双歧杆菌对裸鼠腹腔巨噬细胞激活作用的初步观察

用青春型双歧杆菌注射于裸鼠腹腔,分别以中性红吞噬法以及M TT 法检测了裸鼠腹腔巨噬细胞的吞噬能力和能量代谢水平。结果显示双歧杆菌注射组裸鼠腹腔巨噬细胞的吞噬能力和能量代谢水平均显著高于对照组(P< 0.01)。提示青春型双歧杆菌能激活巨噬细胞,增强其吞噬功能,提高其能量代谢水平     

双歧杆菌习腹腔巨噬细胞激活作用的初步观察

用青春型双歧杆菌注射于裸鼠腹腔,分别以中性红吞噬法以及ＭＴＴ法检测了裸鼠腹腔巨噬细胞的吞噬能力和能量代谢水平。结果显示双歧杆菌注射组裸鼠腹腔巨噬细胞的吞噬能力和能量代谢水平均显著高于对照组（Ｐ＜０．０１）。提示青春型双歧杆菌能激活巨噬细胞,增强其吞噬功能,提高其能量代谢水平。     

双歧杆菌对裸鼠腹腔巨噬细胞产生IL—1及IL—6的影响

给裸小鼠腹腔注射活的青春型双歧杆菌,并以小鼠胸腺细胞增殖法及ＥＬＩＳＡ法分别检测了裸鼠腹腔巨噬细胞分泌的ＩＬ１活性及ＩＬ６含量。结果表明：实验组裸鼠腹腔巨噬细胞分泌的ＩＬ１活性以及ＩＬ６含量均显著高于对照组,两者均具有统计学意义（ｐ＜００１）。这提示青春型双歧杆菌可激活巨噬细胞产生ＩＬ１以及ＩＬ６,它们在该菌调节机体免疫反应中可能起一定作用。     

青春型双歧杆菌微生态制品对小鼠腹腔巨噬细胞酸性磷酸酶含量的影响

我们观察了青春型双歧杆菌微生态制品ＤＭ８５０４,对小鼠腹腔巨噬细胞酸性磷酸酶的影响。以青春型双歧杆菌ＤＭ８５０４,０．２ｍｌ（含活菌１８×１０９）。注入小鼠腹腔。每天１次连续５次,对小鼠腹腔巨噬细胞酸性磷酸酶变化进行连续定量测定,并于终止注射后第３天取腹腔巨噬细胞涂片,以酶染色方法进行半定量观察。结果表明,于注射开始后第２天直至注射停止后第５夫,实验组小鼠腹腔巨噬细胞酸性磷酸酶含量均高于对照组,说明ＤＭ８５０４可激活小鼠巨噬细胞,提高酸性磷酸酶的含量及这种作用可维持的天数。     

双歧杆菌DNA对巨噬细胞MAPK的影响

目的 探索青春型双歧杆菌的DNA对巨噬细胞丝裂素活化的蛋白激酶（MAPK）活性的影响。方法 以激光共聚焦显微镜定量测定小鼠腹腔巨噬细胞MAPK家系中ERK1／2、JNK和p38的含量。结果 双歧杆菌DNA注射组小鼠腹腔巨噬细胞ERK1／2的平均荧光强度明显高于对照组（P〈0．01）,而JNK和p38的平均荧光强度在2组间则差异无显著性（P〉0．05）。结论 青春型双歧杆菌的DNA能提高巨噬细胞ERK1／2的活性,这可能是其激活巨噬细胞的途径之一。     

双歧杆菌DNA对巨噬细胞PKC的影响

目的探索青春型双歧杆菌的DNA对巨噬细胞PKC家族的影响.方法以激光共聚焦显微镜定量测定小鼠腹腔巨噬细胞PKCα、PKCβⅠ、PKCβⅡ、PKCγ、PKCε和PKCζ的含量.结果双歧杆菌DNA注射组小鼠腹腔巨噬细胞PKCα和PKCβⅡ的平均荧光强度明显高于对照组（P＜0.01）,而PKCβⅠ、PKCγ、PKCε和PKCζ的平均荧光强度在2组间则差异无显著性（P＞0.05）.结论青春型双歧杆菌的DNA能活化巨噬细胞的PKCα和PKCβⅡ.     

双歧杆菌的完整肽聚糖对巨噬细胞产生IL-18的影响

目的探讨分叉双歧杆菌的完整肽聚糖对巨噬细胞功能的调节作用.方法以完整肽聚糖注射于小鼠腹腔,用ELISA法测定小鼠腹腔巨噬细胞产生的IL-18的含量.结果完整肽聚糖注射组小鼠腹腔巨噬细胞产生的IL-18的含量显著高于对照组(P＜0.01).结论分叉双歧杆菌的完整肽聚糖能激活巨噬细胞,并使之分泌多量的IL-18.     

双歧杆菌对实验性大肠癌诱导型一氧化氮合酶表达的影响

目的:用免疫组化法观察大肠癌移植瘤诱导型一氧化氮合酶(iNOS)的表达。方法:以大肠癌裸鼠移植瘤为动物模型,将青春型双歧杆菌注射于裸鼠腹腔。结果:显示双歧杆菌注射组大肠癌移植瘤iNOS的表达率、表达强度和阳性细胞数量均显著高于肿瘤对照组(P<0.01)。结论:青春型双歧杆菌能增强大肠癌移植瘤iNOS的蛋白表达水平。它的表达可能介导了双歧杆菌诱导大肠癌移植瘤细胞的凋亡。     

双歧杆菌的DNA对小鼠腹腔巨噬细胞游离Ca2+离子水平的影响

目的 从信号转导途径探索青春型双歧杆菌的DNA激活巨噬细胞的机制。方法 用钙离子荧光指示剂Fluo-3／AM负载小鼠腹腔巨噬细胞,以激光共焦镜显微镜观察不同浓度的DNA对巨噬细胞内游离Ca^2＋离子的浓度变化。结果 所用刺激浓度的DNA均能显著升高巨噬细胞内游离Ca^2＋．离子的浓度,并且随着刺激物浓度的逐增,巨噬细胞内游离Ca^2＋离子的上升速度以及达到峰值的荧光值也逐渐升高（P〈0．01）。结论 青春型双歧杆菌的DNA能提高巨噬细胞内游离Ca^2＋离子的浓度,并呈剂量依赖性。     

双歧杆菌对实验性大肠癌PCNA和bcl-2表达的影响

目的：观察青春型双歧杆菌对大肠癌移植瘤增殖细胞核抗原（ＰＣＮＡ）和ｂｃｌ－２基因蛋白表达水平的影响。方法：以大肠癌裸鼠移植瘤为动物模型,用免疫组化法。结果：显示双歧杆菌注射组大肠癌移植瘤ＰＣＮＡ阳性细胞密度以及ｂｃｌ－２蛋白表达率、表达强度、阳性细胞密度均显著低于肿瘤对照组（Ｐ＜０．０１）。结论：青春型双歧杆菌能明显降低大肠癌的增殖活性,同时使其ｂｃｌ－２基因的表达下调。     

青春双歧杆菌DM8504菌株对荷瘤小鼠体内NO诱生作用的研究

应用处死的青春双歧杆菌ＤＭ８５０４菌株皮下免疫荷瘤小鼠,按Ｇｒｉｅｓ反应原理测定一氧化氮（ＮＯ）的含量。结果表明,双歧杆菌能提高荷瘤小鼠体内ＮＯ的含量,较对照组显著升高,肿瘤组织的坏死程度在处理组与对照组之间也具有显著性差异。提示：在双歧杆菌抗肿瘤作用中,除ＴＮＦ—α外,ＮＯ也发挥重要作用。     

青春双歧杆菌对2型糖尿病模型大鼠免疫功能和肾脏的影响

目的通过观察青春双歧杆菌对2型糖尿病模型大鼠血清中细胞因子IL-2、IL-6和IFN-γ活性的影响,以及血清及尿中的NO与ET-1的变化,探讨青春双歧杆菌对2型糖尿病模型免疫功能和肾脏的影响。方法采用青春双歧杆菌灌胃2型糖尿病模型大鼠,取血液和尿液,ELISA法检测细胞因子IL-2、IL-4、IL-6、IFN-γ和ET-1活性,硝酸酶还原法测定NO水平。结果青春双歧杆菌提高IL-2、IL-4水平,降低IL-6、IFN-γ和ET-1活性,NO水平在病程中动态变化。结论青春双歧杆菌具有平衡2型糖尿病模型大鼠免疫功能,抑制ET-1,调节NO水平的作用,从而预防肾小球硬化的发生。     

The effect of vaccination with the lysate of heat-shocked tumor cells on nitric oxide production in BALB/c mice with fibrosarcoma tumor

The aim of this study was to investigate the effect of heat shock protein-70 (HSP-70) on splenocyte proliferation and nitric oxide (NO) production in the BALB/c mice fibrosarcoma tumor model. To do so, HSP-70 was induced in the lysate of heat-shocked tumor cells and WEHI-164 cells (mouse fibrosarcoma cell line) were injected subcutaneously into the right flank of inbred BALB/c mice to establish a tumor model. Three animal bearing tumor groups were applied: the test group; vaccinated with HSP-70 enriched tumor lysate; control group I, vaccinated with tumor lysate only; and control group II, which received PBS. Using immunoblot analysis, an increase of HSP-70 expression was detected in the lysate of heat-shocked cells in comparison with non-heat-shocked cells. The effect of the test lysate on NO production was measured both in vitro and in vivo in the peritoneal macrophages and splenocytes of tumor bearing mice, respectively. The result showed a significant increase in NO production both in vitro by peritoneal macrophages and in vivo after immunization with HSP-70 enriched tumor lysate. In addition, tumor growth was significantly postponed and the proliferation of splenocytes was increased in the test group. Our results indicate that the lysate of heat-shocked tumor cells was more potent than that of non-heat-shocked tumor cells in inducing anti-tumor immunity. Since production of NO by HSP-activated antigen presenting cells (APCs) is likely to affect innate immunity and tumor growth, the probable mechanism of postponing tumor growth would be NO production by innate immune cells. These findings provide a useful therapeutic model for developing novel approaches to cancer treatments.     

Enhanced utilization and altered metabolism of arginine in inflammatory macrophages caused by raised nitric oxide synthesis

Nitric oxide (NO) production was increased in macrophages during inflammation. Casein-elicitation of rodents causing a peritoneal inflammation offered a good model to study alterations in the metabolism of L-arginine, the precursor of NO synthesis. The utilization of L-arginine for NO production, arginase pathway and protein synthesis were studied by radioactive labeling and chromatographic separation. The expression of NO synthase and arginase was studied by Western blotting.Rat macrophages utilized more arginine than mouse macrophages (228+/-27 versus 71+/-12.8pmol per 10(6) macrophages). Arginine incorporation into proteins was low in both species (<15% of labeling). When NO synthesis was blocked, arginine was utilized at a lower general rate, but L-ornithine formation did not increase. The expression of enzymes utilizing arginine increased. NO production was raised mainly in rats (1162+/-84pmol citrulline per 10(6) cells) while in mice both arginase and NO synthase were active in elicited macrophages (677+/-85pmol ornithine and 456+/-48pmol citrulline per 10(6) cells).We concluded, that inflammation induced enhanced L-arginine utilization in rodent macrophages. The expressions and the activities of arginase and NO synthase as well as NO formation were increased in elicited macrophages. Specific blocking of NO synthesis did not result in the enhanced effectivity of the arginase pathway, rather was manifested in a general lower rate of arginine utilization. Different rodent species reacted differently to inflammation: in rats, high NO increase was found exclusively, while in mice the activation of the arginase pathway was also important.     

Nitric oxide production and iNOS mRNA expression in mice induced by repeated stimulation with live Fusobacterium nucleatum

There have been few studies on the detection of direct nitric oxide (NO) production and interferon-gamma (IFN-gamma) in vivo without using animal cell culture. We questioned whether NO and IFN-gamma could be produced at the site of infection. The peritoneal cavity of mice was used as the local infection model. NO and IFN-gamma in abdominal washings from these mice were measured directly at various times after injection of Fusobacterium nucleatum, a gram-negative rod periodontal pathogen. The mice were divided into three groups: those treated with live bacteria (LB), those treated with heat-killed bacteria (HKB) and those untreated: normal (N). These mice were compared on the basis of cell filtration, NO and IFN-gamma production by injection of live bacteria (LFn) or heat-killed bacteria (HKFn). In the LB group, the total cell number increased corresponding to an increase in neutrophils after injection of both LFn and HKFn. A low level of NO was constantly produced in abdominal washings, but a significant amount of NO was synthesized in the LB group only 12 hr to 24 hr after injection of LFn. At the same time iNOS enzyme activity and iNOS mRNA expression were detected. IFN-gamma, which may contribute to enhance NO production, was also secreted at a high level from peritoneal exudate cells (PEC) at 12 hr and 24 hr in the LB group by stimulation of LFn. At 12 hr and 24 hr, iNOS positive cells in the LB group by infection of LFn were identified and shown to contain mostly macrophages. These findings indicate that live bacteria play important roles in NO production by macrophages. It is suggested that NO may contribute to the inflammatory response during F. nucleatum infection in periodontitis.     

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.     

纳米氧化铁双歧杆菌LTA对实验性胃癌抑制效果的影响

目的探讨纳米氧化铁（Fe3O4）双歧杆菌脂磷壁酸（Lipoteichoic acid,LTA）对实验性胃癌体内抑制效果的影响及其抑瘤途径。方法用人胃癌BGC823细胞建立裸鼠移植瘤模型,用硝酸还原酶法检测各剂量组裸鼠腹腔巨噬细胞分泌NO和iNOS的含量,用免疫组化法检测移植瘤内VEGF、Survivin和TAMs的表达。结果纳米Fe3O4-LTA各组与阴性对照组相比,NO生成量增加、iNOS活性增高,差异有非常显著性（P〈0.01）。移植瘤内VEGF、Survivin和TAMs蛋白表达显著低于阴性对照组（P〈0.01）。结论纳米Fe3O4-LTA对人胃癌裸鼠移植瘤的生长具有明显的抑制作用,其抑瘤机制可能与激活巨噬细胞,使其分泌多种具有杀瘤作用的活性因子以及下调胃癌内VEGF、Survivin和TAMs的表达,进而抑制其血管形成有关。     

In vitro and in vivo induction of nitric oxide by murine macrophages stimulated with Bordetella pertussis

Abstract Nitric oxide (NO) exhibits potent antimicrobial activity in vitro. The function of NO in host defenses in vivo, however, is presently unclear. Experiments were undertaken to determine the production of NO in vitro from murine peritoneal and alveolar macrophages, and murine macrophage cell line (J774A.1) stimulated with Bordetella pertussis or pertussis toxin (PT). In addition, we determined circulating levels of NO in the sera and bronchoalveolar lavage (BAL) fluids of mice infected intranasally with B. pertussis . The results of this study showed that in vitro murine peritoneal macrophages induce production of NO in response to B. pertussis and PT. In addition, murine macrophage cell line, J774A.1 also induces NO production after stimulation with B. pertussis . NO production was also detected in alveolar macrophages from mice infected intranasally with B. pertussis . Finally, a significant increment of circulating levels of NO was noted, in the sera but not in the BAL fluids, of mice infected intranasally with B. pertussis .