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Role of NF-κB in the Regulation of Non-apoptotic, Non-necrotic Neutrophil Death Induced by ONO-AE-248

Author: LiGuoLi
Tutor: LiuJiaJia
School: Luzhou Medical College
Course: Medical Immunology
Keywords: Non-apoptotic necrotic cell death Neutrophil NF-κB DFF40 ONO-AE-248 Apoptosis DNA fragmentation Signal Transduction
CLC: R392.12
Type: Master's thesis
Year: 2008
Downloads: 28
Quote: 0
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Abstract


Objective: neutrophils (polymorphonuclear neutrophil, PMN) is the body's most important inflammatory cells at sites of inflammation may lead to excessive accumulation of own tissue the secondary damage cells and cause inflammation-related diseases. Characteristics of terminally differentiated neutrophils, once the differentiation and maturation of its spontaneous apoptosis (spontaneous apoptosis) program, this mechanism, we start to play an important contribute to the inflammation without injury convergence and maintain the internal environment stable role. However, recent results indicate that apoptosis is not the only form of programmed cell death. The preliminary study of the subject found that prostaglandin E2 receptor subtype 3 (EP3) agonists the ONO-AE-248 can induce human neutrophils \biochemical signals within cells play an important role in spontaneous apoptosis caspase-3 activation of caspase-8 and p38-MAPK is not ONO-AE-248, show that they are not involved in non-apoptotic cell death of neutrophils signaling pathway, is highly suggestive of this new type of cell death in the presence of the unique cell biochemical signaling pathways. Nuclear transcription factors (nuclear factor-κB, NF-κB) is a key factor gene expression in the regulation of the inflammatory response and the apoptotic process. ONO-AE-248-induced neutrophil order to further study the molecular mechanisms of non-apoptotic non necrotic death, the study will examine the nuclear transcription factor NF-play an important regulatory role in spontaneous apoptosis of neutrophils κB in the role of this particular form of cell death. Through research, to explore the molecular mechanism of normal neutrophil apoptosis ONO-AE-248-induced neutrophil apoptosis in non-necrotic death from the level of signal transduction, and thus to confirm whether this form of cell death Further laboratory evidence is provided a novel form of cell death. Methods: Ficoll density gradient separation, purification healthy human the outer peripheral blood neutrophils, ONO-AE-248, LPS, TNF-α and without any irritants were added culture, non-necrotic cells to build a non-apoptotic death, apoptosis delay apoptosis accelerated and spontaneous apoptosis of the cell model. Light microscopy after trypan blue dye test and blend of Wright-Giemsa staining to detect activity and purity of the freshly isolated neutrophils; detected by flow cytometry (FCM) analysis, comparing the non-apoptotic necrotic cell death and apoptosis rate of spontaneous apoptosis and DNA karyotype analysis; agarose gel electrophoresis of low molecular weight DNA of non-apoptotic non necrotic cell death group and spontaneous apoptosis of genomic DNA fragmentation (DNA fragmentation); extract ONO-AE -248 non-apoptotic necrotic cell death, LPS delayed apoptosis, TNF-α, apoptosis accelerated neutrophil spontaneous apoptosis group total protein, SDS-PAGE observe histone expression; using anti-IκBα polyclonal antibody and the internal reference β-actin monoclonal antibody by Western blotting to detect and compare IκBα protein levels in a different model of death, so as to analyze the activation state of the NF-κB; RT-PCR to detect the gene product of the NF-κB regulation of XIAP ( X-linked inhibitory apoptosis protein), DAD1 (defender against apoptotic death), FLIP (FLICE inhibitory protein) and DFF40 (DNA fragmentation factor 40) mRNA expression. Results: Light microscopy shows: freshly isolated, the purified neutrophil activity and purity gt; 96%; 2. Use of flow cytometry of neutrophil spontaneous apoptosis and non-apoptotic non-necrotic death hypodiploid detect neutrophil apoptosis only after ONO-AE-248 stimulated 26% hypodiploid DNA histogram peak is not obvious; spontaneous apoptosis compared with 67% apparent hypodiploid peak (P lt; 0.05). Low molecular weight DNA agarose gel electrophoresis, spontaneous apoptosis group generated an integer multiple of the 180 bp DNA ladder (DNA ladder), and non-apoptotic group appeared the short dispersion DNA electrophoresis bands, mainly distributed in the larger molecular weight segments. After 12 h of culture, the group of death model group neutrophil cell lysis, total protein was extracted by SDS-PAGE and compared with each other. The results show the different degrees of change in the different stimuli stimulus, the nature and quantity of the neutrophil protein expression has occurred. 5 different time periods to extract the neutrophils protein, detected by Western blotting technique and compare IκBα protein levels. The results showed: LPS and TNF-α can promote IκBα degradation, IκBα expression levels decreased LPS and TNF-α can promote NF-κB activation; ONO-AE-248 group compared with spontaneous apoptosis IκBα no significant change in expression levels. 6. RT-PCR analysis of gene products regulated by NF-κB of XIAP, DAD1 and a FLIP mRNA expression levels. The results show: XIAP, DAD1 and the FLIP three gene expression in LPS group increased, ONO-AE-248, the three gene expression of TNF-α and spontaneous apoptosis group did not change significantly (P gt; 0.05). 7. RT-PCR analysis of DNA fragmentation factor the DFF40 gene expression level found in spontaneous apoptosis, increased expression of TNF-α group, LPS group no significant changes in expression levels of ONO-AE-248 to stimulate group decreased significantly (P lt; 0.01). Conclusion: 1. Nuclear transcription factor NF-κB is not involved in the ONO-AE-248-induced human neutrophil apoptosis in non-necrotic cell death signal transduction pathways. DNA breakage factor DFF40 downregulation ONO-AE-248-induced non-apoptotic non necrotic death of neutrophils is not one of the main reasons small DNA fragments. 3. Experimental results of this study suggest that neutrophils the body neutrophil spontaneous apoptosis may not be the only form of injury convergence.

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