THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 10⁶ cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-¹²⁵I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-¹²⁵I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.     

THE INTERACTION OF CATIONIC LIPOSOMES CONTAINING ENTRAPPED HORSERADISH PEROXIDASE WITH CELLS IN CULTURE

Cationic liposomes composed of sphingomyelin, cholesterol, and stearylamine were prepared with horseradish peroxidase trapped inside. Stable particles were formed in which 10–12% of the enzymic activity appeared to be located at, or near, the outer surface of the liposome. Adsorption and uptake of liposomes by HeLa cells were followed cytochemically by electron microscopy and quantitated by enzyme assay and by the distribution and fate of particles labeled with [¹⁴C]cholesterol and [¹²⁵I]horseradish peroxidase. The particles were adsorbed by HeLa cells at least 300 times as efficiently as was free horseradish peroxidase. Many of the particles remained at the cell surface, but numerous membrane-bound cytoplasmic inclusions were observed to contain peroxidase-staining material. In addition, many areas of the cell membrane gave a positive staining reaction. It was concluded that many particles (presumably the larger ones) did not gain access to the interior of the cells, many were phagocytized, and some enzyme was transferred to the cell membrane, perhaps as a result of fusion of the liposomal membrane with the cell membrane.     

APPEARANCE AND DISTRIBUTION OF FERRITIN IN MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF HETEROLOGOUS ERYTHROCYTES

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.     

小鼠骨胳肌在切腱、协同肌切腱和肉毒杆菌毒素中毒后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用生物化学方法测定离体小鼠比目鱼肌对 HRP 的胞纳作用。结果表明,在切断神经或切腱后引起萎缩的肌肉侧或在协同肌切腱后引起代偿性肥大的肌肉侧,与它们各自对照的正常肌肉侧相比,都可发生对 HRP 胞纳的明显增加。而在肉毒杆菌毒素中毒后引起萎缩的肌肉侧,和它正常的对照肌肉侧相比,却意外地不发生这种胞纳摄取的明显增加。本文的实验结果进一步证实。肌肉的胞纳增加并不一定导致肌纤维的变性和萎缩(例如代偿性肥大的肌肉);而肌纤维的变性和萎缩亦不一定需要肌肉的胞纳增加为前提(例如肉毒杆菌毒素中毒的肌肉)。本工作结果还提示:肌肉的胞纳增加有其神经原性和肌原性因素。本文还对肌肉胞纳增加的可能机制进行了讨论。     

鸡前、后背阔肌去神经后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用辣根过氧化物酶(HRP)作为大分子标记物,以组织化学和生物化学方法观察了鸡前后背阔肌在去神经后的胞纳现象。结果表明,在去神经后发生肥大的前背阔肌和去神经后发生萎缩的后背阔肌同样都出现胞纳的明显增加。组织化学所观察的结果表明,浸泡在含 HRP 的任氏液中的有完整神经支配的前、后背阔肌只有极少数的肌纤维摄取 HRP,而在去神经的前、后背阔肌中则有不少的肌纤维内部出现 HRP 染色反应。这种反应在有的纤维表现为弥散性染色,有的表现为浓的 HRP 反应颗粒。生物化学的结果显示,去神经后的前、后背阔肌中 HRP 的相对含量分别比有神经支配的对照肌肉明显地增多54%和87%,我们在鸡前背阔肌用组织化学和生物化学所得的实验结果与 Thesleff 等人提出的关于肌肉萎缩机制的假设显然不符。本工作证实了肌肉的胞纳作用的增加并不一定最终导致肌纤维的变性和萎缩。     

子宫内膜异位症小鼠腹腔巨噬细胞吞噬功能的变化规律

目的研究子宫内膜异位症发生进程中腹腔巨噬细胞吞噬功能的变化规律。方法构造小鼠子宫内膜异位症模型,用流式细胞术检测造模前后腹腔巨噬细胞吞噬荧光微球数,计算吞噬率和吞噬指数代表巨噬细胞的吞噬能力。结果巨噬细胞吞噬率和吞噬指数分别为：未造模组[（10．1±0．82）％,0．17±0．01]、造模后第1d[（32．78±2．43）％,0．60±0．02]、第2d[（33．82±1．23）％,0．61±0．02]、第3d[（35．93±2．81）％,0．72±0．03]、第4d[（27．92±1．24）％,0．51±0．03]、第5d[（24．34±0．91）％,0．40±0．02]、第6d[（17．91±1．03）％,0．28±0．01]、第9d[（17．56±0．80）％,0．26±0．01I、第12d[（19．42±1．02）％,0．36±0．01]、第15d[（26．78±2．05）％,0．54±0．02]、第18d[（27．46±1．61）％,0．50±0．02]、第21d[（25．99±2．31）％,0．54±0．03]。造模后的小鼠腹腔巨噬细胞吞噬能力均显著高于未造模组,且呈现出三个阶段的改变：第1—5d为第1阶段,第6-12d为第2阶段,第15—21d为第3阶段,第1和第3阶段显著高于第2阶段（P〈0．01）。结论 子宫内膜异位症小鼠腹腔巨噬细胞的吞噬功能增强,且随时间呈现规律性变化特征,可能与子宫内膜的清除和异位存活有关。     

LOSS OF IRON FROM MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF [55FE]FERRITIN AND [55FE]FERRITIN RABBIT ANTIFERRITIN COMPLEXES

Mouse peritoneal macrophages in culture for 24 h were exposed to horse [⁵⁵Fe]ferritin and rabbit antihorse [⁵⁵Fe]ferritin antibody complex and the amount of ⁵⁵Fe in the medium was assayed up to 2 days after the pulse uptake. Cell survival was assayed by photographing the same areas of the tissue culture Petri dish on successive days and by counting cell numbers per unit area. In experiments in which quantitative assay for cell death is negligible, about 10–20% of the iron ingested by pinocytosis or phagocytosis is released to iron-free medium containing either freshly dialyzed or deironized newborn calf serum (10%). Over the 2-day postpulse period, iron loss is linear. This loss of iron to the medium is significantly reduced by adding iron-saturated newborn calf serum in the postpulse recovery period. A significant portion of the iron released to the medium is bound to transferrin. When human serum is used in the tissue culture system, similar quantities (10–25%) of the ingested iron are lost to the medium 2 days after the pulse.     

戊巴比妥对大鼠骨胳肌辣根过氧化物酶(HRP)的轴突摄取和逆行传送作用

腓肠肌内注射HRP后,用生物化学法测定坐骨神经、L_(4-6)节段背根和腹根神经的HRP含量。在戊巴比妥连续全身麻醉大鼠的HRP含量明显低于不麻醉的大鼠,而肌肉不活动(TTX中毒和切腱)大鼠神经组织中的HRP含量无甚变化。刺激神经不能改变麻醉大鼠的HRP含量。上述结果提示:除麻醉剂造成的肌肉不活动因素外,戊巴比妥对大鼠骨胳肌HRP的轴突摄取和逆行传送具有另外的抑制作用。已有研究报道:破伤风和单纯性疱疹病毒脑炎都是由于它们的毒素或病毒,通过外周神经摄取然后逆行传送到各级中枢而致病的。     

IN VITRO PRODUCTION OF COLONY-STIMULATING ACTIVITY. I. EXPOSURE OF MOUSE PERITONEAL CELLS TO ENDOTOXIN

Cell suspensions, obtained from bone marrow, spleen, thymus, lung, liver, and from peritoneal washings, were incubated in vitro with low concentrations of endo-toxin and the supernatant media assayed for colony-stimulating activity (CSA). Peritoneal cells were markedly responsive. The kinetics of CSA production in vitro by peritoneal cells were not remarkably different from that seen in vivo following intravenous administration of endotoxin. The activities of CSA prepared from peritoneal cells and serum were compared following serial dilution; both gave a similar linear relationship when plotted as a function of log-concentration. The bulk of the CSA was produced by adherent peritoneal cells. Separation of peritoneal cells by velocity sedimentation showed that the CSA-producing cell had a sedimentation velocity of 7 mm/hr. Cells with this sedimentation velocity were found to be large mononuclear cells which demonstrated adherence and phagocytosis.     

硬头鳟巨噬细胞的体外长期培养(简报)

鱼类的巨噬细胞和高等脊椎动物的一样,在吞灭入侵的病原体方面起着极其重要的作用。探索在体外长期培养巨噬细胞的方法,有助于研究巨噬细胞的机能。Braun-Nesje等虽已从硬头鳟(Salmo gairdneri)等鲑科鱼类的头肾中分离出大量的巨噬细胞,并在体外培养了3个月之久,但因细胞不分裂,无法传代。本研究改用组织块培养法和饲养层技术探索长期培养巨噬细胞的方法,并获得成功。迄今巨噬细胞已在体外培养22个月,传代48次。细胞的原代培养分为三组。前两组是单独的脾或头肾的组织培养;第三组是脾与头肾的混合组织培养。培养液是Leibovitz’s L-15,外加20%胎牛血清,100 IU/ml青霉素和100μg/ml链霉素。巨噬细胞的吞噬活力用酵母菌Candida     

长爪沙鼠腹腔巨噬细胞对流行性出血热病毒的敏感性

本文就长爪沙鼠腹腔巨噬细胞(MΦ)对流行性出血热病毒(EHFV)的敏感性进行了试验,用3株野鼠型(A9、R3、76-118)和1株家鼠型EHFV(R22),并用对EHFV敏感的Vero-E6细胞作对照,结果沙鼠腹腔巨噬细胞感染EHFV后,第1代第8天前即可查见明显的特异性荧光,第16天左右达高峰,病毒滴度≥10~(-7.5),与Vero-E6细胞比较,培养上清液的病毒滴度,沙鼠MΦ较Vero-E6细胞高1～4个对数,当感染病毒量很低时,在Vero-E6细胞内测不出特异性抗原,而在沙鼠MΦ内病毒仍可繁殖,滴度达10~(-5.(?)),中和试验、间接免疫荧光检测和荧光阻断试验均证明,沙鼠MΦ内繁殖的病毒确实为EHFV。     

TRANSFECTION OF NORMAL HUMAN EPIDERMAL KERATINOCYTES WITH LIPID/DNA COMPLEXES IN VITRO

Highly proliferative normal human epidermal keratinocytes (NHK) were isolated from human foreskin biopsies, cultivated in serum-free medium and characterized by flow cytometry. The expression of cytokeratin 19, cytokeratin 14 and vimentin indicated that the suspension contained a high percentage of undifferentiated cells of the basal epidermal layer. The NHK were transfected in vitro with lipid/DNA complexes made of Effectene or Lipofectamine and different reporter genes. The transfection efficiency of Effectene/DNA complexes was 20fold higher compared to Lipofectamine. Transfected keratinocytes continued to grow and developed within 2 weeks a cellular multilayer (3-D culture). Areas of transfected cells were detected within this layer.     

COMPARATIVE ANALYSIS OF THE CONCENTRATION OF INJECTED HORSERADISH PEROXIDASE IN CYTOPLASMIC GRANULES OF THE KIDNEY CORTEX, IN THE BLOOD, URINE, AND LIVER

The concentration of horseradish peroxidase in total particulate fractions from the kidney cortex did not change much during the first few hours after injection, as long as most of the injected protein was not yet cleared from the blood. It decreased at a rate of 6–8% per hr afterwards. The concentration of peroxidase in total particulate fractions increased in proportion to the load (dose) over a wide range, suggesting that a constant fraction of the protein was reabsorbed by micropinocytic vesicles into the tubule cells from the glomerular filtrate. The amount of peroxidase excreted in the urine also increased in proportion to the injected dose. The proportion of peroxidase taken up by the liver, however, decreased several times when the dose was increased. A marked decrease of protein uptake into the kidney cortex and an increase of urinary excretion were observed when rats received a second, equal dose of peroxidase 4 hr after the first injection, and the rate of clearance of peroxidase from the blood was decreased after the second injection. The liver, on the other hand, took up almost twice as much peroxidase after two injections as after one. The uptake of peroxidase by the kidney cortex increased with age. Cytochemical observations on the preferential absorption of peroxidase by certain cell types and segments of the renal tubules in relation to dose are reported.     

THE IN VITRO FORMATION OF ACETYL-ASPARTYL-PEPTIDO-SEROTONIN COMPLEXES BY PIG HYPOTHALAMIC TISSUE EXTRACTS

Peptido-serotonin complexes are formed on incubation of pig hypothalamic tissue extracts with acetyl-aspartate, the amino acids glu. ser, gly and ala. an ATP-regenerating system and serotonin. The purification and partial characterisation of two acetyl-aspartyl-peptido-serotonin complexes is described.     

RESPONSE OF MOUSE BONE MARROW COLONY FORMING UNITS IN DIFFERENT STAGES OF THE CELL CYCLE TO IN VITRO INCUBATION WITH MITOMYCIN-C

A short-term in vitro method was employed to study the Mitomycin-C sensitivity of normal mouse bone marrow CFU without triggering the G₀-phase cells into the proliferative cycle. Comparison was made of the toxicities of the drug against cells in different phases of the cell cycle including G₀. Mitomycin-c killed CFU both in and out of the S-phase. No significant difference could be found between its toxicities against normal and proliferating CFU; along the exponential part of the survival curve 1·6 μg/ml concentration of the drug reduced survival to 10%. Although in the normal bone marrow only a few CFU are in the S-phase and are killed by the agent, presence of the sensitive G₀ cells produce a significant amount of non-S-phase mortality. Among the proliferating CFU population the non-S-phase lethality is less due to the absence of G₀ cells. About 75% of the S-phase cells are killed after incubation with 1 μg/ml drug; outside the S-phase, the lethality is about 40–50%. The studies indicate that the G₀ cells which are situated near the G₁-S boundary are almost as sensitive to the drug as other non-S-phase cells like G₁ cells. The clinical significance of the findings is discussed.     

KINETICS OF CELL PROLIFERATION IN THE PRE-IMPLANTED MOUSE EMBRYO IN VIVO AND IN VITRO

The cell proliferation of pre-implanted mouse embryos was investigated after development in vivo and in vitro. The studies were started at the pronuclear stage, 2 h post conception (p.c.) and continued until the hatching of blastocysts, 120–144 h p.c. The number of cell nuclei, the DNA content of each nucleus, the mitotic index and the labelling index were determined. From these data it was possible to calculate the length of the cell generation cycle and its various phases. With the exception of the first cell cycle the S-phase was constant. The G₁- as well as the G₂-phase varied in length during the different cell cycles. From 31–72 h p.c. the increase in cell number was exponential. After cultivation in vitro this increase was smaller than in vivo. At later periods the proliferation rate decreased with proceeding development. In late blastocysts most of the cells were in the G₁-phase. The development of the embryos was somewhat faster in vivo than in vitro. But in principle conditions were comparable.     

IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5,7-DIHYDROXYTRYPTAMINE12

[¹⁴C]5,6-Dihydroxytryptamine ([¹⁴C] 5,6-DHT) and [¹⁴C]5,7-dihydroxytryptamine ([¹⁴C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [¹⁴C]5,6-Dihydroxyindole acetic acid ([¹⁴C]5.6-DHIAA) and [¹⁴C]5,7-dihydroxyindole acetic acid ([¹⁴C]5,7-DHIAA) were detected as MAO metabolites by TLC besides non-identified components. The conversion of [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics (K_m 5,7-DHT: 0.5 × 10^?3M; K_m 5,6-DHT: 1.25 × 10^?3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of MAO A. Incubation of rat brain homogenates with [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT or with the MAO metabolites [¹⁴C]5,6-DHIAA and [¹⁴C]5,7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5,6-DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O₂] in the incubation medium slightly attenuated the inactivation of MAO by 5,6-DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT, e.g. its corresponding o-quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6-DHT. The lack of inactivation of MAO by 5,7-DHT in vitro correlated with a low degree of radioactivity binding (from [¹⁴C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l -ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7-DHT.     

顺天胶囊对小鼠免疫功能的影响

本文探讨了顺天胶囊(Shun Tian Capsule;STC)在体外对小鼠淋巴免疫功能的影响,实验结果表明,本品浓度在0.25～1.0μml~(-1)范围内明显促进ConA和LPS对淋巴细胞增殖反应,增强诱导淋巴细胞产生IL-2和INF-α。提示,本品具有明显增强免疫功能的作用,最适剂量为0.5～0.75 ug.ml~(-1),并呈浓度依赖性的双向作用。     

THE MECHANISM OF SPERM HEAD TRANSFORMATION DURING MOUSE OOCYTE FERTILIZATION IN VITRO

用Ｈｏｅｃｈｓｔ３３３４２标记及氨银反应的方法在光镜和电镜水平上研究不同发育阶段的小鼠卵母细胞转化精核的能力。生发泡期卵母细胞质不能诱发精子组蛋白替代鱼精蛋白及精核解聚。生发泡破裂后,卵母细胞获得使精核解聚的能力,但直到卵母细胞完成成熟前,雄原核均不能形成。进一步研究表明,卵母细胞成熟过程中持续的蛋白质合成是雄原核形成所必需的,鱼精蛋白质磷酸化是精核解聚中的关键步骤。     

幽门螺杆菌表面抗原免疫保护作用的体外与活体研究

目的：调查幽门螺杆菌（Hp)几种表面蛋白体外对T细胞增殖的影响和在小鼠体内的免疫保护作用。方法：评价Hp全菌抗原、尿原酶（Urease)、黏附素（hpaA)、外膜蛋白25（Hop25)和38（Hop38)对人外周血T细胞及小鼠CD4^ T细胞增殖的影响;与佐剂合用,评价上述重组蛋白对小鼠Hp感染的免疫预防作用。结果：Urease和Hop25可刺激人及鼠T细胞增殖,hapA只能刺激Hp^ PBL增殖,而Hop38则有毒性作用;Hop25和Hop38均可产生60％的完全保护,hpaA可产生100％的部分保护即降低细菌定植密度,而Urease只能产生40％的部分保护。结论：外膜蛋白可能是一组高效的Hp疫苗免疫原;其长期免疫效果及对T细胞功能的活体调节作用尚需进一步评价。国际上尚未见相关报道。