-AB(10mM)刺激RAW264.7細胞,通過免疫印跡法檢測細胞上清液和胞漿胞核內HMGBl的變化；構建PARP-1siRNA質粒和對照的sc siRNA質粒,瞬時轉染到RAW264.7細胞中,用100ng/ml的LPS處理轉染后的細胞,通過免疫印跡法檢測細胞上清液和胞漿胞核內HMGBl的變化。 結果：LPS刺激RAW264.7細胞后,PARP-1活性升高,刺激4小時后活性達到高峰：在LPS+DMSO刺激后4、8、16、24小時,細胞培養(yǎng)上清中HMGBl的含量增加,刺激后2、4、8小時,細胞漿中HMGBl的含量增加,胞核內HMGBl含量降低,加用3-AB刺激和轉染PARP-1siRNA沉默PARP-1表達可以抑制上述反應。 結論：LPS刺激RAW264.7細胞后PARP-1活性增加,抑制PARP-1的活性或沉默其表達,可以減少HMGBl向胞漿移位和向細胞外釋放。PARP-1參與了LPS誘導的HMGBl核漿移位和釋放的調節(jié)。 第二部分PARP-1通過促進HMGB1乙酰化介導LPS誘導的HMGB1核漿移位 目的：研究LPS誘導的RAW264.7細胞PAR-1對HMGB1乙�；潭鹊挠绊�,以及PARP-1對RAW264.7細胞內乙酰化酶和去乙�；富钚缘挠绊�,探討PARP-1在炎癥反應中的作用。 方法：用3-AB抑制PARP-1活性或將PARP-1沉默后,通過免疫沉淀法純化HMGB1蛋白,然后用western blot的方法檢測HMGB1的乙�；揎�,用比色法檢測RAW264.7細胞內HAT和HDAC活性。 結果：LPS刺激RAW264.7細胞后,細胞內HMGB1的乙�；皆黾�,抑制PARP-1活性或沉默PARP-1的表達降低胞內HMGB1乙酰化水平,降低HAT活性、提高HDAC活性。 結論：PARP-1通過提高胞內HAT活性促進HMGB1的乙酰化,介導HMGB1核漿移位。 第三部分HMGBl蛋白NLS的PAR修飾位點突變對其核漿移位的影響 目的：研究HMGBl蛋白NLS的PAR修飾位點突變對HMGBl核漿移位的影響,探討PARP-1在炎癥反應中的作用。 方法：用3-AB抑制PARP-1活性或將PARP-1沉默后,通過免疫沉淀法純化HMGBl蛋白,然后用western blot的方法檢測HMGBl的核糖基化,對HMGBl蛋白NLS的PAR修飾位點進行點突變,構建HMGBl-GFP融合蛋白,轉染RAW264.7細胞,LPS激活細胞,用western blot的方法檢測LPS活化后胞漿和胞核內HMGB1的表達,熒光顯微鏡下直接觀察胞漿胞核內HMGBl表達；NLS的PAR修飾位點突變后用3-AB抑制PARP-1活性,用western blot的方法檢測胞漿和胞核內HMGBl的表達。 結果：LPS刺激RAW264.7細胞后,細胞內HMGBl的核糖基化水平增加,抑制PARP-1活性或沉默PARP-1的表達降低胞內HMGBl核糖基化水平,HMGBl的NLS序列的PAR修飾位點突變抑制HMGBl向胞漿移位,NLS的PAR修飾位點突變后,抑制PARP-1的活性對HMGBl核漿移位沒有影響。 結論：NLS的PAR修飾位點在HMGBl核漿移位的調節(jié)中占主導地位。 第四部分PARP-1對LPS誘導的膿毒癥模型小鼠的影響 目的：探討PAPR-1對膿毒癥小鼠血清HMGB1含量以及對膿毒癥小鼠生存時間的影響。 方法：SPF級昆明雄性小鼠60只,隨機分為4組：對照組(PBS組),溶劑對照組(LPS+DMSO組),3-AB干預組(LPS+3-AB組),AZD2281干預組(LPS+AZD2281組),每組15只。腹腔注射LPS法制作小鼠膿毒癥模型。對照組腹腔注射PBS,余下三組先在腹腔分別注射DMSO、3-AB、 AZD2281,30min后腹腔內注射LPS做膿毒癥模型。完成后8h分別處死各組小鼠3只,心臟采血,離心后分離血清,行western blot檢測血清中HMGB1含量,余下小鼠觀察120h,記錄死亡時間和數(shù)目。 結果：LPS刺激后小鼠血清中HMGB1的含量增加,LPS+3-AB組和LPS+AZD2281組在刺激后8h血清中HMGB1含量明顯低于LPS+DMSO,差異有顯著性(P0.05)。LPS致膿毒癥導致了明顯的致死效應,經(jīng)3-AB和AZD2281治療后顯著延長了小鼠的生存時間,PBS對照組觀察期內無死亡,LPS+DMSO組生存時間明顯短于LPS+3-AB組和LPS+AZD2281組。 結論：抑制PARP-1的活性可下調膿毒癥小鼠血清HMGB1水平、提高存活率。
[Abstract]:Effect of the first part of parp - 1 on the migration and release of HMGBl induced by LPS

AIM : To study the time regularity of the activity of RAW264.7 cells activated by LPS and the effect of parp - 1 on HMGBl secretion in RAW264.7 cells .

Methods : The time regularity of the activity change of RAW264.7 cells in RAW264.7 cells after LPS stimulation was determined by cell ELISA .
RAW264.7 cells were stimulated with LPS ( 100 ng / ml ) + DMSO . LPS + 3 - AB ( 10 mM ) , and the changes of HMGBl in the supernatant and cytoplasm of cells were detected by Western blotting .
The cells transfected into RAW264.7 cells were transiently transfected into RAW264.7 cells and transfected with 100 ng / ml LPS . The changes of HMGBl in the supernatant and cytoplasm of the cells were detected by Western blotting .

Results : After stimulation of RAW264.7 cells with LPS , the activity of HMGBl increased and the activity reached its peak after 4 hours . The content of HMGBl in the cell culture increased after 4 , 8 , 16 , 24 hours after LPS + DMSO stimulation . HMGBl content in the cell plasm increased 2 , 4 , 8 hours after stimulation .

Conclusion : After LPS stimulation of RAW264.7 cells , the activity of parp - 1 is increased , and the activity or silencing of parp - 1 is inhibited . HMGBl can be reduced to the cytoplasm and released to the outside of the cell . The parp - 1 is involved in the regulation of the migration and release of HMGBl induced by LPS .

The second part of parp - 1 mediated LPS - induced migration of the nuclei by promoting the acetylation of the human body

AIM : To study the effect of LPS - induced RAW264.7 cell PAR - 1 on the degree of acetylation of the cells , and to investigate the effect of parp - 1 on the activity of acetylated enzyme and deacetylating enzyme in RAW264.7 cells .

Methods : After 3 - AB was used to inhibit the activity or silencing of parp - 1 , the protein was purified by immuno - precipitation method , then the acetylization modification was detected by western blot , and the activity was detected by colorimetric method in RAW264.7 cells .

Results : After stimulation of RAW264.7 cells with LPS , the level of acetylation in the cells increased , the expression of parp - 1 was decreased , and the activity of HDAC was increased .

Conclusion : It is possible to promote the acetylation and mediate the migration of the cytoplasm by increasing the activity of the in - the - cell hat .

The Effect of Mutation of PAR Modified Sites of HMGBl in the Third Part on the Nuclear Slurry Displacement

Objective : To study the effect of the mutation on HMGBl nuclear pulp in HMGBl protein ( HMGBl ) .

Methods : HMGBl protein was purified by immuno - precipitation method . HMGBl - GFP fusion protein was purified by Western blot . HMGBl - GFP fusion protein was constructed by Western blot . The expression of HMGBl - GFP was detected by Western blot .
The expression of HMGBl in cytoplasm and nucleus was detected by western blot .

Results : After LPS stimulation of RAW264.7 cells , the level of HMGBl in the cells increased , the level of HMGBl of HMGBl was decreased and HMGBl ' s nuclear glycosylation level was decreased .

Conclusion : The PAR modification sites in HMGBl are dominant in the regulation of HMGBl nuclear pulp migration .

Effects of the fourth part of parp - 1 on LPS - induced sepsis model mice

Objective : To study the effect of PAPR - 1 on the serum levels of serum protein in septic mice and the survival time of septic mice .

Methods : 60 SPF Kunming mice were randomly divided into 4 groups : control group ( PBS group ) , solvent control group ( LPS + DMSO group ) , 3 - AB intervention group ( LPS + 3 - AB group ) , AZD2281 intervention group ( LPS + AZD2281 group ) .

Results : In the serum of LPS + 3 - AB group and LPS + AZD2281 group , the content of high - protein in serum of LPS + 3 - AB group and LPS + AZD2281 group were significantly lower than that in LPS + DMSO group ( P0.05 ) . The survival time of mice was significantly prolonged after treatment with 3 - AB and AZD2281 . The survival time of LPS + DMSO group was shorter than that in LPS + 3 - AB group and LPS + AZD2281 group .

Conclusion : Inhibition of the activity of parp - 1 can down - regulate the level of serum protein in septic mice and improve the survival rate .
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363

【參考文獻】

相關期刊論文 前2條

1 Hidehiro Sawa;Takashi Ueda;Yoshifumi Takeyama;Takeo Yasuda;Makoto Shinzeki;Takahiro Nakajima;Yoshikazu Kuroda;;Blockade of high mobility group box-1 protein attenuates experimental severe acute pancreatitis[J];World Journal of Gastroenterology;2006年47期

2 ;Delayed ethyl pyruvate therapy attenuates experimental severe acute pancreatitis via reduced serum high mobility group box 1 levels in rats[J];World Journal of Gastroenterology;2008年28期

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本文編號：2090857