用细胞游走抑制试验研究带移植肝癌大鼠的免疫状态

引言致敏淋巴细胞和抗原接触时能释出淋巴因子,使巨噬细胞或白细胞的游走受到抑制。这现象最早为Rich和Lewis(1932)所发现。1962年George 和Vanghan建立了细胞游走抑制试验的毛细管法,成为研究细胞免疫的一个新方法。一般认为这方法和迟发性过敏反应比较相符(Thor等,1968;Rosenberg和David,1970;Ramsey等,1976),可用于检测淋巴细胞对抗原的致敏性和效应反应。目前已应用于细胞免疫和肿瘤免疫方面。方法学上有巨噬细胞游走抑制试验(Macrophage mi-gration inhibition test,MMIT)和白细胞     

应用流式细胞仪研究抗IL-8单抗对IL-8激活人颗粒细胞活力的中和作用(英文)

以IL-8免疫的BALB/C小鼠脾细胞与Sp2/0或653小鼠骨髓瘤细胞融合构建了淋巴细胞杂交瘤克隆I8-S2和I8-63。ELISA叠加试验(ELISA Additivity Test)表明这两杂交瘤克隆分泌的单抗分别识别IL-8分子的不同表位。IL-8能激活人颗粒细胞,引起细胞内Ca~(2 )浓度([Ca~(2 )]_i)上升。通过流式细胞仪分析[Ca~(2 )]_i的变化,发现两个克隆单抗对IL8激活细胞的活力具有不同的中和作用。克隆I8-S2具有很强的中和作用,而克隆18-63则不然。上述结果提示IL-8的激活细胞活力局限于该分子的某表位。     

体外诱发抗体生成过程中淋巴细胞的变化

An in vitro system for induction of antibody responses of human cells has been established in our lab. B cell enriched fractions from excised human tonsils or trauma spleen were cultured for 7-14 days with tetanus toxoid or HBsAg vaccine with or without human T cell conditioned medium (C. M.) or a mixture of low concentrations of PWM and LPS (MTG). Positive antibody responses could be detected in cultures. Cells taken from different culture periods were subjected to FACS analysis in order to expound cellular changes during antibody induction periods so as to improve the in vitro antibody induction system. The results were described as follows: 1. Variations in total percentages of T cells during culturing periods seemed to be related its initial percentages. Cells with bigger initial percentages tended to decrease first and finally maintained at about 30%. While cells with smaller initial percentages tended to increase and finally also maintained at 30%. 2. CD4+ Th cells and CD8+ Ts cells from tonsils and spleen behaved somewhat differently. In tonsil cell cultures the percentages of CD4+ cells were often bigger than the percentages of CD8+ cells throughout the culture period. However, the inverted proportions of CD4+/CD8+ were shown in spleen cell cultures, especially in the culture with C. M. The possible relationships between the variations in CD4+/CD8+ proportions described as above and the intensities of antibody responses were discussed. Additionally, adding 1-Leucine-Methyl Ester showed no effects either on CD8+ or CD4+ cell percentages. 3. B cell (SIg+) percentages in both tonsil and spleen cultures were quite stable throughout the culture period, about 60% of total cells. CD19, a marker of B cell, was only present in part of the cultured SIg+ cells. The significance of the variations in CD19+, SIg+ cells was unclear. CD5+ B cells were known as cells secreting autoantibodies. Our results showed that these cells consistently maintained a relatively low percentage in the whole antibody induction period. 4. The reasonableness standard we used for "gating" in FACS analysis was discussed.     

应用流式细胞仪定量评估人—鼠杂交瘤株细胞抗体生成功能稳定性的研究

用FACS分析和Luria-Delbrǔck方程计算淋巴细胞杂交瘤株细胞丧失抗体生成功能的突变机率(μ),以评估杂交瘤的稳定性。结果表明μ值大,则杂交瘤不稳定,易丧失抗体生成功能,反之则杂交瘤比较稳定。实验证明所建立的方法能反映杂交瘤稳定性的程度,具有实验少人为主观因素干扰,统计意义强,适用范围广等优点。     

一种改良的检出抗体生成细胞的溶血空斑技术

本文报道了一种改良的检出抗体生成细胞的溶血空斑技术。在Cunningham 玻片小室法基础上,对玻片小室的制作,空斑的计数等方面进行了改进。通过对大鼠抗DNP 半抗原的抗体生成细胞的动力学检测,表明本文所述方法是简便可行的。此外对半抗原与SRBC 的连结和影响实验的一些因素——补体、抗TgG 抗血清等进行了探讨。     

用抗独特型单抗体外诱发抗天花粉蛋白的二次抗体应答

在前阶段工作中已获得16个抗天花粉蛋白的单抗。用其中的一个IgE类单抗TE 1免疫Wistar大鼠,通过大鼠-小鼠杂交瘤技术得到了抗独特型单抗。现研究抗独特型单抗AId 6c5在体外诱发二次抗体应答中所起的作用。实验结果表明:1.当将AId 6c5和天花粉蛋白初次免疫8周后的小鼠脾细胞和肠系膜淋巴结细胞一起培养时,能引起二次抗体应答,说明AId 6c5能代替抗原的刺激作用,如果培养系统中同时存在AId 6c5和天花粉蛋白(其剂量能引起体外二次抗体应答),则可出现某种程度的抑制。由于AId 6c5是单克隆抗体,提示一种AId能在不同情况下起刺激或抑制作用。3.应用竞争结合试验阐明AId 6c5除和TE 1外,还和另外两个单抗——TE 4(IgE)和2 A1(IgG1)——起反应,先前的工作证明这三个单抗和另外4个单抗都识别天花粉蛋白上的同一抗原决定簇。但AId 6c5对识别这同一决定簇的其余4个单抗反应很弱。以上说明a.IgE和其它Ig类别具有共同或交叉的独特型,b.也说明识别同一决定簇的IgE具有不同的独特型。4.将TE 1等三个单抗和AId 6c5预先作用后能抑制这三个单抗和天花粉蛋白的结合,说明AId 6c5所识别的独特型,位于抗原结合部位内,至少??是很靠近抗原结合部位的。     

小鼠的自身反应性T淋巴细胞系及其功能

本文报道了用同基因脾细胞和抗原在体外不断再刺激天花粉蛋白免疫过的C57BL/6J小鼠的T淋巴细胞,能刺激自身反应性的T细胞在体外增殖并长期存活。实验结果表明它们的增殖是依赖于同基因脾细胞的再刺激,C57BL/6J(H一2~b),B 10 ScSn(H-2~b)和129(H-2~b)小鼠的脾细胞都能引起它们明显的增殖,但对C3H/He(H-2~K)和Balb/c(H-2~b)小鼠的脾细胞很弱,说明识别的可能是H-2~b抗原。应用未经免疫的C 57 BL/6 J小鼠的脾和淋巴结T淋巴细胞,采用同样的体外刺激方法,未能引起它们对同基因脾细胞的增殖。从而提示自身反应性T细胞是存在于正常机体内的一种能识别自身抗原的T淋巴细胞。在无外来抗原刺激时,它们可能是处于静止或不激活状态;在外来抗原诱发免疫过程中,它们也随了抗原特异的淋巴细胞一同被激活,并可能起调节作用。它们在免疫系统中的地位还有待进一步阐明。     

用体外免疫的人淋巴细胞建立抗破伤风类毒素的杂交瘤克隆

我们应用破伤风类毒素体外免疫的人扁桃体细胞和人-鼠异源骨髓瘤RF 系细胞进行融合,从中筛选到一株杂交瘤细胞89112—50,并通过克隆化筛选获得了两个亚克隆,其分泌的抗体是抗原特异的,和三种抗原(OVA、TC-S、FYG)都不交叉,分泌抗体的功能也比较稳定,在培养瓶内连续扩增传代13次后,仍维持相当高的抗体分泌能力,在常规传代培养过程中所收集的培养物上清液中抗体的含量平均为69.6μg/ml。     

应用流式细胞仪定量评估人—鼠杂交瘤株细胞抗体生...

The paper described a method of quantitative stability analysis of antibody production by hybridoma clones with flow cytometry (FACS). Through FACS analysis the frequency of antibody-negative variants was measured. The rate of generation of antibody-negative variants/cell/generation (mu) could be calculated through Luria-Delbrück fluctuation analysis. The results showed that the value of mu was correlated to the antibody production of hybridoma clones: the larger was the mu value, the more unstable was the antibody production by the hybridoma clones. The experimental results suggested that the clone was stable if its mu value was on the magnitude of 10(-3), while the clone was not stable if its mu value was on the magnitude of 10(-2). According to requirements of single cell for FACS analysis and presence of a variety of methods for dispersing cell aggregates to single cells, the authors regard that this method is fully suitable for analysis of cells in suspension or even in the form of aggregate.