FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

The relationship between apoptosis of neutrophils and the change of their intracellular free Ca2+ concentration [Ca2+]i was studied. FMLP and A23187 were used to elevate the [Ca2+]i while BAPTA was used to deplete it. Fluorescence microscope, flow cytometry and gel electrophoresis were used to study the percentage of cell apoptosis and the change of f-actin during apoptosis. The results showed that the apoptosis was obviously inhibited by fMLP and A23187, while accelerated by BAPTA. The detection of f-actin showed that the f-actin depolymerized obviously during apoptosis. The elevation of [Ca2+]i inhibit the actin depolymerization while depletion of [Ca2+]i accelerated it. This result indicated that the apoptosis of neutrophil was obviously inhibited by [Ca2+]i elevation but accelerated by [Ca2+]i depletion.     

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。     

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.     

嗜热芽孢杆菌XJT—9503高温中性蛋白酶的研究

嗜热芽孢杆菌ＸＪＴ－９５０３菌的发酵液经硫酸铵和丙酮分级沉淀分离纯化得到聚丙烯酰胺凝胶电泳均一的高温中性蛋白酶制品。ＳＤＳ－ＰＡＧＥ则得酶分子量为３００００。当以酪蛋白为底物时,酶反应最适温度为６５℃,最适ＰＨ为７,在ＰＨ６．５－９范围内稳定。在６５℃、０．０２ＭＰＨ７．５的磷酸缓冲液中的半衰期为５４ｍｉｎ。金属离了铜、汞、铝强列抑制酶活,钙离子、镁离子对酶活有促进作用     

一种嗜热厌氧纤维素降解细菌的分离纯化方法

根据纤维素降解细菌对不溶性纤维素底部的粘附作用,利用Ｈｕｎｇａｔｅ厌氧操作技术直接以不溶性纤维素粉为基质进行滚管,分离和纯化获得嗜热厌氧纤维素降解细菌。     

嗜盐放线菌分离方法

盐环境放线菌是一类独特的微生物资源。介绍了盐环境放线菌的种类,生理特性,影响嗜盐放线菌分离效果的基本因素,分离方法存在的问题,以及嗜盐放线菌分离的最新研究进展。推荐了3种适于分离嗜盐放线菌的培养基及分离方法。认为15%或以上NaCl浓度、一定比例的复合盐和培养基成分是高选择性分离嗜盐放线菌的关键。     

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.     

嗜碱放线菌分离方法的研究

本文从土样的处理、分离培养基的选择、抑菌剂的选择和培养基的配制方法等方面研究了嗜碱放线菌的分离方法。察氏琼脂、淀粉酪素琼脂甘油精氨酸琼脂的分离效果较好;土样用十二烷基磺酸钠处理,分离培养基中加重酪酸钾,放线菌酮能更有效地分离嗜碱放线菌;用缓冲溶液配制培养基不影响分离效果,且能使培养基的pH保持相对稳定。     

Examination of the Inhibitory Effect of Norathyriol in Formylmethionyl-Leucyl-Phenylalanine-Induced Respiratory Burst in Rat Neutrophils

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O₂^˙−) generation and O₂ consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O₂^˙− generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca²⁺]_i and the formation of inositol trisphosphate (IP₃) were significantly inhibited by norathyriol (30 μM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 μM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O₂^˙−, however, the inhibition of O₂^˙− generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.     

Cultured Rat and Purified Human Pneumocystis carinii Stimulate Intra-but not Extracellular Free Radical Production in Human Neutrophils

The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation. It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils. 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intra-cellular response of superoxide production, and 3) opsonized P. carinii. purified from human lung also stimulated human neutrophils to produce intracellular free radicals.     

Superoxide Production by Stimulated Neutrophils: Temperature Effect

Activation of neutrophils results in a one-electron reduction of oxygen to produce the superoxide anion and other oxygen-derived, microbicidal species. Evidence from many kinetic studies of oxygen-derived radicals generated by stimulated neutrophils in vitro shows that radical production is optimal at 37°C but only lasts several minutes and then rapidly subsides. These findings support the widely held perception that the neutrophil's “oxidative burst” is a transitory event that peaks within minutes of stimulation and ends shortly thereafter. However, while some studies have shown that under controlled conditions stimulated neutrophils can generate superoxide continuously for several hours, others have observed that the superoxide formation by neutrophils stimulated in buffer at 37°C does not persist. To reconcile the conflicting findings and to better understand neutrophil function, we have reinvestigated the effect of temperature on the kinetics of radical generation by PMA-stimulated cells. Electron paramagnetic resonance spectroscopy coupled with spin-trapping and SOD-inhibitable ferricytochrome c reduction were used to monitor superoxide production by neutrophils stimulated at either 25°C or 37°C in RPMI 1640 medium or in Hank's balanced salt solution. When oxygen was supplied continuously, neutrophils stimulated at 25°Cin buffer or in medium generated superoxide for several hours but at 37°C. particularly in HBSS, O₂-formation strikingly and rapidly decreased. This cessation of superoxide generation was reversible by lowering the temperature back to 25°C. These data imply that in vivo neutrophils may be capable of generating oxy-radicals for prolonged periods. In part, our results may also explain the often observed termination of neutrophil-derived radical formation in vitro and help to dispel the perception that neutrophil-derived oxy-radical production is an ephemeral phenomenon.     

Suppression of Anti-Candida Activity of Murine and Human Neutrophils by Glucocorticoids

Effects of glucocorticoid (GC) compounds on inhibitory activity of neutrophils to mycelial growth of Candida albicans were examined by in vitro crystal violet staining method with 14 hr co-culture. Both GC hormones (hydrocortisone ≥6 × 10^–7 m and corticosterone ≥10^–6 m ) and anti-inflammatory GC agents (prednisolone ≥10^–7 m and dexamethasone ≥10^–8 m ) significantly suppressed anti-Candida activity of murine casein-induced neutrophils. Anti-Candida activity of human neutrophils prepared from peripheral blood was also suppressed by hydrocortisone (≥6 × 10^–7 m ). These GC compounds did not affect the Candida growth in the absence of neutrophils. Steroidal compounds without anti-inflammatory activity, cholesterol, cholic acid, aldosterone did not suppress neutrophil activity. These results suggest that GCs at their physiological or clinical concentration may suppress anti-Candida activity of neutrophils in vivo.     

A Method for Separation of Granulocytes From Normal Human Blood Using Hidroxiethrl Starch

A comparison of the granulocyte yield obtained from normal buffy coats following the addition of different concentrations of hydrojtyethyl starch (H.E.S.) was made. Hydrcocyethyl starch proved to be at least as effective as Dextran both in regard to the percentage of granulocytes recovered and the speed of sedimentation of the red blood cells.     

A Modified Wright's Method for Staining Blood Smears

After the blood smear is treated for the proper length of time with Wright's stain, neutral distilled water is used for diluting the stain. After the slide has been treated with neutral distilled water until the smear becomes pinkish it is then treated with pure absolute methyl alcohol which destains the plasma. Neutral distilled water is again kept on the mount until the corpuscles are well stained. Then the slide is dehydrated with absolute ethyl alcohol, cleared with clove oil and completed in the usual way.

Blood smears of different groups of vertebrates were uniformly brilliantly stained with the above technic.

Several lots of Wright's dry stain have been tested with the modified technic and no difficulties have been encountered in its application.     

Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O₂^.-) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.     

用ESR自旋捕集法研究吸烟烟气处理的脂质体对大鼠粒细胞产生O_2~-的影响

吸烟烟气能引发卵磷脂脂质体的脂质过氧化。若将吸烟烟气处理20s的脂质体作用完整的大鼠粒细胞(RPN),用ESR自旋捕集方法发现,当作用时间在25min内(脂浓度1.0mg/ml)或脂浓度小于15.0mg/ml(而作用时间均为15min)时,这种过氧化的脂质体能增加RPN呼吸爆发产生O_2~-的量。而没有经吸烟烟气处理的新制脂质体,在脂浓度大于0.2mg/ml(作用时间15min)时,都不同程度地抑制RPN产生O_2~-。     

用ESR自旋捕集法研究吸烟烟气处理的脂质体对大鼠粒细胞产生O_2~-的影响

吸烟烟气能引发卵磷脂脂质体的脂质过氧化。若将吸烟烟气处理20s的脂质体作用完整的大鼠粒细胞(RPN),用ESR自旋捕集方法发现,当作用时间在25min内(脂浓度1.0mg/ml)或脂浓度小于15.0mg/ml(而作用时间均为15min)时,这种过氧化的脂质体能增加RPN呼吸爆发产生O_2~-的量。而没有经吸烟烟气处理的新制脂质体,在脂浓度大于0.2mg/ml(作用时间15min)时,都不同程度地抑制RPN产生O_2~-。     

Activation of the neutrophil respiratory burst by chemoattractants: Regulation of the N-formyl peptide receptor in the plasma membrane

The N-formyl peptide receptor mediates a number of host defensive responses of human neutrophils that result in chemotaxis, secretion of hydrolytic enzymes, and superoxide generation. Inappropriate activation or defective regulation of these responses can result in pathogenic states responsible for inflammatory disease. The receptor is a 50 to 70-kD, integral plasma membrane glycoprotein with intracellular and surface localization. Its abundance in the membrane is regulated by membrane flow and recycling processes. Cytoskeletal interactions are believed to control its organization in the plane of the membrane and interaction with other proteins. The receptor's most important interaction is with guanyl nucleotide binding proteins that serve as signal transduction partners ultimately leading to activation of effector responses. Because the interaction of the receptor with G proteins is necessary for transduction, control of this interaction may be at the root of understanding the molecular control of responses in these cells. This review briefly summarizes some of the molecular properties, dynamics, and interactions of this receptor system in human neutrophils and discusses how these characteristics may pertain to the activation and control of superoxide generation.