甲壳胺对自由基损伤小鼠腹腔巨噬细胞的作用

<正>人类生存的环境中充斥着不计其数的自由基,人体自由基损伤已经成为一种常见损伤。自由基对人体的损害主要有三个方面:一是可以使细胞膜破坏;二是使血清抗蛋白酶失去活性;三是损伤基因导致细胞变异的出现和蓄积。目前,开     

严重烫伤后小鼠腹腔巨噬细胞PKC的变化及调控

目的：观察小鼠烫伤后不同时相腹腔巨噬细胞的细胞膜及细胞质蛋白激酶C(PKC)的变化及应用特异性调控剂H 7后腹腔巨噬细胞内核因子 κB(NF κB)活性,从信号转导的角度阐明巨噬细胞功能紊乱的机制.方法：以15%体表面积Ⅲ度烫伤小鼠为模型,收集腹腔巨噬细胞,同位素掺入法测膜浆PKC活性,电泳迁移率改变分析法检测NF κB的活性.结果：烫伤后巨噬细胞细胞膜及细胞质PKC活性升高,其中胞膜PKC活性与TNF α mRNA的变化呈正相关.给予H 7后NF κB活性下降.结论：烫伤后小鼠腹腔巨噬细胞PKC、NF κB的活性明显被激活,PKC NF κB信号通路参与了TNF α表达的调控.     

蛋白激酶C激活剂和抑制剂对小鼠腹腔巨噬细胞释出肿瘤坏死因子的影响

本文研究蛋白激酶C（PKC）激活剂TPA和抑制剂H－7，槲皮素对痤疮丙酸杆菌（Propionibacterium acnes,PA)启动的小鼠腹腔巨噬细胞（macrophage,MΦ）释出肿瘤坏死因子（TNF）的影响，表明TPA可诱导PA启动的M分泌TNF，其作用可被H－7和槲皮素抑制，LPS体外和体内诱生TNF的作用亦可被两者抑制，提示PKC和PA启动的M分泌TNF过程中具有关键性作用。     

灵芝多糖对小鼠腹腔巨噬细胞蛋白激酶C活性的影响

目的　观察灵芝多糖在体外对小鼠腹腔巨噬细胞(MΦ)蛋白激酶C(PKC)活性是否有影响。方法　采用反相离子对高效液相色谱法测定PKC活力。结果　灵芝多糖GLB7( 40mg·L-1)能引起小鼠腹腔MΦ中PKC活性明显升高 ,30min达峰值 ,2h恢复到基础水平 ;GLB7还可引起MΦ中PKC发生质膜转位 ,并拮抗Staurosporine对MΦ中PKC的抑制作用。结论　灵芝多糖的免疫增强作用与其增强PKC活性有关     

CD40-CD40配体相互作用对培养的人单核细胞二酰基甘油-蛋白激酶C信号通路及胞内Ca^2＋的影响

目的：探讨CD40-CD40配体(CD40L)相互作用是否能激活人外周血单核细胞(PBMC)内二酰基甘油(DAG)-蛋白激酶C(PKC)信号通路．方法：细胞内DAG含量采用放射酶标记、薄层层析和放射自显影方法检测．细胞PKC活性及胞内游离钙分别采用[γ-^32P]ATP磷酸转移法和Fluo-3荧光负载流式细胞术检测．结果：CD40L以剂量依赖方式刺激人外周血单核细胞合成DAG,并具有双时限性变化,第一峰值在20s,第二峰值在10min时出现．然后DAG水平缓慢下降,至少持续20-30min．单核细胞蛋白激酶,C总活性受CD40L刺激后明显增加,峰值在12min,持续20min以上．并且这种作用主要是胞浆PKC活性向胞膜PKC活性转位所致．CD40L,能刺激胞内游离Ca^2 出现短暂的快速升高,继之为持续阶段．移去细胞外Ca^2 ,胞内快速阶段无影响,而持续阶段明显受到抑制．抗CD40抗体能显著抑制CD40L引起的胞内DAG-PKC信号通路激活及[Ca^2 ]i的动态变化．结论：CD40-CD40L相互作用能激活人外周血单核细胞[Ca^2 ]i动态变化及二酰基甘油-蛋白激酶C信号通路．     

水溶性和非水溶性甲壳胺对小鼠的急性毒性比较

目的观察水溶性和非水溶性甲壳胺对小鼠灌胃及腹腔注射给药的急性毒性反应,对其安全性进行初步评价比较。方法两种甲壳胺以最大浓度及最大灌胃体积2次/d灌胃给药或1次/d腹腔注射给药后,连续观察14 d,记录各组小鼠的体重、活动及急性毒性反应情况。结果小鼠按1 d灌胃给药的最大给药量4800 mg/kg给药、相当于成人(60 kg)每天用药量120倍,一次性腹腔注射量为2400 mg/kg,两种甲壳胺给药的各组小鼠均未出现毒性反应及死亡,与对照组相比亦无明显体重差异。结论水溶性和非水溶性甲壳胺按成人日剂量用药均无急性毒性反应。     

甲壳胺体外抗菌活性的研究

采用两倍稀释法对临床分离的 4种细菌进行了体外抗菌试验。结果显示 ,甲壳胺有较强的抗菌活性     

甲壳胺胶囊对小鼠免疫功能的影响

目的探讨甲壳胺胶囊对小鼠免疫功能的影响。方法免疫实验采用0.045、0.45、1.35g/kg剂量的甲壳胺胶囊,小鼠经口灌胃30d,分别测定免疫器官脏器/体质量比值、半数溶血值、抗体生成细胞数、ConA诱导的小鼠脾淋巴细胞转化实验、迟发型变态反应和碳廓清实验、小鼠腹腔巨噬细胞吞噬鸡红细胞实验、NK细胞活性。结果与阴性对照组比较,1.35g/kg剂量能提高小鼠的半数溶血值(HC50)、小鼠抗体生成细胞数,1.35、0.45g/kg剂量能增强小鼠的迟发型变态反应能力。3个剂量对小鼠体质量增长、胸腺体质量比值、脾脏体质量比值、小鼠单核-巨噬细胞碳廓清能力、Co-nA诱导的小鼠脾淋巴细胞转化能力、小鼠腹腔巨噬细胞吞噬鸡红细胞的能力、NK细胞活性均无影响。结论甲壳胺胶囊有增强免疫力的作用。     

吗啡长时程作用下小鼠脑组织磷酸肌醇含量和PKC活性的变化

目的旨在进一步阐明阿片依赖中PLC-PKC信号系统的变化与作用。用小鼠吗啡依赖模型,观察脑组织磷酸肌醇含量、PKC活性变化。结果发现:纹状体IP及IP3、大脑皮质IP含量明显升高,二者磷酸肌醇总量显著增加;胞质可溶相PKC活性在大脑皮质及小脑明显升高,在纹状体显著下降。膜相PKC活性在纹状体明显升高,在小脑和海马明显降低;PKC抑制剂可抑制吗啡依赖的产生。结果提示,吗啡依赖小鼠纹状体磷酸肌醇含量的增加意味着PLC的活化,这可能与纹状体中PKC的激活有关,而该PKC的激活可能参与吗啡依赖的形成。     

灵芝多糖对小鼠腹腔巨噬细胞活性氧自由基的影响

为进一步探讨灵芝多糖 ( GLP)免疫调节作用机理 ,采用激光扫描共聚焦显微镜技术 ,动态监测灵芝多糖均一体 GLB7对小鼠腹腔巨噬细胞 ( M )活性氧自由基含量的影响 .以 GLB7( 2 0 mg· L-1)刺激 M ,发现探针 DCHF- DA的荧光强度明显下降 ,与静息水平相比 ,平均下降 ( 36± 6) % ( n=6,P<0 .0 5) ;同时还能拮抗 PMA引起的 M 呼吸爆发 ,荧光强度下降幅度为 ( 1 9± 4) % ( n=6,P<0 .0 5) .结果证明 :GLB7能减少 M 内活性氧自由基的生成 ,具有清除活性氧的作用 ,这也是 GLB7产生免疫增强和抗衰老作用的重要途径 .     

Quercetin uptake and metabolism by murine peritoneal macrophages in vitro

Quercetin (Q), a bioflavonoid ubiquitously distributed in vegetables, fruits, leaves, and grains, can be absorbed, transported, and excreted after oral intake. However, little is known about Q uptake and metabolism by macrophages. To clarify the puzzle, Q at its noncytotoxic concentration (44μM) was incubated without or with mouse peritoneal macrophages for different time periods. Medium alone, extracellular, and intracellular fluids of macrophages were collected to detect changes in Q and its possible metabolites using high-performance liquid chromatography. The results showed that Q was unstable and easily oxidized in either the absence or the presence of macrophages. The remaining Q and its metabolites, including isorhamnetin and an unknown Q metabolite [possibly Q⁻ (O-semiquinone)], might be absorbed by macrophages. The percentage of maximal Q uptake by macrophages was found to be 2.28% immediately after incubation; however, Q uptake might persist for about 24 hours. Q uptake by macrophages was greater than the uptake of its methylated derivative isorhamnetin. As Q or its metabolites entered macrophages, those compounds were metabolized primarily into isorhamnetin, kaempferol, or unknown endogenous Q metabolites. The present study, which aimed to clarify cellular uptake and metabolism of Q by macrophages, may have great potential for future practical applications for human health and immunopharmacology.     

Different roles of PKC and PKA in effect of interferon-y on proliferation and collagen synthesis of fibroblasts

AIM: To study the signal roles of protein kinase C (PKC) and protein kinase A (PKA) in the influence of interferon-γ(IFN-γ) on proliferation and collagen synthesis of flbroblasts derived from hypertrophic scar (HS-FB) and normal skin (NS-FB). METHODS: HS-FB and NS-FB were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM). Activity of PKC and PKA were assayed by transferring phosphorus (32P) into substrate after treatment with IFN-γ1000 kU/L at 10, 30, 60, and 120 min. Cell proliferation was determined with MTT assay. The collagen synthesis was measured with [3H]proline incorporation and Type Ⅲ pre-collagen was determined with radioimmunoassay. RESULTS: After exposure to IFN-γ1000 kU/L for 30 min, PKC activity of HS-FB and NS-FB increased from 2.57±0.14 and 2.13±0.12 nmol·min-1·g-1 of control to 3.75±0.19 and 3.36±0.16 nmol·min-1·g-1 respectively (P<0.05). After exposure to IFN-y 1000 kU/L for 60 and 120 min, PKA activities of HS-FB increased gradually from 0.82±0.04 nmol·m     

壳聚糖DCX-16对术后腹膜粘连的防治作用

目的:探讨壳聚糖DCX-16对术后腹膜粘连的防治作用。方法:采用盲肠浆膜擦伤的方法建立大鼠腹膜粘连的动物模型。分别在创伤局部给予5 mL·kg-1的生理盐水、透明质酸钠(50 mg·kg-1)和壳聚糖DCX- 16(15,50,150,500 mg·kg-1),观察术后d 14腹膜粘连和切口愈合情况。选用最有效浓度的壳聚糖DCX-16, 比较其与生理盐水、透明质酸钠对大鼠术后d 3,7,14,21腹膜粘连形成的影响。结果:术后d 14对照组(用生理盐水)粘连发生率100％。粘连程度壳聚糖DCX-16各剂量组与对照组差异非常显著(P<0．01),与透明质酸钠组无显著差异(P>0．05)。取150 mg·kg-1壳聚糖DCX-16为最佳有效剂量,其可明显减轻大鼠术后 d 3创面出血和术后d 7,14,21时的腹膜粘连。结论:壳聚糖DCX-16可明显减少术后腹膜粘连的发生及严重程度。     

仿刺参糖胺聚糖对小鼠腹腔巨噬细胞功能的影响

目的 研究仿刺参糖胺聚糖（HGAG）对小鼠腹腔巨噬细胞（Mφ）功能的影响,探讨其对小鼠的免疫调节作用。方法 不同浓度HGAG作用于体外培养Mφ,MTT比色法测定代谢活力;中性红法测定吞噬功能;试剂盒测定Mφ中乳酸脱氢酶（LDH）活性;Griess法测定NO生成量;ELISA法测定培养上清中TNF-α、IL-1β的分泌水平。结果 HGAG在0.1~25 μg·mL?1范围内对Mφ的功能有一定的促进作用。在1~10 μg·mL?1范围内与空白对照组相比可显著增强Mφ代谢活力和吞噬能力（P<0.01）,提高LDH活力和NO生成量（P<0.05）,显著增加培养上清中TNF-α和IL-1β（P<0.01）的浓度。与阳性对照组相比,在1~10 μg·mL?1范围内对Mφ吞噬功能以及TNF-α、IL-1β的生成有更好的促进作用（P<0.05）,在高浓度（100μ g·mL?1）Mφ的各项功能指标均低于空白对照组。结论 HGAG在1~10 μg·mL?1浓度范围内可显著促进Mφ活性,并在一定程度上优于阳性对照组。表明HGAG能够激活Mφ,促进Mφ的功能,增强机体免疫。     

水飞蓟宾对小鼠腹腔巨噬细胞释放纤维化因子的影响

目的:研究水飞蓟宾对小鼠腹腔巨噬细胞释放纤维化因子的影响。方法:小鼠腹腔巨噬细胞先后用卡西霉素和脂多糖刺激培养24h诱导纤维化因子。巨噬细胞培养上清中促胶原合成活性和转化生长因子β活性分别采用³H-脯氨酸掺入法和雕肺上皮Mv-1-Lu细胞测定。结果:水飞蓟宾(6.25-50μg／mL)以浓度依赖方式抑制巨噬细胞产生胶原刺激活性和转化生长因子β₁。结论:水飞蓟宾减少巨噬细胞释放促纤维化因子可能是其保肝抗硬化机制之一。     

水飞蓟宾对小鼠腹腔巨噬细胞释放纤维化因子的影响

目的：研究水飞蓟宾对小鼠腹腔巨噬细胞释放纤维化因子的影响。方法：小鼠腹腔巨噬细胞先后用卡西霉素和脂多糖刺激培养24h诱导纤维化因子。巨噬细胞培养上清中促胶原合成活性和转化生长因子p活性分别采用^3H-脯氨酸掺入法和雕肺上皮My-1-Lu细胞测定。结果：水飞蓟宾（6．25—50μg／mL）以浓度依赖方式抑制巨噬细胞产生胶原刺激活性和转化生长因子β1。结论：水飞蓟宾减少巨噬细胞释放促纤维化因子可能是其保肝抗硬化机制之一。     

3，4—二苯基—3—吡咯啉—2—酮衍生物对小鼠腹腔巨噬细胞环氧酶1和2的抑制作用

目的:建立腹腔巨噬细胞模型评价化合物对环氧酶1和2的抑制作用.方法:放免法测定calcimycin、脂多糖刺激细胞将内源性花生四烯酸转化为6-keto-PGF_(1α)或PGE_2的量.GAPDH和环氧酶1/2的RNA用RT-PCR测定.结果:Rofecoxib选择性抑制脂多糖诱导的PGE_2合成,IC_(50)为(4.7±0.5)nmol/L,而抑制calcimycin诱导的6-keto-PGF_(1α)合成最大抑制率为17.3％.吲哚美辛对环氧酶1和2的IC_(50)分别为(4.7±1.1)nmol/L和(7.1±1.2)nmol/L.17个化合物中,系列Ⅱ化合物的活性与rofecoxib相当.磺酰胺基和内酰胺环的相对位置对抑制环氧酶2的活性很重要.结论:模型适用于药物设计的体外评价.3,4-二苯基-3-吡咯啉-2-酮衍生物有望成为新的环氧酶2选择性抑制剂.     

Influence of cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in mice

2,4,6-Trinitrophenyl (TNP) hapten-labeled peritoneal macrophages (Mf) given intravenously (iv) to recipients are poor inducers of contact sensitivity (CS) reactions unless Mf donors are pretreated with low doses of cyclophosphamide (CY). In vivo CY is converted into active alkylating metabolites, phosphoramide mustard (PM) and acrolein (ACR).Our experiments aimed to test how in vitro treatment of non-immunogenic Mf with different concentrations (10^?5 to 10^?7 M) of CY metabolites will influence their immunogenicity and other biological functions. Instead of chemically unstable PM, we used structurally and functionally similar nitrogen mustard (NM).Our experiments show that treatment of Mf with ACR or NM stimulates the in vitro production of pro-inflammatory IL-6 and IL-12 and down-regulates anti-inflammatory IL-10 and TGF-β cytokines. In vivo non-immunogenic TNP-Mf become capable of inducing CS reactions in two situations: first, after treatment with NM or ACR and second, when cell recipients are received iv before Mf transfer of monoclonal antibodies against IL-10 and/or TGF-β (500 μg per animal). Treatment with NM, but not with ACR, was also an efficient stimulus for production by Mf of significantly increased levels of reactive oxygen intermediates (ROIs).In summary, our experiments show that CY metabolites can significantly increase the specific immune response as well as nonspecific innate reaction (ROIs production) and support the notion that CYand its metabolites can be a promising accessory tool when upregulation of the immune response is desired.