有机锗(Ge-401)对小鼠B淋巴细胞产生抗体的调节

本文报道了有机锗(Ge-401)对Balb/C小鼠产生抗SRBC抗体能力的调节。Balb/C小鼠先经SF_1(人精浆经Sephadex G-100分离得到的第一组分)抑制其免疫功能,然后实验小鼠经腹腔注射Ge-401,每天一次连续7天,对照组小鼠则以等量生理盐水代替。在实验第7天用SRBC免疫小鼠,于免疫后第4天进行溶血空斑试验和血清抗SRBC抗体凝集效价测定。结果表明,Ge-401能明显提高实验组小鼠的溶血空斑数和抗体凝集效价,与对照组相比,p<0.01。提示Ge-401能增强免疫功能抑制状态下小鼠的免疫应答能力——B细胞产生特异性抗体的能力。     

空斑形成细胞测定技术的进展及临床免疫应用

一、简介空斑形成细胞(Plaque-forming cell.PFG)测定技术又称溶血空斑技术,为Jerne等在1963年建立,主要用于体外单个抗体分泌细胞的检测。PFC技术具有敏感、特异、简便、快速的优点,因而在免疫学各领域得到广泛应用,成为免疫学研究不可缺少的工具之一。Jerne等建立的方法为平皿法,即用羊红细胞(SRBC)免疫小鼠,经一段时间取脾制成细胞悬液,加入含SRBC的琼脂介质中,倾到平皿,孵育后加入补体。由于抗体分泌细胞分泌抗SRBC抗体,致敏了周围的SRBC,经补体作用使SRBG溶解,形成溶血空斑。     

黄芪多糖对去T细胞小鼠促进抗体产生机理的探讨

黄芪多糖(APS)100mg/kgx6天 ip 可以增加正常小鼠脾脏重量,而对去胸腺与抗胸腺细胞血清处理过的小鼠(T_x-ATS)的作用明显降低。APS 能够显著提高正常小鼠 SRBC 免疫后脾细胞直接溶血空斑(d-PFC)和间接溶血空斑(i-PFC)数量,但对 T_x-ATS 小鼠不能促进d-PFC 数升高。APS 可以提高 T_x-ATS 小鼠在 LPS 免疫后 d-PFC 数,这个作用同 APS 对正常小鼠 SRBC 免疫后的效应相比显著降低。这些结果提示,APS 促进抗体形成作用机理可能是通过 T 细胞介导的。     

获得性免疫缺陷综合征病人的抗原特异性多克隆的B细胞应答

获得性免疫缺陷综合征(AIDS)是一种复杂的免疫异常症候群。本文作者检查了58例AIDS和Kaposi氏肉瘤患者的B细胞功能。体外检测结果证明AIDS/KS患者的B细胞在体外失去产生绵羊红细胞(SRBC)抗体的能力。取患者末梢血单个核细胞(PB-MC),用RPMl1640液体外培养,加SRBC进行免疫,用Jerne溶血空斑法测定B细胞     

精制厌氧棒状杆菌细胞壁的佐剂作用及其对巨噬细胞的激活性能

本文主要观察经冷冻高压破碎菌体、SDS处理以及蛋白酶消化提纯的精制厌氧棒菌细胞壁的佐剂作用;用溶血空斑法测定抗体形成细胞数以及间接血凝法测定免疫小鼠血清抗体,证明其对抗原SRBC及BSA具有明显佐剂作用;此外亦能恢复带瘤小鼠(艾氏腹水癌)的吞噬功能。     

日本血吸虫感染宿主的免疫抑制现象

本文报告用巨噬细胞移动抑制试验,溶血空斑试验及T细胞辅助活力测定观察小鼠感染日本血吸虫后对植物血凝素、血吸虫成虫抗原、羊红细胞及半抗原-载体(TNP-童虫)免疫应答的抑制现象。MMIT的结果表明:对PHA的应答于感染后28天出现抑制;对血吸虫成虫抗原的应答于感染后2周内正常,第3周开始出现抑制,14周后又基本恢复正常。溶血空斑试验结果表明,感染后头3周对SRBC的应答水平递增,第3周达最高水平,第4周开始应答逐渐下降。不同H-2的纯系小鼠感染日本血吸虫后对SRBC的应答规律基本相似,但于感染后同一时间内不同H-2的小鼠应答水平随小鼠品系的不同而不同。T细胞辅助活力测定的结果表明:小鼠感染后3周内对TNP-童虫的应答逐渐增加,第3周最高,之后逐渐下降。上述结果提示感染宿主存在免疫应答的抑制现象。这种抑制现象不仅表现在T细胞对促有丝分裂因子PHA和血吸虫成虫抗原的应答上;也表现在B细胞对胸腺依赖抗原SRBC的应答上,以及在T-B细胞协作产生抗半抗原抗体时,T细胞辅助应答也受到抑制的影响。     

红花多糖的免疫活性作用

用SRBC免疫的C57BL小鼠脾脏作试验,观察到红花多糖制剂有增强免疫应答作用。动物用SRBC致敏后,连续4天分别以三种不同剂量(15、45、135毫克/公斤体重)的红花多糖制剂作腹腔注射,第5天取各鼠脾细胞做溶血空斑试验。结果表明:红花多糖均有刺激PFC的作用,其促进作用随剂量增加而明显增加,在剂量增加至135毫克/公斤时,PFC值比对照组高58％。用安配常数统计法     

试管内诱生抗体的研究——Ⅱ.对实验条件的进一步探讨

本文对体外诱生溶血空斑形成细胞(PFC)的实验条件进行了进一步研究,包括筛选新生小牛血清、挑选绵羊红细胞(SRBC)的供羊;和诱生抗体反应的最适SRBC剂量,以及选择适合于本实验的小鼠品系。结果证明不同批的小牛血清支持PFC反应的能力及不同个体的SRBC诱生PFC反应的能力存在着差异;每毫升脾细胞培养物中加入50微升1％SRBC悬液及用615小鼠作为脾脏细胞的供体较为合适。还对贮存不同时间的SRBC和用SRBC吸收过的小牛血清进行了比较。通过选用合适的条件,能够获得稳定的实验结果和诱生出较高的PFC反应。文内分析和讨论了可能引起实验失败的原因。     

介绍一种简便测定γ-干扰素活性的方法

<正> Virelizier等人报道γ-干扰素有较明显抑制溶血空斑的形成,为此我们采用稍加改良的空斑减少法,测定γ-干扰素活性。 先给实验组小鼠肌肉注射不同稀释度的Hu-IFN-γ,同时从尾部静脉注入10％SRBC 0.2ml。对照组只注入10％SRBC 0.2ml。免疫4天后,将小鼠拉颈处死,取脾脏磨制成含5×10~6细胞/ml的生理盐水悬液。在直径9cm平皿内倒入0.8％底层琼脂糖9ml。另取一试管,装入0.4％琼脂糖1.5ml,置50℃水浴中保温,然后加入10％SRBC 0.15ml,5×10~6/ml脾细胞是液0.15ml,混匀,迅速倒入已铺     

抗HLA抗原的抗体细胞空斑试验

本文作者建立了一种能够检测出产生抗HLA抗体的单个细胞的空斑试验。试验原理是用绿色荧光素(碳氧荧光素乙酰乙酸盐,CFDA)染活细胞,红色荧光素(Propidum iodide PI)染空斑内由抗体和补体作用溶解的细胞,在荧光显微镜下计数。标本干燥后可长期保存而不发生退色等改变。空斑形成的主要原理是细胞溶解,靶细胞释放荧光素,摄取PI而形成空斑。镜下可见桔红色的斑点,在其周围有带绿色荧光素的细胞围绕。靶细胞的来源是经EB病毒刺激后转化的淋巴母细胞。该细胞在加有L-谷氨酰胺和热灭活10%胎牛血清RPMI 1640培养液中培养,在其对数生长期收获,再用CFDA进行染色。抗体分泌细胞是通过鼠—鼠杂交瘤     

Molecular mechanism of SSFA2 deletion inhibiting cell proliferation and promoting cell apoptosis in glioma

Gliomas are the most common primary brain malignant tumors in humans. Glioblastoma multiforme(GBM) is the most malignant intracranial tumor with a relatively poor prognosis. There promote us to find effective anti-cancer therapies to reduce cancer mortality. By using bioinformatic analysis, we found SSFA2 as a gene with elevated expression in the glioma tissues. We detected the expression of SSFA2 in glioma tissues and in the glioma cell lines, as well as in normal brain tissues. SSFA2 expression was higher in glioma tissues, especially in glioblastoma multiforme than normal brain tissues. Subsequently, we found that down-regulate SSFA2 in glioma cell lines can regulate the cell cycle to reduce the proliferation ability and induce the early apoptosis rate in shSSFA2 cells relative to control cells. Moreover, we found that down-regulate SSFA2 in glioma cell line U87(shSSFA2-U87) inhibited the growth effectiveness compared to the control cell line U87. These result reveals us that SSFA2 may act as oncogene to promote the progression of glioma. For further research specific mechanisms of SSFA2 in gliomas, we used the gene chip to detect the downstream gene in U87. We found that 30 genes also may be as target gene of SSFA2, and we testify the protein expression by western-blot. The result reveal that IL1A, IL1B and CDK6 as target gene of SSFA2 to regulate the progression of glioma. These finding suggest that SSFA2 could be a new therapeutic target for gliomas.     

The adaptation of the plaque reduction assay for measuring specific cytotoxic cells in limiting dilution cultures

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained.     

A feline kidney cell line-based plaque assay for feline calicivirus,a surrogate for Norwalk virus

Feline calicivirus (FCV) has been used by researchers as a surrogate for Norwalk virus (NV), since they share a similar genomic organization, physicochemical characteristics, and are grouped in the same family, Caliciviridae. Unlike NV, however, FCV can grow in established cell lines and produce a syncytial form of cytopathic effect. In this report, we describe the development and standardization of a plaque assay for FCV using monolayers of an established line of feline kidney (CrFK) cells in 12-well cell culture plates. The assay method has demonstrated reproducibility, ease of performance and resulted in clear plaque zones, readable in 24 h after virus inoculation. The infectivity titre of the virus by this plaque assay agreed well with tissue culture infectious dose(50) (TCID(50)) determinations. The described plaque assay would be a valuable tool in conducting various quantitative investigations using FCV as a model for NV and Norwalk-like viruses (NLV).     

The haemolytic plaque reduction technique to measure cytotoxicity with hybridoma cells as a target

The use of hybridoma cells as targets to measure cell-mediated cytotoxicity has been established. The ability of target hybridoma cells to form haemolytic plaques has been used as an indicator of target viability and therefore, cytotoxicity has been detected by plaque reduction (PR). Allogeneic responses, spontaneous cytotoxic responses and anti-hapten responses have been measured by the PR assay. Compared with the standard [51Cr]chromate release assay, small numbers of target cells (400 or less) can be used in the PR assay and this results in a greatly enhanced sensitivity in detecting small amounts of cytotoxicity. The ability to construct a range of hybridoma targets with the appropriate cell surface determinants presents a new approach, of general applicability, to the sensitive detection of cytotoxic lymphocytes.     

A quantitative comet infection assay for influenza virus

The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2-6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells.     

Development of an integrated cell culture--real-time RT-PCR assay for detection of reovirus in biosolids

The current method for viral detection in biosolids is a plaque assay, as specified by the EPA in the 40 CFR Part 503 rule. Development of an integrated cell culture-polymerase chain reaction (ICC-PCR) assay has allowed detection of viruses that are under-detected and undetected by the plaque assay. This study examined the efficiency of the ICC-PCR method to detect mammalian orthoreovirus, a virus typically under-detected in biosolids. Biosolid samples seeded with mammalian orthoreovirus type 1 (Lang) detected to 3 x 10(5) plaque forming units (pfu) with a plaque assay, 10(2)pfu equivalents with real-time RT-PCR and no incubation, and 10(8)pfu equivalents with real-time RT-PCR after 7 days incubation. More infectious virus was detected using ICC-real-time RT-PCR than a plaque assay. Twenty-four environmental samples from three locations around the United States did not plaque with the EPA method; however the ICC-PCR detected infectious reovirus in 13 of the samples. Raw biosolids samples accounted for 12 of the positive samples, and 1 positive was from an aerobically digested sample.     

土槿皮乙酸诱导脑胶质瘤细胞株U87阻滞于M期并发生凋亡

目的:通过研究土槿皮乙酸对脑胶质瘤细胞株U87增殖与凋亡的影响,探讨土槿皮乙酸对脑胶质瘤的潜在应用价值。方法:用不同浓度土槿皮乙酸处理U87细胞,并于不同时点观察细胞形态改变;采用MTS法对U87细胞进行细胞活力测定;采用流式细胞术和Western blot法检测土槿皮乙酸对细胞周期的影响;采用Annexin V/PI双染流式细胞术检测细胞凋亡;利用Western blot法检测caspase通路相关蛋白(cleaved PARP、caspase-3、procaspase-9和caspase-8)的变化情况。结果:土槿皮乙酸明显抑制脑胶质瘤U87细胞的活力,能使细胞阻滞于M期,并通过caspase通路诱导细胞发生凋亡。结论:土槿皮乙酸能使脑胶质瘤细胞株U87细胞阻滞于M期,并诱导其凋亡。     

A plaque assay for all cells secreting Ig of a given type or class

A modification of the hemolytic plaque assay using protein A-coated red cells is described which makes use of the fact that the Fc portion of IgG binds to protein A. A number of murine plasmacytomas secreting different classes of Ig have been tested for plaque formation with these indicator red cells. In the presence of complement-binding antibodies specific for the corresponding class of secreted Ig, between 10 and 70 % of all plated myeloma cells formed plaques. The assay shows a prozone effect in excess of antibody, suggesting that complexes of antibody and secreted Ig effect lysis of the target cells. This assay can be used to enumerate cells secreting any molecules for which complement-binding antibodies are available.     

Production of human immune interferon (Hu IFN-gamma) studied at the single cell level. Origin, evidence for spontaneous secretion and effect of cyclosporin A

A reverse hemolytic plaque assay has been developed which specifically detects secretion of human immune interferon (Hu IFN-gamma) at the single cell level. Unstimulated peripheral blood lymphocytes from healthy adult volunteers spontaneously secreted IFN-gamma. Stimulation of these cells with concanavalin A, phytohemagglutinin, or the UCHT1 monoclonal anti-human T cell antibody significantly increased the number of IFN-gamma-secreting cells. The cell producing IFN-gamma, both spontaneously and after UCHT1 antibody stimulation, is an OKT3+,4+,8-,HLA-DR-T lymphocyte as determined at the single cell level. Finally, cyclosporin A, a potent and selective immunosuppressive drug for T cells, strongly inhibited the secretion of IFN-gamma as assayed at the cell level. This IFN-gamma reverse hemolytic plaque assay has great potential for the further study of IFN-gamma both in physiological and pathological conditions.     

A modified plaque test for the detection of cells forming antibody to alloantigens

Cells secreting antibody to H-2 alloantigens can be detected in vitro by the alloantibody plaque assay. This test has been modified to allow the earlier and more objective determination of plaques by staining the lysed target cells with trypan blue. Details are given of optimum conditions necessary for the assay.