FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

本文对fMLP诱导的嗜中性白细胞胞内钙浓度变化与凋亡的关系进行了研究。用膜受体激动剂fMLP和钙离子载体A23187诱导细胞内钙浓度升高,BAPTA螯合胞质钙。运用荧光显微镜,流式细胞仪,电泳等方法对培养细胞的凋亡百分率及细胞骨架变化进行了研究。结果表明:fMLP和A23187均有效地抑制了凋亡,而BAPTA促进了凋亡。对骨架测定表明随细胞凋亡微丝解聚明显,胞内钙升高抑制骨架解聚,胞内钙降低促进其解聚。故嗜中性白细胞凋亡过程中伴随有微丝的解聚,胞内钙浓度升高时凋亡被有效抑制,胞内钙浓度降低时促进了凋亡。     

嗜中性白细胞呼吸爆发与胞内外钙信号的关系研究

以膜受体激动剂ｆＭＬＰ和ＰＫＣ激活剂ＰＭＡ为刺激剂,ｆｕｒａ－２为荧光探针,分别用化学发光和荧光方法研究了嗜中性白细胞呼吸爆发与胞内外钙信号的关系。并以ｆｌｕｏ－３为荧光探针,在激光共聚焦显微镜上观测了呼吸爆发时的胞内钙信号的时间与空间变化。ｆＭＬＰ能够迅速引起胞内钙变化,而ＰＭＡ不引起胞内钙变化。在呼吸爆发启动时间上,ｆＭＬＰ明显短于ＰＭＡ,且呼吸爆发的强度更高,持续时间较短。比较胞内钙信号与呼吸爆发,胞内钙变化在启动时间,到达峰值时间和持续时间上均短于呼吸爆发时间。胞内无钙时,呼吸爆发完全被抑制。胞外无钙时,呼吸爆发强度比有钙时低７０％左右。激光共聚焦显微镜观测发现：细胞在ｆＭＬＰ作用之后胞内钙库释放钙进入胞浆并向胞外流动。当胞外有钙时,胞内钙浓度降至最低后,由于外钙内流会使其再次缓慢上升;当胞外无钙时,胞浆钙浓度降至最低后不会再回升。结果提示胞内钙信号对细胞进入呼吸爆发有重要控制作用,而胞外钙主要用于维持细胞的呼吸爆发。     

一种改进的分离嗜中性白细胞的方法

报道一种从全血中分离嗜中性白细胞的方法.人血经过葡聚糖沉降,淋巴细胞分离液梯度分离,特殊分离液的洗涤和红细胞的溶胀后,得到了嗜中性白细胞.经瑞特氏染色和台盼蓝染色证明细胞纯度与存活率均在95％以上.以化学发光和细胞色素c还原法证明了细胞的高活力与膜受体的完整性.与国外的分离方法相比较,该方法简便易行,且分离效果好,是一种高效、经济的分离方法.     

猴免疫缺陷病毒引起多形核嗜中性白细胞凋亡的可能机理

本实验目的是研究猴免疫缺陷病毒（SIV）引起多形核嗜中性白细胞（PMNs）凋亡的机理。实验用PCR技术扩增gag基因,用Western blot法测定p53和bcl-2基因的表达。结果显示PMNs在被SIV感染后随着保温时间的延长存活率下降,在感染后24h可以从PMNs中扩增出gag基因。PMNs中p53基因的表达在感染后24h增加。同时bcl-2基因的表达在对照组和SIV感染组都增加,但在SIV感染组bcl-2蛋白的表达明显低于对照组。结果揭示SIV能够感染PMNs,p53和bcl-2基因表达的改变可能是SIV感染PMNs引起细胞凋亡的机理。     

猴免疫缺陷病毒引起多形核嗜中性白细胞凋亡的可能机理

本实验目的是研究猴免疫缺陷病毒(SIV)引起多形核嗜中性白细胞(PMNs)凋亡的机理.实验用PCR技术扩增gag基因,用Western blot法测定p53和bcl-2基因的表达.结果显示PMNs在被SIV感染后随着保温时间的延长存活率下降,在感染后24h可以从PMNs中扩增出gag基因.PMNs中p53基因的的表达在感染后24h增加.同时bcl-2基因的表达在对照组和SIV感染组都增加,但在SIV感染组bcl-2蛋白的表达明显低于对照组.结果揭示SⅣ能够感染PMNs,p53和bcl-2基因表达的改变可能是SⅣ感染PMNs引起细胞凋亡的机理.     

猴免疫缺陷病毒引起多形核嗜中性白细胞凋亡的可能机理（英文）

本实验目的是研究猴免疫缺陷病毒(SIV)引起多形核嗜中性白细胞(PMNs)凋亡的机理.实验用PCR技术扩增gag基因,用Western blot法测定p53和bcl-2基因的表达.结果显示PMNs在被SIV感染后随着保温时间的延长存活率下降,在感染后24h可以从PMNs中扩增出gag基因.PMNs中p53基因的的表达在感染后24h增加.同时bcl-2基因的表达在对照组和SIV感染组都增加,但在SIV感染组bcl-2蛋白的表达明显低于对照组.结果揭示SⅣ能够感染PMNs,p53和bcl-2基因表达的改变可能是SⅣ感染PMNs引起细胞凋亡的机理.     

炎症介质通过延迟嗜中性粒细胞凋亡促进内皮细胞的损伤

本研究采用分离纯化的人外周血NTP,以LPS/TNF激活后5:1和10:1与内皮细胞共同培养或隔离培养,加或不加10ng/ml IL-6、10%(v/v)烧伤血清,24h后观察NTP凋亡对体外培养内皮细胞损伤程度的影响及其关系。结果表明激活的NTP对内皮细胞没有明显损伤,但是加入IL-6和烧伤血清后,NTP凋亡延迟,内皮细胞受到损伤,表现为发生坏死,并且NTP对内皮细胞的损伤需要两种细胞的直接接触。     

炎症介质通过延迟嗜中性粒细胞凋亡促进内皮细胞的损伤

本研究采用分离纯化的人外周血NTP,以LPS／TNF激活后5：1和10：1与内皮细胞共同培养或隔离培养,加或不加10ng/ml IL-6、10％（v/v）烧伤血清,24h后观察NTP凋亡对体外培养内皮细胞损伤程度的影响及其关系。结果表明激活的NTP对内皮细胞没有明显损伤,但是加入IL－6和烧伤血清后,NTP凋亡延迟,内皮细胞受到损伤,表现为发生坏死,并且NTP对内皮细胞的损伤需要两种细胞的直接接触。     

磷脂酶A2活性对中性粒细胞凋亡的影响

细胞凋亡（Ａｐｏｐｔｏｓｉｓ）是一种机体保持内环境稳定的特殊方式。正常情况下,中性粒细胞（ＰＭＮ）绝大部分通过凋亡而被清除,避免因坏死而造成组织损伤。我们在研究磷脂酶２（ＰＬＡ２）激活介导创伤和感染的机理时,发现其活性介导ＴＮＦ对ＰＭＮ的激发作用。其它证据也显示ＰＬＡ２及其代谢产物在细胞凋亡过程中发挥作用,我们推测ＰＬＡ２活性对ＰＭＮ凋亡或坏死的影响,可能是控制炎症反应的主要途径。这方面的工作尚少见,本文初步报告如下。１　材料和方法（１）材料和主要试剂　雄性Ｗｉｓｔａｒ大鼠由本院动物中心提供。Ｐ…     

白细胞介素1β转化酶家庭与细胞凋亡

白细胞介素１β转化酶家族在细胞凋亡过程中起关键作用身心健康已发现５个成员：ＩＣＥ，ＣＰＰ３２，Ｎｅｄｄ－２／Ｉｃｈ－１，Ｉｃｈ－２ＩＣＥｒｅｌⅢ和ＩＣＥｒｅｌⅢ，它们的高表达能引起细胞调邙，同时也受一些物质的调控。     

Dihydroxyoctadecamonoenoate esters inhibit the neutrophil respiratory burst

The leukotoxins [9(10)-and 12(13)-EpOME] are produced by activated inflammatory leukocytes such as neutrophils. High EpOME levels are observed in disorders such as acute respiratory distress syndrome and in patients with extensive burns. Although the physiological significance of the EpOMEs remains poorly understood, in some systems, the EpOMEs act as a protoxin, with their corresponding epoxide hydrolase metabolites, 9,10-and 12,13-DiHOME, specifically exerting toxicity. Both the EpOMEs and the DiHOMEs were also recently shown to have neutrophil chemotactic activity. We evaluated whether the neutrophil respiratory burst, a surge of oxidant production thought to play an important role in limiting certain bacterial and fungal infections, is modulated by members of the EpOME metabolic pathway. We present evidence that the DiHOMEs suppress the neutrophil respiratory burst by a mechanism distinct from that of respiratory burst inhibitors such as cyclosporin H or lipoxin A4, which inhibit multiple aspects of neutrophil activation.     

Activation of the human neutrophil respiratory burst with zymosan-activated serum

Zymosan-activated serum (ZAS) stimulated a time- and concentration-dependent generation of superoxide anion (O₂^?) by human neutrophils. O₂^? production was rapid with maximum generation occurring 2 minutes after cell exposure to ZAS. O₂^? generation is markedly reduced if cells are not preincubated with cytochalasin B prior to contact with ZAS. The amount of O₂^? produced by ZAS stimulated neutrophils was enhanced in the presence of extracellular calcium. However, the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), caused a dose-related inhibition of ZAS-elicited O₂^? production. Neutrophils pretreated with ZAS were desensitized to the subsequent exposure to this stimulus. The fact that pretreatment of neutrophils with ZAS did not diminish the capacity of these cells to generate O₂^? in response to 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 5(5),12(R)-dihydroxy-6,14-cis-8,10-transeicosatetraenoic acid (LTB₄), demonstrates the stimulus specific nature of ZAS-induced desensitization. Thus, ZAS, which contains the complement-derived neutrophil activator, C5a, a naturally occurring phlogistic mediator, represénts a relevent probe for investigating neutrophil function.     

The bactericidal effects of the respiratory burst and the myeloperoxidase system isolated in neutrophil cytoplasts

Neutrophil polymorphonuclear leucocytes kill bacteria by oxygen-dependent and oxygen-independent mechanisms. Many potentially toxic mechanisms have been described, but the complexity of the phagosomal environment and the synergy between oxidative and non-oxidative systems hamper the investigation of individual bactericidal mechanism in whole cells. Neutrophil cytoplasts are greatly depleted of granule proteins and permit the investigation of the bactericidal effects of the respiratory burst in isolation. In this study they have been used to examine the role of the respiratory burst and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus. Cytoplasts generated oxygen radicals at comparable rates to human neutrophils and phagocytosed but did not kill S. aureus. The selective reconstitution of the myeloperoxidase-hydrogen peroxide-halide system by coating bacteria with myeloperoxidase conferred on cytoplasts the ability to kill intracellular bacteria. However, extracellular killing by diffusible bactericidal factors was not detected in this system.     

Small G-proteins Ras,Rac and Rho in the regulation of the neutrophil respiratory burst induced by formyl peptide

Formylated peptides specifically activate many of the neutrophil functions; their action is mediated via formyl peptide receptors (FPRs). FPRs belong to the family of receptors having seven transmembrane-spanning domains and coupled with G-proteins (GPCR). About a dozen of highly homologous genes of FPRs were found to be localized in mouse chromosome 17. By binding with labeled N-formyl-Met-Leu-Phe (fMLF), FPRs are classified as receptors with high (FPR1) and low (FPR2 and FPR3/FPRL1) affinity to formyl peptide. Binding of formyl peptide with FPRs triggers the complex signaling events, the most studied are: activation of phospholipase C (PLC) with subsequent calcium signaling; launching of mitogen activated protein kinases (MAPKs) cascade pathway, and activation of phosphoinositol-3-kinase (PI3K) cascades. As we have shown previously, the priming of the respiratory burst of mice neutrophils occurs under the cell activation by fMLF in high doses only, i.e., it is necessary to activate low affinity FPRs. Besides, the usage of the specific MEK and p38MAPK inhibitors induced significant suppression of the response to 1 μM fMLM, while the response to 50 μM fMLF increased in the presence of the inhibitors. We suggest that there is a signal divergence upon activation of high and low affinity fMLF receptors, and small G protein dependent signaling pathways could be alternative to activate NADPH oxidase. Here we demonstrate that Ras-proteins participate in the respiratory burst activation, especially in activation via the high affinity fMLF receptors. Activation of the Rho- and Rac-proteins induced the down-regulation of the respiratory burst under the stimulation of high affinity FPRs. The inhibition of the Rho-proteins almost completely suppressed the respiratory burst activated via the high and low affinity receptors, probably due to inability to assemble of the cytoskeleton proteins and NADPH oxidase components.     

Elevated neutrophil respiratory burst activity in essential hypertensive patients

Neutrophils play a significant role in maintaining the integrity of innate immunity via their potent respiratory burst activity. However, the uncontrolled activation of respiratory burst in neutrophils also attributes to chronic diseases such as primary hypertension and atherosclerosis. In our study, we have investigated the activation of respiratory burst function of neutrophils harvested from essential hypertensive patients. In the presence of stimuli PMA and opsonized zymosan (OZ), hypertensive patients’ neutrophils secrete significantly higher amount of superoxide anions compared to normotensive control. Although the magnitude of activation varies between both groups, yet the kinetics of activation is similar. When normotensive control’s neutrophils were pre-treated with hypertensive serum, the cells failed to migrate toward fMLP which indicates the impairment of the migration property. In conclusion, the respiratory burst activity of neutrophils is affected by hypertension and their elevated superoxide anions production could be an aggravating factor in hypertension-related complication.     

Hepatocyte growth factor stimulates neutrophil degranulation but not respiratory burst

Neutrophil function is regulated in part by cytokines with growth factor activities for different cell types. Hepatocyte growth factor (HGF) is a cytokine produced during injury to the liver and other organs. Neutrophils are numerous in such tissue injury sites and may be influenced by HGF. In the present study the effect of HGF on neutrophils was investigated. The data show that HGF at 1-10 ng/ml increased lysosomal enzyme release from both specific and azurophilic granules of cytochalasin-B treated neutrophils. The release of specific granule contents in response to N-formyl-methionyl-leucylphenylalanine was also increased by HGF. In contrast there were no significant effects of HGF on neutrophil respiratory burst, adherence or locomotion. It is concluded that HGF modulates neutrophil granule exocytosis.     

Diverse effects of neutrophil integrin occupation on respiratory burst activation.

Integrin occupation can alter the function of neutrophils (PMN), but the mechanism(s) involved is still unclear. This study demonstrated that the occupation of PMN integrins (especially those of the beta(3) subfamily) strongly enhances TNF stimulation of the respiratory burst but down-regulates that induced by PMA, fMLP, Con A, and serum treated zymosan. Treatment of PMN with genistein, staurosporine, and wortmannin, inhibitors of tyrosine kinases, protein kinase C, and phosphotidylinostol 3-kinase (PI 3-kinase) respectively, completely blocked the TNF-stimulated respiratory burst in PMN. Genistein and wortmannin enhanced the PMA-stimulated respiratory burst but only in cells adherent to RGD peptide. These findings suggest that PMN integrins (beta(3) subfamily) can generate signals that regulate the PMN agonist responses, probably through the activities of tyrosine kinases and PI 3-kinase.     

Carbamylated albumin is a potent inhibitor of polymorphonuclear neutrophil respiratory burst

Carbamylation is a post-translational modification of proteins characterized by the binding of cyanate to amino groups, increased in renal failure. Pathophysiological consequences of carbamylation and adverse effects of carbamylated proteins on cell functions are poorly understood. We studied the influence of carbamylated albumin on polymorphonuclear neutrophil (PMN) O(2)(-) production. Carbamylated albumin significantly decreased O(2)(-) production in PMNs stimulated by type I collagen, but not by phorbol 12-myristate 13-acetate or tumor necrosis factor-alpha. This effect was related to inhibition of p(125)FAK phosphorylation. Such an alteration of neutrophil oxidative functions might explain characteristic complications of renal failure, such as increased occurrence of inflammation or infections.     

细胞凋亡与疾病的相关性

细胞凋亡是一种由基因控制的细胞自主性死亡过程。细胞凋亡在维持机体内环境的稳定方面起着重要作用。在细胞代谢过程中,一旦出现细胞凋亡失衡,将会导致许多疾病的发生。简单介绍细胞凋亡的基本情况及与疾病的关系。