体内表达谷胱甘肽过氧化物酶-7基因对骨髓抑制的防护作用研究

目的 探讨体内表达谷胱甘肽过氧化物酶-7基因(Gpx-7)对5-氟尿嘧啶(5-Fu)引发小鼠骨髓抑制的防护作用.方法 以尾静脉注射5.Fu(250 mg/kg)的方法建立小鼠骨髓抑制和再生模型.5-Fu注射后第1天,通过电穿孔转染法将pcDNA3.1-Gpx7重组质粒50 μg注入小鼠胫前肌(实验组),以pcDNA3.1空质粒载体50μg电穿孔转染作为对照组,每组各30只小鼠.5-Fu注射前和注射后3、7、11、14 d,2组各断颈处死6只小鼠,计数小鼠外周血白细胞数、血小板数和单条腿骨髓细胞总数;然后分别取5-Fu注射后7、14 d断颈处死小鼠各3只进行骨髓细胞集落形成试验;并分别取5-Fu注射前和注射后3、7、11、14d断颈处死的小鼠各1只制备完整大腿骨石蜡组织切片,观察骨髓组织形态学的变化.结果 注射5-Fu后11和14 d,实验组外周血白细胞数分别为(3917±733)和(6857±1878)/μl,均明显高于同时点对照组[分别为(2683±920)和(4017±1011)/μl,均P<0.05)];注射5-Fu后11 d,实验组外周血小板数为(150.8±64.3)万/μl,明显高于同时点对照组[(63.0±60.3)万/μl,P<0.05)];注射5-Fu后不同时间2组间单条腿骨髓细胞总数差异无统计学意义.注射5-Fu后7、14 d,2组之间骨髓细胞集落形成数差异无统计学意义.实验组注射5-Fu后11 d小鼠的骨髓组织形态恢复程度与对照组注射5-Fu后14 d小鼠类似:注射5-Fu后14 d小鼠的骨髓组织形态接近注射前.结论 体内表达Gpx-7基因可以促进被化疗药物5-Fu抑制的小鼠骨髓再生.     

Effect of hashish compounds on mouse peritoneal macrophages.

Light microscopy reveals an induction of extensive vacuolation in the macrophage after exposure to either delta 1-tetrahydrocannabinol or cannabidiol. Numerous small vacuoles appear in the cell periphery as early as 15 minutes after exposure (at 37degrees C.) to either of the compounds in 20 per cent newborn calf serum-Dulbecco's modified Eagle medium. The small lucent vacuoles coaleasce and yield enormous vacuoles which dominate the the cytoplasm. At approximately 3 hours, many of the vacuoles seem to burst with a concomitant expulsion of cell interior. The effect of hashish compounds on macrophages is essentially irreversible; exposure for 15 minutes to 10(-5) m of delta 1-THC or cannabidiol and a thorough wash in 20 per cent serum-medium, suffices to trigger the sequence of vacuolation and total cell death in the culture. Two major processes involving early reorganization of cellular membranes have been observed using electron microscopy. One relates to the formation of numerous autophagic vacuoles full of organelles, the other relates to the appearance of cytoplasmic inclusions representing extensive destruction of intracellular constituents. Both types of cytoplasmic change have been observed in alveolar macrophages of hashish smokers. Thus, the conditions in the in vitro studies are similar to conditions in people exposed to hashish smoke.     

脂多糖诱导的小鼠腹腔巨噬细胞基因表达谱分析

目的　利用基因芯片技术分析脂多糖 (LPS)活化的小鼠腹腔巨噬细胞基因表达谱 ,以更全面地了解LPS诱导的巨噬细胞反应。方法　以未刺激的和用 1mg/LLPS刺激的小鼠腹腔巨噬细胞制备3 3 P标记的cDNA探针 ,分别与含有 1176个已知基因的小鼠cDNA芯片杂交。结果　活化组和未刺激组间的 2倍差异表达基因为 118个 ,3倍差异表达基因为 6 9个 ,其中 4 4个上调 ,2 5个下调。转录因子、细胞内信号调节蛋白、炎症细胞因子和细胞凋亡相关基因的转录发生明显的调节变化。结论提供了LPS活化的巨噬细胞综合基因表达信息 ,并筛选出一些新的可能与LPS活化相关的基因。     

Effects of interleukin-10 on chemokine KC gene expression by mouse peritoneal macrophages in response to Candida albicans

Chemokine KC has been considered to be a murine homologue of human GRO/MGSA and was identified as chemoattractant for monocytes and neutrophils. This study examined the expression of KC mRNA in thioglycollate-elicited mouse peritoneal macrophages that were stimulated in vitro with Candida albicans (CA). Also examined were the inhibitory effects of IL-10 on the CA-induced expression of KC gene by Northern blot analysis. CA was found to induce chemokine gene expression in a gene-specific manner, CXC chemokine IP-10 mRNA expression was not detected in CA-stimulated macrophages. Maximum KC mRNA expression was observed approximately 2 hr after adding CA. The inhibitory action of IL-10 to CA-induced KC mRNA expression on mouse peritoneal macrophages was independent on concentration and stimulation time of IL-10 and was observed approximately one hour after adding IL-10 and CA simultaneously. IL-10 produced a decrease in the stability of KC mRNA, and CA-stimulated macrophages with cycloheximide blocked the suppressive effect of IL-10. These results suggest that CA also induces chemokine KC from macrophages, and IL-10 acts to destabilize CA-induced KC mRNA and de novo synthesis of an intermediate protein is a part of the IL-10 suppressive mechanism.     

Levamisole causes differential cytokine expression by elicited mouse peritoneal macrophages.

Thioglycolate-elicited peritoneal macrophages from normal C57B1/6J mice were examined in vitro for bacterial lipopolysaccharide (LPS)-stimulated interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF) production. Macrophages from mice administered a single oral dose of levamisole (3 mg/kg) 1 to 4 days prior to macrophage harvest demonstrated a twofold enhancement of IL-1 production compared to vehicle-treated mice. In contrast, IL-6 production and TNF production by the same macrophages were inhibited up to 36 and 62%, respectively, compared to production by macrophages harvested from vehicle-treated mice. Similar results were observed when IL-1 production and TNF production were followed in peritoneal exidate cells directly stimulated with levamisole in vitro. The ex vivo LPS-stimulated IL-1 production was enhanced 4 days after macrophage elicitation, whereas TNF and IL-6 production returned to baseline by 72 h after macrophage recruitment and augmentation. No evidence could be found for the presence of inhibitors of TNF or IL-6. The specificity of the IL-1, IL-6, and TNF bioactivities was demonstrated by neutralization with specific antisera. Immunoprecipitation studies of supernatants from biosynthetically labeled macrophages also revealed augmented IL-1 production and decreased IL-6 and TNF, indicating that levamisole may have affected cytokine production at the translational level. Kinetics studies revealed that ex vivo release of IL-6 and TNF by macrophages from levamisole-dosed mice was delayed compared to production of these cytokines by macrophages harvested from mice given vehicle only. The results may explain, in part, the reported ability of levamisole to ameliorate cases of rheumatoid arthritis or other autoimmune and inflammatory diseases by affecting the relative levels of cytokines produced by macrophages recruited to sites of injury, which are associated with inflammation and acute-phase protein synthesis.     

Effect of histamine on the gene expression and biosynthesis of complement components C2, factor B and C3 in mouse peritoneal macrophages.

The gene expression and biosynthesis of C2, factor B and C3 have been investigated in vitro in mouse resident peritoneal macrophages after incubation with histamine. C2- and factor B-specific mRNA and the amount of the immunoprecipitated C2 and factor B were decreased by 10(-4) M and 10(-8) M histamine. These effects can be abrogated by the H2 antagonist cimetidine and mimicked by the H2 agonists impromidine and 4-methylhistamine. Since the H1 antagonist chlorpheniramine and the H1 agonists PEA and 2-methylhistamine have little effect on C2 and are ineffective on factor B, a strong H2 receptor dependence of the inhibition of C2 and factor B gene expression and biosynthesis is suggested. Conversely, the C3 gene expression and biosynthesis can be influenced through both H1 and H2 receptors, e.g. elevated by histamine + cimetidine, PEA and 2-methylhistamine through H1 receptors, and inhibited by histamine + chlorpheniramine, impromidine and 4-methylhistamine through H2 receptors. The data obtained by quantification of C2, factor B and C3 mRNA concentration of peritoneal macrophages suggest that the regulation of biosynthesis of these complement components by histamine in mouse peritoneal macrophages is under pretranslational control.     

Effect of lipopolysaccharide on transport and metabolism of arginine in mouse peritoneal macrophages.

The transport activity of arginine in mouse peritoneal macrophages was strongly induced when they were cultured with 1 ng/ml bacterial lipopolysaccharide (LPS) for 12 h. Arginine in the medium decreased whereas ornithine in the medium increased during the culture. This time-dependent change of arginine to ornithine was accelerated by LPS. However, the activity of arginase in the macrophages did not change during the culture with or without LPS and release of arginase from the cells to the medium was not detected. It is suggested that the transport of arginine and ornithine was a rate-limiting step in arginine-to-ornithine conversion in the macrophage culture medium. A possible role of the induction of arginine transport activity in the macrophage cytocidal activity due to arginine depletion and nitric oxide production is discussed.     

Increased superoxide anion production and glutathione peroxidase activity in peritoneal macrophages from autoimmune-prone MRL/Mp-Ipr/lpr mice

We studied the release of superoxide anion (O-2) in peritoneal macrophages from autoimmuneprone MRL/Mp-lpr/lpr (MRL/l) mice. Compared to resident peritoneal macrophages from control MRL/Mp-+/+ (MRL/n) mice, macrophages from MRL/l mice exhibited an age-related increase of spontaneous and PMA-induced O-2 secretion in association with the development of the autoimmune process. Analysis of the kinetic parameters of NADPH oxidase in macrophages revealed that MRL/l macrophages were in a primed state, as shown by the decreased Km value of the oxidase for NADPH. Furthermore, we studied several key enzymes for their ability to scavenge the oxygen radicals in the macrophages. Among the enzymes studied, only glutathione peroxidase (GSH Px) activity was increased in peritoneal macrophages from MRL/l mice and this change was closely correlated with the increase in O-2 production. Thus, GSH Px activity in macrophages seems to play an important role in macrophage functions under increased oxidative stress.     

Fish oil dietary supplementation reduces Ia expression in rat and mouse peritoneal macrophages

Preliminary studies suggest that administration of fish oil fatty acids may be beneficial in several immunological diseases; therefore, we studied the effect of fish oil dietary supplementation on the expression of Ia in stimulated murine peritoneal macrophages. Rats (n = 19) and mice (n = 27) on standard rodent feeding were separated in experimental (E) and control (C) groups that received fish oil or saline solution, respectively, daily for 4 weeks by esophageal gavage. Cholesterol serum levels were significantly lowered by fish oil (E vs C, P less than 0.01). E and C groups were injected intraperitoneally with Listeria monocytogenes (LM) and peritoneal cells were harvested 4 and 7 days after infection. Decreased expression of Ia induced by LM was found in rats (C = 49.68 +/- 5.09%, E = 16.95 +/- 4.3%, P less than 0.01) and mice (C = 47.38 +/- 7.63%, E = 26.66 +/- 1.92%, P less than 0.01). Animals with a more pronounced depression of serum cholesterol (reduction of 44.04 +/- 1.52% of baseline levels) had more depression of Ia expression (6.47 +/- 1.22%, P less than 0.001 vs control). Reduction of Ia expression was not related to PGE2 production by peritoneal cells. Reduction of Ia expression by fish oil could induce down-regulation of antigen presentation and alloreactivity.     

Surface expression and rapid internalization of macrosialin (mouse CD68) on elicited mouse peritoneal macrophages

Macrosialin, the mouse homolog of human CD68, is a heavily glycosylated transmembrane protein found almost exclusively in macrophages. Its function remains uncertain. It has a high affinity for oxidized low-density lipoprotein (LDL) in ligand blots and antibodies against the human homolog, CD68, inhibit the binding of oxidized LDL to a human monocyte-derived cell line (THP-1). However, there is still controversy as to whether macrosialin, found predominantly in late endosomes, is expressed at all on the plasma membrane. The present studies, done in thioglycollate-elicited peritoneal macrophages, confirm that macrosialin is predominantly intracellular but show clearly that 10-15% of it is expressed on the cell surface. Exchange with intracellular pools occurs at an extremely high rate. The results are compatible with a surface function, including internalization of bound ligands or adhesion to surfaces.     

Survival of Enterococcus faecalis in mouse peritoneal macrophages

Enterococcus faecalis was tested for the ability to persist in mouse peritoneal macrophages in two separate studies. In the first study, the intracellular survival of serum-passaged E. faecalis 418 and two isogenic mutants [cytolytic strain FA2-2(pAM714) and non-cytolytic strain FA2-2(pAM771)] was compared with that of Escherichia coli DH5alpha by infecting BALB/c mice intraperitoneally and then monitoring the survival of the bacteria within lavaged peritoneal macrophages over a 72-h period. All E. faecalis isolates were serum passaged to enhance the production of cytolysin. E. faecalis 418, FA2-2(pAM714), and FA2-2(pAM771) survived at a significantly higher level (P = 0.0001) than did E. coli DH5alpha at 24, 48, and 72 h. Internalized E. faecalis 418, FA2-2(pAM714), and FA2-2(pAM771) decreased 10-, 55-, and 31-fold, respectively, over the 72-h infection period, while internalized E. coli DH5alpha decreased 20, 542-fold. The difference in the rate of survival of E. faecalis strains and E. coli DH5alpha was most prominent between 6 and 48 h postinfection (P = 0.0001); however, no significant difference in killing was observed between 48 and 72 h postinfection. In the second study, additional E. faecalis strains from clinical sources, including DS16C2, MGH-2, OG1X, and the cytolytic strain FA2-2(pAM714), were compared with the nonpathogenic gram-positive bacterium, Lactococcus lactis K1, for the ability to survive in mouse peritoneal macrophages. In these experiments, the E. faecalis strains and L. lactis K1 were grown in brain heart infusion (BHI) broth to ensure that there were equal quantities of injected bacteria. E. faecalis FA2-2(pAM714), DS16C2, MGH-2, and OG1X survived significantly better (P < 0.0001) than did L. lactis K1 at each time point. L. lactis K1 was rapidly destroyed by the macrophages, and by 24 h postinfection, viable L. lactis could not be recovered. E. faecalis FA2-2(pAM714), DS16C2, MGH-2, and OG1X declined at an equivalent rate over the 72-h infection period, and there was no significant difference in survival or rate of decline among the strains. E. faecalis FA2-2(pAM714), MGH-2, DS16C2, and OG1X exhibited an overall decrease of 25-, 55-, 186-, and 129-fold respectively, between 6 and 72 h postinfection. The overall reduction by 1.3 to 2.27 log units is slightly higher than that seen for serum-passaged E. faecalis strains and may be attributable to the higher level of uptake of serum-passaged E. faecalis than of E. faecalis grown in BHI broth. Electron microscopy of infected macrophages revealed that E. faecalis 418 was present within an intact phagocytic vacuole at 6 h postinfection but that by 24 h the infected macrophages were disorganized, the vacuolar membrane was degraded, and the bacterial cells had entered the cytoplasm. Macrophage destruction occurred by 48 h, and the bacteria were released. In conclusion, the results of these experiments indicate that E. faecalis can persist for an extended period in mouse peritoneal macrophages.     

Interleukin-13 primes iNO synthase expression induced by LPS in mouse peritoneal macrophages

Th2 cytokines such as interleukin-13 (IL-13) have both, stimulatory and inhibitory effects on effector functions of macrophages. Reactive nitrogen species are classically induced in Th1 cytokines and/or lipopolysaccharides (LPS) activated macrophages and this response is inhibited by IL-13. In contrast, IL-13 primes macrophages to produce NO in response to LPS when IL-13 treatment happens prior to LPS exposure. This mechanism occurs through a complex signalling pathway, which involves the scavenger receptor CD36, the LPS receptor CD14 and the nuclear receptor PPARgamma. The enhancement of NO production is the consequence of iNOS induction at mRNA and protein levels. The increase of the NO production induced by LPS in IL-13 pre-treated macrophages is found to potentiate the inhibition of Toxoplasma gondii intracellular replication. These results reveal a novel IL-13 signalling pathway that primes the antimicrobial activity of macrophages induced by LPS caused by overexpression of the iNOS-NO axis.     

Differences in peroxidase localization of rabbit peritoneal macrophages after surface adherence

Unlike resident peritoneal macrophages, which contain peroxidase in the rough endoplasmic reticulum (RER) and perinuclear cisternae (PN), macrophages elicited into the rabbit peritoneal cavity by various stimulants lack the enzyme. Since we had previously found that such peroxidase reactivity rapidly appears in the RER and PN of blood monocytes after surface adherence in vitro, we wondered whether the enzyme could be similarly produced in elicited macrophages by adherence. Cells from peritoneal exudates (96 hours after endotoxin injection) were harvested, suspended in culture medium, and allowed to adhere to fibrin-coated or plastic surfaces. Following culture for various intervals, they were fixed, incubated for peroxidase, and examined by electron microscopy. We observed that these elicited cells, which initially contained no cytochemically detectable peroxidase, acquired peroxidatic activity in the RER and PN within 2 hours after adherence in culture. Thus macrophages, like blood monocytes, may rapidly acquire peroxidase reactivity as a consequence of plasma membrane: external surface interaction. In view of this finding, it would seem unwise to use peroxidase localization as the basis for advocating the existence of two separate lines of peritoneal macrophages, as has been proposed by previous investigators.     

Some effects of lignocaine on cultured mouse peritoneal macrophages

Macrophages were exposed to lignocaine in perifused and non-perifused cultures using media with or without foetal calf serum. Effects on morphology, viability, phagocytosis and the release of enzymes were assessed. During the period of contact with lignocaine there was a selective release of -glucuronidase. After washing enzyme release continued over a period of 7 hours and, in the absence of foetal calf serum, a decrease in the total -glucuronidase content was found in non-perifused, cultures. Although lignocaine-treated cells phagocytosed particulates the rate of enzyme release was reduced compared with normal cells when subsequently exposed to quartz.