發(fā)布時(shí)間：2018-11-08 10:13

【摘要】： 目的:研究BCG-CpG-DNA的免疫毒性作用,為BCG-CpG-DNA臨床前安全性評(píng)價(jià)提供依據(jù)。 方法:采用異常毒性試驗(yàn)、急性毒性試驗(yàn)、28天毒性試驗(yàn)初步觀察BCG-CpG-DNA毒性;并通過(guò)計(jì)算免疫器官臟器系數(shù),檢測(cè)淋巴細(xì)胞轉(zhuǎn)化能力、細(xì)胞因子釋放(Elospot法)、T細(xì)胞亞群變化(免疫熒光法)、NK細(xì)胞殺傷活性(LDH法),外周血抗雙鏈DNA自身抗體水平(Elisa法),與溶劑對(duì)照組比較,進(jìn)一步觀察BCG-CpG-DNA重復(fù)給藥對(duì)小鼠免疫功能的影響。 結(jié)果:(1)異常毒性試驗(yàn):實(shí)驗(yàn)期間,所有動(dòng)物一般行為活動(dòng)、外觀體征、進(jìn)食和飲水活動(dòng)等均正常;所有動(dòng)物全部健康存活;動(dòng)物均有體重增加;大體解剖肉眼觀察各臟器均無(wú)異常。(2)急性毒性試驗(yàn):實(shí)驗(yàn)期間,所有動(dòng)物一般行為活動(dòng)、外觀體征、進(jìn)食和飲水活動(dòng)等均正常;所有動(dòng)物全部健康存活;動(dòng)物均有體重增加;大體解剖,臟器無(wú)病變,各組脾重?zé)o統(tǒng)計(jì)學(xué)差異(P0.05)。(3)28天毒性試驗(yàn):①外觀體征、體重觀察:在給藥期間及恢復(fù)期,實(shí)驗(yàn)組和對(duì)照組動(dòng)物均活動(dòng)正常,進(jìn)食敏捷、毛貼身有光澤,糞便正常;動(dòng)物體重各組間無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。②血液學(xué)指標(biāo):4次檢測(cè)結(jié)果表明,BCG-CpG-DNA主要影響小鼠白細(xì)胞總數(shù)、淋巴細(xì)胞絕對(duì)值、中性粒細(xì)胞絕對(duì)值及相應(yīng)的百分比,對(duì)其他指標(biāo)無(wú)明顯影響。③血生化學(xué)指標(biāo):4次檢測(cè)結(jié)果表明,BCG-CpG-DNA對(duì)小鼠外周血生化指標(biāo)無(wú)明顯影響。④大體解剖與臟器系數(shù):大體解剖觀察,各組動(dòng)物的主要器官均未見明顯病理性改變;免疫中期即免疫后10天,BCG-CpG-DNA中、高劑量組脾臟重量系數(shù)高于溶劑對(duì)照組,有顯著性差異(P=0.03,0.05);免疫末期即末次免疫后3天,BCG-CpG-DNA低、中、高劑量組脾臟重量系數(shù)高于溶劑對(duì)照組,有顯著性差異(P=0.009,0.007,0.007);恢復(fù)期即免后21天,BCG-CpG-DNA低、中、高劑量組脾臟重量系數(shù)、肝重系數(shù)、胸腺重系數(shù)和腎臟重系數(shù),各劑量組和溶劑對(duì)照組均無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);⑤局部刺激反應(yīng):在給藥期間及恢復(fù)期,實(shí)驗(yàn)組和對(duì)照組動(dòng)物的給藥部位表觀無(wú)異常反應(yīng),大體解剖后,給藥部位也無(wú)異常反應(yīng)。(4)免疫功能檢測(cè):BCG-CpG-DNA重復(fù)給藥,劑量分別達(dá)到0.7mg、1.75mg和3.5mg,對(duì)細(xì)胞免疫功能的影響主要有:增強(qiáng)淋巴細(xì)胞轉(zhuǎn)化功能;降低中、高劑量組CD3+T細(xì)胞含量;提高體內(nèi)分泌IFN-γ和IL-4的細(xì)胞數(shù)二者的比例;增強(qiáng)NK細(xì)胞殺傷能力,不同實(shí)驗(yàn)組與對(duì)照組差異均有顯著意義(P0.05);經(jīng)過(guò)三周恢復(fù)期,變化的指標(biāo)均恢復(fù)正常。 結(jié)論:BCG-CpG-DNA在異常毒性試驗(yàn)、急性毒性試驗(yàn)、28天毒性試驗(yàn)均未見明顯毒性反應(yīng);BCG-CpG-DNA重復(fù)給藥累計(jì)達(dá)0.7mg、1.75mg和3.5mg的情況下,小鼠表現(xiàn)為免疫功能增強(qiáng),但對(duì)免疫器官和免疫功能無(wú)不良影響,無(wú)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- 緯 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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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