结核病新疫苗的免疫毒理学评价方法研究

Immunotoxicological evaluation about new tuberculosis vaccines

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 develop a method for the immunotoxicological evaluation of vaccines in a guinea pig model,and to preliminarily evaluate the immunotoxicological effects of a new tuberculosis vaccine（smegmatis vaccine）.Methods（1） Thirty guinea pigs were prepared the allergy models,and 15 as negative controls.The total IgG1 antibody in serum and the content of allergic media in the bronchoalveolar lavage fluid and the culture supernatants from the peripheral blood basophils and mast cells in peritoneal lavage fluid in vitro simulated were detected by ELISA.（2） The guinea pigs were divided into 3 groups as follow： allergy model,smegmatis vaccine and negative control.Their immunotoxicological effects were preliminarily evaluated using the methods developed above.Results The optimal experimental conditions for the detection of total IgG1 antibody in sera from guinea pigs included that 6 μg/ml goat anti-guinea pig IgG1 antibody was coated,the serum sample was diluted 1∶105,and horseradish peroxidase labeled goat anti-guinea pig IgG1 antibody was diluted 1∶20 000.In the culture supernatant of peripheral blood basophils simulated in vitro in the allergy model group and negative control group,the histamine contents were（99.37±15.34） nmol/L and（29.94±5.86） nmol/L,and leukotriene levels were（13.09±3.87） pg/ml and（2.22±0.53） pg/ml,respectively.In the supernatant of bronchoalveolar lavage fluid,the histamine contents were（73.42±18.60） nmol/L and（31.00±12.09） nmol/L,and leukotriene levels were（123.20±16.57） pg/ml and（39.05±16.94） pg/ml,respectively.In the culture supernatant of peritoneal lavage fluid simulated in vitro,the histamine contents were（39.59±12.94） nmol/L and（14.35±5.85） nmol/L,and leukotriene levels were（6.79±2.58） pg/ml and（3.09±1.18） pg/ml,respectively.Both of the contents of histamine and leukotriene in three kinds of samples mentioned above were significantly different between the allergy model group and control group（histamine：t=12.60,t=5.41,t=5.20,P0.05;leukotriene：t=8.46,t=9.15,t=3.85,P0.05）.The abnormal immunotoxicological effects were not observed in the safety evaluation of smegmatis vaccine by the methods above.Conclusion The method for immunotoxicological evaluation of vaccines was preliminarily established.The smegmatis vaccine proved preliminarily to be safe.