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目的建立疫苗评价用豚鼠免疫毒理学评价方法,并应用建立的评价指标对结核病新疫苗(耻垢疫苗)的免疫毒性进行初步评价。方法制备豚鼠过敏模型30只,阴性对照15只。建立豚鼠血清总IgG1抗体检测和外周血嗜碱粒细胞体外刺激培养上清、支气管肺泡灌洗液上清,以及腹腔灌洗液中肥大细胞体外刺激培养上清中的过敏性介质(组胺和白三烯)含量的检测方法。将豚鼠分成3组,分别为过敏模型组、耻垢疫苗组和阴性对照组,每组6只,应用已建立的以上几种评价指标初步进行了结核病新疫苗(耻垢疫苗)的免疫毒性评价。结果以6μg/ml羊抗豚鼠IgG1抗体包被,待测血清稀释1∶105,以及辣根过氧化物酶(HRP)标记的羊抗豚鼠IgG1抗体稀释1∶20 000的实验条件为豚鼠血清总IgG1抗体的最佳检测方法。外周血嗜碱粒细胞体外刺激培养上清中,豚鼠过敏模型组和对照组中组胺含量分别为(99.37±15.34)nmol/L和(29.94±5.86)nmol/L;白三烯含量分别为(13.09±3.87)pg/ml和(2.22±0.53)pg/ml。支气管肺泡灌洗液上清组胺含量分别为(73.42±18.60)nmol/L和(31.00±12.09)nmol/L,白三烯含量分别为(123.20±16.57)pg/ml和(39.05±16.94)pg/ml。腹腔灌洗液中肥大细胞体外刺激培养上清中组胺含量分别为(39.59±12.94)nmol/L和(14.35±5.85)nmol/L,白三烯含量分别为(6.79±2.58)pg/ml和(3.09±1.18)pg/ml。以上3种不同组织来源的样本中,过敏模型组的组胺和白三烯含量与对照组相比差异均有统计学意义(组胺:t=12.60,t=5.41,t=5.20,P<0.05;白三烯:t=8.46,t=9.15,t=3.85,P<0.05)。应用以上方法评价结核病新疫苗未见免疫学毒理学异常反应。结论实验初步建立了免疫毒理学评价方法,以该方法评价耻垢疫苗未见免疫毒理学异常反应。
Objective To establish a method for evaluating the immunotoxicology of guinea pigs for vaccine evaluation and evaluate the immunotoxicity of the new vaccine against tuberculosis (smegma vaccine) with the established evaluation indexes. Methods Guinea pig allergic model 30, negative control 15. To establish a guinea pig serum total IgG1 antibody detection and peripheral blood basophils in vitro stimulation of culture supernatant, bronchoalveolar lavage fluid supernatant, and peritoneal lavage fluid mast cells in vitro stimulation of culture supernatant of allergic mediators (histamine and Leukotriene) content of the detection method. The guinea pigs were divided into three groups: allergic model group, smegma vaccine group and negative control group, with 6 in each group. The immunotoxicity evaluation of the new TB vaccine (smegma vaccine) was initially carried out by using the above mentioned evaluation indexes . Results The experimental conditions were as follows: 6μg / ml goat anti-guinea pig IgG1 antibody, 1: 105 serum dilution and 1: 20 000 horseradish peroxidase (HRP) labeled goat anti-guinea pig IgG1 antibody. IgG1 antibody best detection method. The levels of histamine in the model group and the control group were (99.37 ± 15.34) nmol / L and (29.94 ± 5.86) nmol / L, respectively. The levels of leukotrienes in the supernatant of the peripheral blood basophils were (13.09 ± 3.87) pg / ml and (2.22 ± 0.53) pg / ml, respectively. The contents of histamine in bronchoalveolar lavage fluid were (73.42 ± 18.60) nmol / L and (31.00 ± 12.09) nmol / L, respectively, and the leukotriene contents were 123.20 ± 16.57 pg / ml and 39.05 ± 16.94, pg / ml. The amount of histamine in cultured supernatant stimulated by mast cells in peritoneal lavage fluid were (39.59 ± 12.94) nmol / L and (14.35 ± 5.85) nmol / L respectively, and the leukotriene contents were (6.79 ± 2.58) pg / ml And (3.09 ± 1.18) pg / ml. The histamine and leukotriene in the allergic model group were significantly different from those in the control group (histamine: t = 12.60, t = 5.41, t = 5.20, P < 0.05; leukotrienes: t = 8.46, t = 9.15, t = 3.85, P <0.05). The application of the above method to evaluate the new TB vaccine did not show immunological toxicological abnormalities. Conclusion The method of immunotoxicological evaluation was initially established in this experiment. No immunotoxicological abnormalities were observed in the smegma vaccine.