血清淀粉样P成分的DNA结合特性

目的 检测血清淀粉样P成分(SAP)与不同DNA的结合活性并研究SAP与DNA的结合是否有利于此类抗原的清除，探讨它在SLE发生发展中的作用及意义。方法 用亲和层析和凝胶过滤方法自小鼠和人血清中纯化SAP，分别提取来自细菌、质粒、酵母、小鼠淋巴细胞等不同DNA，用DOT-EIA方法检测SAP与它们的结合活性，用巨噬细胞吞噬实验观察SAP与DNA结合后对DNA清除的影响。结果 人和小鼠SAP均与细菌DNA结合最强，其次为质粒、酵母DNA，与淋巴细胞DNA和小牛胸腺DNA的结合较弱，与单链λDNA结合最弱；与来源于活化状态小鼠淋巴细胞DNA的结合力高于非活化状态；SAP与活化淋巴细胞DNA结合后可促进巨噬细胞对DNA的吞噬。结论 SAP与不同DNA结合活性各异。     

活性DNA诱导小鼠SLE样综合征的量效关系及其特点

目的：研究活性DNA的剂量与其诱导小鼠SLE样综合征的关系，并对其特点进行全面研究。方法：从ConA活化的小鼠脾淋巴细胞中提取DNA，以不同剂量的活性DNA免疫同系小鼠，用ELISA方法测定IgG类抗dsDNA、抗组蛋白抗体的动态变化以及产生抗体的亚型，用免疫荧光检测抗核抗体核型和肾脏免疫复合物沉积。结果：10μg活性DNA能100％诱导抗dsDNA、抗组蛋白抗体生成，诱导的抗体以IgGI类为主，且小鼠肾脏存在大量免疫复合物沉积。5μg活性DNA诱导小鼠ANA阳性率为25％。结论：10μg活性DNA即可诱导小鼠SLE样综合征，主要诱导自身体液免疫应答。     

波形蛋白的警报素功能鉴定

目的：鉴定胞外可溶性波形蛋白（Vim）促进相关细胞增殖、活化、趋化的能力。方法：体外检测Vim刺激大鼠脾细胞的增殖情况。体内检测Vim免疫小鼠的外周血淋巴细胞数及Vim抗体水平。收集小鼠腹腔巨噬细胞与不同浓度Vim共培养,检测其对巨噬细胞吞噬鸡红细胞能力的影响。以Transwell小室法检测Vim对NIH3T3成纤维细胞的趋化作用。结果：体外实验Vim剂量依赖性地促进大鼠脾细胞增殖,16 μg/ml浓度的Vim刺激预致敏或未致敏脾细胞增殖率分别高达（196.0±9.7）%、（208.9±4.6）%,且该二者无显著性差异。体内实验单用Vim免疫小鼠的外周血淋巴细胞浓度（10.9 L -1 ）激增（5.74±0.51 vs 1.69±0.13）,同时激发了Vim特异性抗体水平（OD值2.31±0.06 vs 0.19±0.08）;且与Vim辅以佐剂免疫小鼠的相应指标均无显著性差异。体外实验Vim浓度依赖性地促进巨噬细胞吞噬鸡红细胞的能力,并能够对NIH3T3成纤维细胞产生趋化作用。结论：胞外可溶性Vim非特异性促进炎症反应相关细胞增殖、活化、趋化,具有警报素功能。     

LPSp辅佐HBsAg诱导机体产生特异性抗体的机制

目的:研究革兰阴性非致病性成团泛菌脂多糖(LPSp)作为佐剂促进乙型肝炎表面抗原蛋白(HBsAg)诱导小鼠产生抗-HBs抗体的作用机制。方法:在体外用LPSp HBsAg、HBsAg、LPSp、生理盐水分别致敏小鼠腹腔巨噬细胞,观察致敏细胞回输体内后诱导HBs抗体产生水平,检测巨噬细胞培养上清中TNF-α活性和NO浓度;观察4组巨噬细胞吞噬功能变化,并检测致敏巨噬细胞诱导局部淋巴结细胞分泌IL-4和IFN-γ的能力。结果:HBsAg LPSp致敏巨噬细胞促进小鼠HBs抗体产生,LPSp致敏的巨噬细胞的吞噬能力显著增强(P<0.05),释放TNF-α和NO水平增加(P<0.05)。LPSp HBsAg致敏巨噬细胞诱导局部淋巴结细胞释放IL-4水平升高(P<0.05),诱导IFN-γ水平与单纯HBsAg致敏组差异无统计学意义(P>0.05)。结论:LPSp的佐剂机制为参与活化巨噬细胞,增强巨噬细胞对抗原的吞噬、消化和处理能力;同时促进HBsAg致敏的巨噬细胞诱导淋巴结内淋巴细胞释放IL-4,增强HBsAg致敏的巨噬细胞诱导Th2淋巴细胞活化,促进B细胞产生抗体。     

狼疮性BXSB小鼠脾脏淋巴细胞增殖与凋亡的初步分析

为了比较全面准确地了解系统性红斑狼疮 (SLE )BXSB小鼠的发病过程中 ,淋巴细胞增殖与凋亡的动力学变化及其机制。采用细胞双色荧光染色的标记技术 ,检测了脾脏淋巴细胞中的增殖细胞和凋亡细胞的百分率 ,并且测定了巨噬细胞吞噬凋亡细胞的能力。结果发现 ,发病的雄性BXSB小鼠和雌性BXSB小鼠脾脏中增殖的CD4 + T淋巴细胞和B淋巴细胞百分率显著高于对照C5 7小鼠 ,而凋亡的B淋巴细胞的百分率显著低于对照C5 7小鼠 ;但是 ,雌雄BXSB小鼠和对照C5 7小鼠巨噬细胞吞噬凋亡细胞的吞噬指数相同。本研究结果表明 ,在BXSB小鼠的SLE发病过程中 ,淋巴细胞的增殖速度异常升高、而凋亡速度下降 ,可能与其脾脏肿大有关 ;而且淋巴细胞的增殖与凋亡的失衡与巨噬细胞的功能无关 ,可能与淋巴细胞内在的异常有关。     

活化淋巴细胞与慢性GVHR诱导的SLE样小鼠模型的比较

目的 :将本室建立的用活化淋巴细胞诱导的系统性红斑狼疮 (SLE)样小鼠模型与国际上公认的用慢性GVHR诱导的SLE样小鼠模型进行比较 ,进一步探索SLE的发病机理。方法 :分别将亲代Balb c小鼠淋巴细胞经静脉和用ConA活化的(Balb c×C5 7BL 6 )F1代小鼠淋巴细胞经皮下途径输入F1代小鼠 ,用ELISA测定IgG类抗dsDNA抗体和抗组蛋白抗体 ,用免疫荧光法检测抗核抗体 (ANA)荧光核型和肾小球内免疫复合物沉积 ,用免疫印迹法检测抗可溶性核抗原 (ENA)抗体。结果 :亲代淋巴细胞免疫F1代小鼠所致的慢性GVHR和活化F1代小鼠淋巴细胞均可诱导F1代小鼠产生高滴度的抗dsDNA抗体、抗组蛋白抗体等ANA ,并且肾脏都有明显的IgG类免疫复合物沉积。但亲代淋巴细胞免疫组ANA核型以颗粒型、核仁型为主 ,ENA多肽谱多在 32、47、6 7kD处显色 ;而活化淋巴细胞免疫组以胞浆型、周边型、均质型为主 ,ENA多肽谱在 2 8、47、6 7kD处显色。结论 :这 2种方法均可诱导出SLE样综合征 ,但其抗核抗体谱有所不同。     

漆黄素对巨噬细胞和T淋巴细胞的免疫调节作用

目的探讨漆黄素(Fisetin,FIS)对小鼠巨噬细胞吞噬功能、NO的释放及对T淋巴细胞体外活化、增殖的影响。方法无菌制备小鼠巨噬细胞悬液及淋巴细胞悬液;荧光微球结合流式细胞术(FCM)分析FIS对巨噬细胞吞噬作用的影响;Griess试剂盒检测巨噬细胞NO的释放;双色荧光抗体染色结合FCM,检测CD3+T细胞CD69的表达水平;CFSE标记技术检测T细胞增殖的情况。结果 2.5μmol/L,5μmol/L,10μmol/LFIS均能明显抑制巨噬细胞的吞噬微球的能力;FIS能抑制LPS和IFN-γ刺激的巨噬细胞的NO的产生(P<0.05);FIS对ConA刺激的T细胞表达CD69有抑制作用,并能有效抑制ConA诱导的T细胞增殖(P<0.01),且均呈剂量依赖性。结论 FIS能显著抑制小鼠腹腔巨噬细胞的吞噬能力和分泌NO的能力,并能够抑制T细胞的活化和增殖,有望发展成为一种新的免疫抑制药物。     

北豆根提取物对小鼠和人淋巴细胞及巨噬细胞作用的体外实验研究

目的：探讨中药北豆根提取物的免疫学调节作用及其作用机制；比较北豆根水提物、醇提物和多糖成分的生物学活性。方法：采用水提取、醇提取及水回流等方法对北豆根进行成分分离。采用MTT法、中性红法检测了北豆根提取物对淋巴细胞增殖的作用以及对小鼠巨噬细胞代谢和吞噬功能的影响。结果：北豆根多糖成分(RMP)具有丝裂原样作用，可刺激小鼠脾细胞及人淋巴细胞增殖，对小鼠巨噬细胞的吞噬功能有一定的促进作用。而北豆根水提物(RMW)和醇提物(RME)对小鼠脾细胞、人淋巴细胞的增殖具有抑制作用，对小鼠巨噬细胞的代谢及吞噬功能也有抑制作用。结论：不同的北豆根成分对免疫细胞有不同的作用，水提物和醇提物对免疫细胞的增殖、代谢和功能具有一定的抑制作用；北豆根多糖成分对淋巴细胞具有丝裂原样作用，能增强小鼠巨噬细胞的吞噬功能，该活性可用于临床辅助治疗肿瘤。     

三七提取物对小鼠淋巴细胞活化、增殖和腹腔巨噬细胞产生NO的影响

目的 分析三七提取物（SE）对小鼠淋巴细胞体外活化、增殖以及对巨噬细胞产生NO的影响，探讨其免疫抑制作用的机制。方法 以多克隆刺激剂刀豆蛋白A（ConA）刺激淋巴细胞活化和增殖，利用荧光标记的单克隆抗体双染技术和流式细胞术检测SE对小鼠CD3^＋T细胞CD69表达的影响；通过CFSE染色流式细胞术检测SE对淋巴细胞增殖的影响；用脂多糖（LPS）或淋巴细胞培养上清液（lymphocytes culture supernate，LCS）体外诱导小鼠腹腔巨噬细胞分泌NO，采用Griess试剂盒检测NO的水平。结果 在ConA刺激6h后小鼠T细胞活化率为59．79％，50、100μg／ml的SE可显著降低其活化率，分别为46．50％和37．73％（P〈0．01）。在培养72h后，不同浓度SE能明显抑制ConA诱导T细胞的增殖。SE在12．5～100μg／ml的浓度范围内对淋巴细胞培养上清和LPS诱导小鼠腹腔巨噬细胞NO的释放具有抑制作用（P〈0．01）。结论 三七提取物对小鼠淋巴细胞的活化、增殖有明显抑制作用，并能抑制巨噬细胞产生NO（P〈0．01）。     

戴氏虫草和粉被虫草多糖对巨噬细胞等活性的影响

目的 研究虫草属新种-戴氏虫草菌丝体胞外水溶性多糖（CDP1）和粉被虫草菌丝体多糖（CPP1）在体外对小鼠腹腔巨噬细胞（Mφ）吞噬功能和脾细胞免疫功能的影响。方法 MTT比色法测定淋巴细胞增殖,孔雀绿比色法测定小鼠腹腔巨噬细胞吞噬功能,中性红比色法测定细胞毒淋巴细胞（CTL）活性,结果 CDP1在正常情况下不能促进小鼠腹腔巨噬细胞的吞噬功能,而CPP1却有促进作用;在免疫抑制的情况下,一定浓度的戴氏虫草菌丝体胞外水溶性多糖和粉被虫草多糖能恢复提高吞噬细胞的吞噬功能,而CPP1却有促进作用;在免疫抑制的情况下,一定浓度的戴氏虫草菌丝体细外水溶性多糖和粉被虫草多糖能恢复提高吞噬功能,它们不仅能促进正常脾活化T淋巴细胞的增殖,而且能恢复环磷酰胺和氢化可的松抑制免疫小鼠脾活化T淋巴细胞的增殖,在合适的浓度下能增高小鼠脾CTL活性,同时在一定浓度下能恢复免疫抑制小鼠脾CTL活性。结论 两者具有增强免疫功能的作用。     

同种异体淋巴细胞诱导的SLE样小鼠“模型”

将亲代BALB/c小鼠淋巴细胞经腹腔或静脉途径输注(BALB/c×C_(57)BL/6)F_1鼠体内,均可诱导出抗dsDNA抗体,抗组蛋白抗体水平的持续升高,以静脉途径更为明显。经16周动态观察表阴:抗dsDNA抗体于第8周达高峰,抗组蛋白抗体于第12周达高峰。抗组蛋白抗体的阳性反应性比抗ds DNA抗体反应高,且出现较早。组织学检查,实验组鼠肾出现慢性炎性浸润,间质细胞增生,基底膜增厚。免疫组化显示,颗粒性免疫复合物沉着于肾小球基底膜及毛细血管内皮区域。     

Interaction of Alveolar Macrophages with Nocardia asteroides: Immunological Enhancement of Phagocytosis, Phagosome-Lysosome Fusion, and Microbicidal Activity

Normal and specifically activated rabbit alveolar macrophages were infected in vitro with Nocardia asteroides GUH-2. In the presence of serum from normal rabbits, no significant differences were noted between normal and activated alveolar macrophages with respect to phagocytosis, incidence of phagosomelysosome fusion, or nocardicidal activity. However, all of these macrophage functions were enhanced by various immunological components. Serum from immunized rabbits enhanced phagocytosis of nocardial cells by activated macrophages, and there was an additional increase in phagocytosis observed when alveolar lining material was present. Complement had no effect on the ability of the macrophages to phagocytize nocardial cells. The greatest percentage of organisms phagocytized was observed when specifically primed lymph node cells, alveolar lining material, and serum from immunized rabbits were present in the incubation medium. N. asteroides GUH-2 inhibited phagosome-lysosome fusion in normal macrophages in the presence of serum from normal rabbits. However, addition of serum from immunized rabbits or the addition of specifically primed lymphocytes increased the amount of phagosome-lysosome fusion, whereas complement had no effect on this fusion process. Nocardial viability was not reduced when either normal or activated macrophages were infected with bacteria in the presence of normal serum, immune serum, or alveolar lining material. However, specifically activated macrophages incubated with primed lymph node cells obtained from immunized rabbits were able to both decrease the number of viable organisms recovered and to increase the incidence and extent of bacterial cell damage. The greatest number of organisms were killed by specifically activated macrophages when the bacterial cells were incubated with primed lymph node cells suspended in immune serum and alveolar lining material. These results indicate that activated macrophages alone are not sufficient to kill ingested N. asteroides GUH-2 and that specifically primed lymphocytes are important in host resistance to nocardial infections.     

Activation of macrophages in an experimental rat model of arthritis induced by Erysipelothrix rhusiopathiae infection.

Infection of Lewis rats with Erysipelothrix rhusiopathiae represents an experimental model system of acute and chronic arthritis. We studied here the acute inflammatory phase with respect to stimulation of macrophages and lymphocytes. Intragluteal injection of viable E. rhusiopathiae (10(2) to 10(4) bacteria) rapidly induced generalized inflammation, loss of body weight, hind leg arthritis, and systemic macrophage activation within 2 to 3 days. The same symptoms could also be evoked by injection of dead E. rhusiopathiae. Ex vivo, peritoneal macrophages released large amounts of tumor necrosis factor alpha on day 2 and interleukin-1 on day 3, whereas production of prostaglandin E2 was delayed to days 5 to 7 and appeared to counteract tumor necrosis factor alpha synthesis. The inflammatory response and development of arthritis were strongly dependent on T lymphocytes, as evidenced by the following findings: (i) lymphocytes released lymphokines that activated macrophages to enhanced mediator release; (ii) treatment of rats with cyclosporin A reduced infection-induced macrophage activation; (iii) mitogen-stimulated thymocyte proliferation was enhanced, indicating an infection-induced maturation-differentiation process in the thymus; and (iv) in T-cell-deficient nude rats, a higher dose of bacteria was required for infection, the inflammatory response was less severe, and only mild, but not chronic, arthritis developed. Thus, an E. rhusiopathiae-induced inflammation in rats provides a useful tool to characterize activated macrophages and T lymphocytes during the development of acute arthritis and its transition into the chronic form.     

T细胞接种对SLE样小鼠防治作用的探讨

本文在空肠弯曲菌（CJ-S131）免疫诱导的小鬼SLE样综合征模型上,观察了T细胞接种（T CellVaccination,TCV）的预防和治疗作用。用于 TCV的细胞为 CJ-S131预致敏的同系小鼠 T细胞。结果显示TCV可明显地抑制CJ-S131诱导的特异性迟发型超敏反应,相反却显著地增强抗dsDNA抗体和抗外膜蛋白抗体生成。因此TCV对SLE样小鼠没有预防及治疗作用。本文对此现象进行了讨论。     

Antineutrophil cytoplasmic antibodies induce reactive oxygen-dependent dysregulation of primed neutrophil apoptosis and clearance by macrophages

This study assessed whether anti-neutrophil cytoplasmic antibodies (ANCAs) interfere with the safe deletion of neutrophils by apoptosis and phagocytic clearance. Tumor necrosis factor (TNF)-primed neutrophils were incubated with normal IgG (N IgG) or ANCA IgG for up to 36 hours. Compared with N IgG, ANCAs accelerated constitutive apoptosis of TNF-alpha primed neutrophils, as assessed by morphology and confirmed by DNA laddering pattern on gel electrophoresis, and accelerated progression to secondary necrosis. The accelerated apoptosis induced by ANCA was dependent on reactive oxygen species generation, as primed neutrophils from patients with chronic granulomatous disease failed to show an effect of ANCAs on apoptosis. However, there was no change in the rate at which neutrophils exhibited annexin V binding, indicating that externalization of phosphatidylserine was not accelerated by ANCAs. Furthermore, when ANCA-treated primed neutrophils were interacted with human or murine peritoneal macrophages after 12 hours there was significantly less phagocytosis by human macrophages and no difference in phagocytosis by murine peritoneal-derived macrophages when compared with N IgG-treated controls. In conclusion, ANCAs accelerate apoptosis and secondary necrosis in TNF-primed neutrophils by a mechanism dependent on the generation of reactive oxygen species, with uncoupling of nuclear and surface membrane changes, resulting in a "reduced window of opportunity" for phagocytic recognition and engulfment before disintegration.     

Kinetics of macrophages and reticuloendothelial cells in mouse lymph nodes during secondary antibody responses to tetanus toxoid

The proliferative activity and rate of cell renewal of, and incorporation of tritiated nucleosides into, macrophages, reticuloendothelial cells and fibroblasts were studied in lymph nodes of mice before and after a regional second antigenic stimulus with tetanus toxoid. Histological and autoradiographic examinations revealed that in non-stimulated nodes of animals primed 6 months previously, none of the above cell-types was found to synthesize DNA. Throughout a period of 10 days following secondary antigenic stimulation via the hind leg foot pads, only four of approximately five thousand of these cells counted in the popliteal lymph nodes of thirty-four mice were labelled initially by [³H]thymidine. Incorporation of [³H]cytidine could, therefore, be interpreted as reflecting almost exclusively RNA synthesis. Mean grain counts of non-proliferating cells 1 hour after injection of this radioactive precursor started to rise first in small lymphocytes, reached a peak around 6 hours following the booster injection of antigen and returned to slightly elevated values at 12 hours. At this time, the mean labelling intensity of macrophages, reticuloendothelial cells and fibroblasts had barely begun to increase. Peak values for the latter cell-types were attained only on the third day after secondary antigenic stimulation. These findings are interpreted as indicating consecutive waves of enhanced RNA synthesis first in lymphocytes and then in macrophages or the other elements mentioned above. This lends further support to the hypothesis that, following a second injection of tetanus toxoid, elevated rates of RNA synthesis in macrophages are not a prerequisite for triggering lymphocytes to enter proliferation and differentiation. Macrophages having ingested various materials showed significantly higher mean grain counts than cells not containing cytoplasmic inclusions. One early response of the macrophage system to the booster injection of antigen consisted of a more rapid and increased turnover, i.e. replacement of unlabelled by labelled cells with increasing time after injection of [³H]thymidine. The results are discussed in relation to macrophage functions in immune responses and possible cellular transformations in the macrophage series.     

雌二醇对小鼠正常淋巴细胞和致敏淋巴细胞活化的调节

为探讨雌二醇(E2)对正常和致敏淋巴细胞活化的调节作用,并分析了E2对ConA活化的小鼠正常和致敏淋巴细胞增值、抗体产生及TGF-β生成的影响。结果显示生理水平尤其是相当于小鼠动情间期水平的E2对致敏脾淋巴细胞增殖具有明显促进作用;对初始脾淋巴细胞增殖没有影响。较高水平E2诱导活化的脾细胞生成TGF-β,促进免疫鼠脾细胞生成特异性抗体;较低水平E2抑制特异性抗体生成,且抑制程度与脾细胞活化状态相关。     

Acquired immune response as a consequence of the macrophage-dependent apoptotic cell clearance and role of the monocyte chemotactic S19 ribosomal protein dimer in this connection

A connection between the apoptotic cell clearance system and the acquired immune system was studied in vivo. When fluorescence-labeled apoptotic HL-60 cells were inoculated into footpads of guinea pigs and rabbits, monocyte/ macrophage infiltration rapidly occurred and subsequently the apoptotic cells as well as the macrophages disappeared from the lesion by 48 hours without any macroscopical signs of inflammation. Inversely, the fluorescent cell debris, which had been engulfed by the macrophages, appeared and chronologically increased in the draining lymphatics and the popliteal lymph nodes by 48 hours. Subsequently, proliferation of T and B lymphocytes in the popliteal lymph nodes was observed. Secondary inoculation of HL-60 cells in the flank skin of guinea pigs on day 3 after the initial inoculation induced an acute immunologic dermatitis with erythema, edema, vascular permeability enhancement, and polymorphonuclear leukocyte infiltration. In vitro characterizations demonstrated the presence of compliment dependent cytotoxic IgM antibody against HL-60 cells in their sera. The infiltration of monocytes/macrophages at the apoptotic cell injection site and the subsequent production of the anti-HL-60 cell IgM antibodies were significantly suppressed by in situ injections of anti-S19 ribosomal protein rabbit antibodies. These results indicated that the serial events with the rapid apoptotic cell clearance by macrophages, the macrophage migration to lymph nodes, and the antigen presentation to T lymphocytes by the macrophages acquire immunity against apoptotic cells. It was also indicated that the S19 ribosomal protein dimer was the major chemotactic factor in the initial monocyte/macrophage infiltration to apoptotic cells.     

Cellular reactions in a SRBC-induced inflammatory exudate

Acute inflammatory exudates which are induced by the intra-peritoneal injection of sheep red blood cells contain a variety of connective tissue cell types. Granulocytes, macrophages and lymphocytes constitute the vast majority of the exudate cells. The peritoneal macrophages are principally involved in the phagocytosis and clearance of the whole SRBC from the peritoneal cavity. The exudate lymphocytes which accumulate during the first 24 hours are derived from a pool with a fast turnover rate and are newly formed. While these lymphocytes undergo an enlargement and increase their basophilic cytoplasm during this response, they fail to show increases in their DNA synthesis while in the inflammatory exudate.