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目的结合多种免疫细胞功能试验,探讨人和食蟹猴外周血单核细胞免疫功能的差异,为临床前药物免疫毒性评价建立起合适的体外试验方法,并且为生物技术药物安全性评价选择动物种属及动物毒性研究外推之人的风险评估提供科学支持。方法 (1)单抗诱导细胞因子释放试验:采用了Lucy Findlay建立的风干包被法及湿包法,人和食蟹猴PBMC细胞与ANC28.1共孵育2 h,24 h,48 h,然后进行TNF-α,IFN-γ的ELSIA检测。(2)T/B细胞增殖试验:收集人和食蟹猴外周血PBMC细胞,给予不同浓度植物血凝素(PHA)和脂多糖(LPS),孵育3 d后,cck-8检测细胞增值率。(3)收集人和食蟹猴外周血PBMC细胞,给与流感疫苗、抑制剂(抗CD20单抗)或细胞培养液。孵育6 d后采用ELISPOT检测抗体的分泌。结果(1)单抗诱导细胞因子释放试验:随ANC28.1作用时间的延长,人PBMC细胞释放的TNF-α及INF-γ逐渐增加。然而,食蟹猴PBMC细胞无TNF-α及INF-γ的释放。(2)T/B细胞增殖试验:人和食蟹猴外周血PBMC细胞给与PHA后细胞数目明显增加,然而人PBMC细胞增殖幅度明显高于食蟹猴。但是在不同浓度LPS刺激下,人和食蟹猴外周血中PBMC增殖趋势非常相似。(3)B细胞抗体形成功能测定:人和食蟹猴外周血PBMC细胞给与流感疫苗刺激后,其分泌相应抗体的细胞数目明显高于阴性对照组。给与抗CD20单抗后,食蟹猴抑制组分泌抗体的细胞数目明显减低。在相同处理条件下,人和食蟹猴分泌抗体的细胞数目增长幅度类似。结论食蟹猴B细胞增殖及B细胞抗体分泌等功能与人比较相似。但是,人和食蟹猴T细胞增殖及细胞因子分泌存在明显差异,人相对食蟹猴而言T细胞的免疫应答功能更为强烈,因此动物数据中T细胞毒性评价外推至人时需要慎重考虑,可能补充体外试验是药物免疫毒性评价的方向。
Objective To explore the difference of immunological function of peripheral blood mononuclear cells from human and cynomolgus monkey combined with a variety of immune cell function tests to establish a suitable in vitro test method for the evaluation of immunotoxicity of preclinical drugs and to select animal species for biotechnological drug safety evaluation And animal toxicity studies to extrapolate the risk assessment to provide scientific support. Methods (1) Monoclonal antibody-induced cytokine release test: Air-dried coating method and wet-pack method established by Lucy Findlay were used to co-culture human and cynomolgus monkey PBMCs with ANC28.1 for 2 h, 24 h and 48 h ELSIA detection of TNF-α, IFN-γ. (2) T / B cell proliferation assay: Peripheral blood PBMCs of human and cynomolgus monkeys were collected and treated with different concentrations of phytohemagglutinin (PHA) and lipopolysaccharide (LPS). After 3 days of incubation, cck-8 was used to detect cell proliferation rate. (3) Human and cynomolgus peripheral blood PBMCs were collected and administered with influenza vaccine, inhibitor (anti-CD20 monoclonal antibody) or cell culture medium. After 6 days of incubation ELISPOT was used to detect the secretion of antibodies. Results (1) Monoclonal Antibody Induced Cytokine Release Test: TNF-α and INF-γ released from human PBMC cells gradually increased with the prolongation of ANC28.1 treatment. However, none of cynomolgus monkey PBMC cells had TNF-α and INF-γ release. (2) T / B cell proliferation test: The number of PBMCs in human and cynomolgus monkey peripheral blood was significantly increased after PHA administration, however, the proliferation rate of human PBMCs was significantly higher than that in cynomolgus monkeys. However, the proliferation of PBMC in human and cynomolgus monkey peripheral blood were very similar at different concentrations of LPS. (3) B cell antibody formation assay: The number of cells secreting the corresponding antibodies after PBMCs from human and cynomolgus monkey peripheral blood were stimulated by influenza vaccine was significantly higher than that of the negative control group. After administration of anti-CD20 mAb, the number of cells secreting antibodies in the cynomolgus monkey inhibition group was significantly reduced. Under the same treatment conditions, the number of human and cynomolgus monkey antibody secreting cells increased in a similar manner. Conclusion The function of B cell proliferation and B cell antibody secretion in cynomolgus monkeys is similar to that in humans. However, T-cell proliferation and cytokine secretion in humans and cynomolgus are significantly different, and human immune response to T-cell is more pronounced relative to cynomolgus monkeys. Therefore, extrapolation of T-cell toxicity in animal data to humans requires careful consideration , May complement the in vitro test is the direction of drug immunotoxicity evaluation.