PARP-1调控LPS诱导的巨噬细胞HMGB1释放的机制及意义研究

发布时间：2018-07-02 20:42

  本文选题：高迁移率族蛋白1 + 多聚(ADP-核糖)聚合酶1　； 参考：《华中科技大学》2012年博士论文

【摘要】：第一部分PARP-1对LPS诱导的HMGBl核浆移位和释放的影响 目的：研究LPS激活RAW264.7细胞PARP-1活性变化的时间规律,以及PARP-1对RAW264.7细胞内HMGBl分泌的影响,探讨PARP-1在炎症反应中的作用。 方法：通过cell ELISA法测定LPS刺激后RAW264.7细胞内PARP-1活性变化的时间规律；用LPS(100ng/m1)+DMSO.LPS+3-AB(10mM)刺激RAW264.7细胞,通过免疫印迹法检测细胞上清液和胞浆胞核内HMGBl的变化；构建PARP-1siRNA质粒和对照的sc siRNA质粒,瞬时转染到RAW264.7细胞中,用100ng/ml的LPS处理转染后的细胞,通过免疫印迹法检测细胞上清液和胞浆胞核内HMGBl的变化。 结果：LPS刺激RAW264.7细胞后,PARP-1活性升高,刺激4小时后活性达到高峰：在LPS+DMSO刺激后4、8、16、24小时,细胞培养上清中HMGBl的含量增加,刺激后2、4、8小时,细胞浆中HMGBl的含量增加,胞核内HMGBl含量降低,加用3-AB刺激和转染PARP-1siRNA沉默PARP-1表达可以抑制上述反应。 结论：LPS刺激RAW264.7细胞后PARP-1活性增加,抑制PARP-1的活性或沉默其表达,可以减少HMGBl向胞浆移位和向细胞外释放。PARP-1参与了LPS诱导的HMGBl核浆移位和释放的调节。 第二部分PARP-1通过促进HMGB1乙酰化介导LPS诱导的HMGB1核浆移位 目的：研究LPS诱导的RAW264.7细胞PAR-1对HMGB1乙酰化程度的影响,以及PARP-1对RAW264.7细胞内乙酰化酶和去乙酰化酶活性的影响,探讨PARP-1在炎症反应中的作用。 方法：用3-AB抑制PARP-1活性或将PARP-1沉默后,通过免疫沉淀法纯化HMGB1蛋白,然后用western blot的方法检测HMGB1的乙酰化修饰,用比色法检测RAW264.7细胞内HAT和HDAC活性。 结果：LPS刺激RAW264.7细胞后,细胞内HMGB1的乙酰化水平增加,抑制PARP-1活性或沉默PARP-1的表达降低胞内HMGB1乙酰化水平,降低HAT活性、提高HDAC活性。 结论：PARP-1通过提高胞内HAT活性促进HMGB1的乙酰化,介导HMGB1核浆移位。 第三部分HMGBl蛋白NLS的PAR修饰位点突变对其核浆移位的影响 目的：研究HMGBl蛋白NLS的PAR修饰位点突变对HMGBl核浆移位的影响,探讨PARP-1在炎症反应中的作用。 方法：用3-AB抑制PARP-1活性或将PARP-1沉默后,通过免疫沉淀法纯化HMGBl蛋白,然后用western blot的方法检测HMGBl的核糖基化,对HMGBl蛋白NLS的PAR修饰位点进行点突变,构建HMGBl-GFP融合蛋白,转染RAW264.7细胞,LPS激活细胞,用western blot的方法检测LPS活化后胞浆和胞核内HMGB1的表达,荧光显微镜下直接观察胞浆胞核内HMGBl表达；NLS的PAR修饰位点突变后用3-AB抑制PARP-1活性,用western blot的方法检测胞浆和胞核内HMGBl的表达。 结果：LPS刺激RAW264.7细胞后,细胞内HMGBl的核糖基化水平增加,抑制PARP-1活性或沉默PARP-1的表达降低胞内HMGBl核糖基化水平,HMGBl的NLS序列的PAR修饰位点突变抑制HMGBl向胞浆移位,NLS的PAR修饰位点突变后,抑制PARP-1的活性对HMGBl核浆移位没有影响。 结论：NLS的PAR修饰位点在HMGBl核浆移位的调节中占主导地位。 第四部分PARP-1对LPS诱导的脓毒症模型小鼠的影响 目的：探讨PAPR-1对脓毒症小鼠血清HMGB1含量以及对脓毒症小鼠生存时间的影响。 方法：SPF级昆明雄性小鼠60只,随机分为4组：对照组(PBS组),溶剂对照组(LPS+DMSO组),3-AB干预组(LPS+3-AB组),AZD2281干预组(LPS+AZD2281组),每组15只。腹腔注射LPS法制作小鼠脓毒症模型。对照组腹腔注射PBS,余下三组先在腹腔分别注射DMSO、3-AB、 AZD2281,30min后腹腔内注射LPS做脓毒症模型。完成后8h分别处死各组小鼠3只,心脏采血,离心后分离血清,行western blot检测血清中HMGB1含量,余下小鼠观察120h,记录死亡时间和数目。 结果：LPS刺激后小鼠血清中HMGB1的含量增加,LPS+3-AB组和LPS+AZD2281组在刺激后8h血清中HMGB1含量明显低于LPS+DMSO,差异有显著性(P0.05)。LPS致脓毒症导致了明显的致死效应,经3-AB和AZD2281治疗后显著延长了小鼠的生存时间,PBS对照组观察期内无死亡,LPS+DMSO组生存时间明显短于LPS+3-AB组和LPS+AZD2281组。 结论：抑制PARP-1的活性可下调脓毒症小鼠血清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 .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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