BCG-CpG-DNA免疫毒性研究

发布时间：2018-11-08 10:13

【摘要】： 目的:研究BCG-CpG-DNA的免疫毒性作用,为BCG-CpG-DNA临床前安全性评价提供依据。方法:采用异常毒性试验、急性毒性试验、28天毒性试验初步观察BCG-CpG-DNA毒性;并通过计算免疫器官脏器系数,检测淋巴细胞转化能力、细胞因子释放(Elospot法)、T细胞亚群变化(免疫荧光法)、NK细胞杀伤活性(LDH法),外周血抗双链DNA自身抗体水平(Elisa法),与溶剂对照组比较,进一步观察BCG-CpG-DNA重复给药对小鼠免疫功能的影响。结果:(1)异常毒性试验:实验期间,所有动物一般行为活动、外观体征、进食和饮水活动等均正常;所有动物全部健康存活;动物均有体重增加;大体解剖肉眼观察各脏器均无异常。(2)急性毒性试验:实验期间,所有动物一般行为活动、外观体征、进食和饮水活动等均正常;所有动物全部健康存活;动物均有体重增加;大体解剖,脏器无病变,各组脾重无统计学差异(P0.05)。(3)28天毒性试验:①外观体征、体重观察:在给药期间及恢复期,实验组和对照组动物均活动正常,进食敏捷、毛贴身有光泽,粪便正常;动物体重各组间无统计学差异(P0.05)。②血液学指标:4次检测结果表明,BCG-CpG-DNA主要影响小鼠白细胞总数、淋巴细胞绝对值、中性粒细胞绝对值及相应的百分比,对其他指标无明显影响。③血生化学指标:4次检测结果表明,BCG-CpG-DNA对小鼠外周血生化指标无明显影响。④大体解剖与脏器系数:大体解剖观察,各组动物的主要器官均未见明显病理性改变;免疫中期即免疫后10天,BCG-CpG-DNA中、高剂量组脾脏重量系数高于溶剂对照组,有显著性差异(P=0.03,0.05);免疫末期即末次免疫后3天,BCG-CpG-DNA低、中、高剂量组脾脏重量系数高于溶剂对照组,有显著性差异(P=0.009,0.007,0.007);恢复期即免后21天,BCG-CpG-DNA低、中、高剂量组脾脏重量系数、肝重系数、胸腺重系数和肾脏重系数,各剂量组和溶剂对照组均无统计学差异(P0.05);⑤局部刺激反应:在给药期间及恢复期,实验组和对照组动物的给药部位表观无异常反应,大体解剖后,给药部位也无异常反应。(4)免疫功能检测:BCG-CpG-DNA重复给药,剂量分别达到0.7mg、1.75mg和3.5mg,对细胞免疫功能的影响主要有:增强淋巴细胞转化功能;降低中、高剂量组CD3+T细胞含量;提高体内分泌IFN-γ和IL-4的细胞数二者的比例;增强NK细胞杀伤能力,不同实验组与对照组差异均有显著意义(P0.05);经过三周恢复期,变化的指标均恢复正常。结论:BCG-CpG-DNA在异常毒性试验、急性毒性试验、28天毒性试验均未见明显毒性反应;BCG-CpG-DNA重复给药累计达0.7mg、1.75mg和3.5mg的情况下,小鼠表现为免疫功能增强,但对免疫器官和免疫功能无不良影响,无免疫毒性作用,安全性良好。
[Abstract]:Objective: to study the immunotoxicity of BCG-CpG-DNA and to provide evidence for pre-clinical safety evaluation of BCG-CpG-DNA. Methods: abnormal toxicity test, acute toxicity test and 28 days toxicity test were used to observe the toxicity of BCG-CpG-DNA. The lymphocyte transformation ability and cytokine release were measured by calculating the organ coefficient of immune organs. The changes of), T cell subsets were detected by Elospot method (LDH assay), and the cytotoxicity of), NK cells by immunofluorescence assay (LDH method). The level of anti-double-stranded DNA autoantibodies in peripheral blood (Elisa method) was compared with that of solvent control group to observe the effect of repeated administration of BCG-CpG-DNA on the immune function of mice. Results: (1) abnormal toxicity test: during the experiment, all animals were normal in general behavior, appearance, eating and drinking, all animals were healthy and survived, all animals had weight gain. (2) Acute toxicity test: during the course of the experiment, all animals were normal in general behavior, appearance, eating and drinking water, all animals survived in health. All animals gained weight; Gross anatomy, viscera no lesion, spleen weight of each group (P0.05). (3) 28 days toxicity test: 1 physical signs, body weight observation: during the administration and recovery period, the experimental group and the control group were all normal activity. Eat quickly, hair close body luster, feces normal; There was no significant difference in body weight among the groups (P0.05). 2 Hematological indexes: the results showed that BCG-CpG-DNA mainly affected the total number of white blood cells, the absolute value of lymphocytes, the absolute value of neutrophils and the corresponding percentage. The results showed that BCG-CpG-DNA had no significant effect on the biochemical indexes of peripheral blood of mice. 4 gross anatomy and organ coefficient: gross anatomical observation. No obvious pathological changes were found in the main organs of the animals in each group. In BCG-CpG-DNA, the spleen weight coefficient of high dose group was higher than that of solvent control group (P < 0. 03). The spleen weight coefficient of the middle and high dose group was significantly higher than that of the solvent control group (P0. 009 0. 007 ~ 0. 007). In the recovery period, 21 days after immunity, there was no significant difference in spleen weight coefficient, liver weight coefficient, thymus weight coefficient and kidney weight coefficient between the low, middle and high dose groups of BCG-CpG-DNA (P0.05). (5) Local stimulation: during administration and convalescence, there was no apparent abnormal reaction in the administration site of the experimental group and the control group, and there was no abnormal response in the administration site after gross anatomy. (4) Immunologic function test: repeated administration of BCG-CpG-DNA; The doses of 0.7 mg and 3.5 mg were 1.75 mg and 3.5 mg, respectively. The main effects on the cellular immune function were as follows: enhancing the lymphocyte transformation function; Reducing the content of CD3 T cells in high dose group, increasing the proportion of IFN- 纬 and IL-4 secreting cells in the body, enhancing the killing ability of NK cells, there were significant differences between the different experimental groups and the control group (P0.05). After three weeks of convalescence, the index of change returned to normal. Conclusion: there were no obvious toxic reactions in abnormal toxicity test, acute toxicity test and 28 day toxicity test of BCG-CpG-DNA. Under the condition of repeated administration of BCG-CpG-DNA (0.7mg / kg) and 3.5mg (1.75mg), the mice showed enhanced immune function, but had no adverse effect on immune organs and immune function, and had no toxic effect and good safety.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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