大黄素对布鲁菌感染小鼠的保护效果及可能机制研究

发布时间：2018-07-04 11:00

本文选题：大黄素 + 布鲁菌　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:通过构建小鼠感染布鲁菌模型,观察单体大黄素对布鲁菌感染小鼠的保护效果,体外培养小鼠巨噬细胞,研究大黄素对布鲁菌侵染小鼠巨噬细胞功能的调节,为寻找新的药物治疗布鲁菌病提供理论和实验依据。方法:1、体内实验:采用VITEK Compact 2全自动微生物分析系统、16SrDNA分子生物学方法,对实验室保存布鲁菌株进行再次鉴定;以不同浓度菌液注射小鼠,通过SAT和脾脏载菌初筛布鲁菌感染小鼠的浓度,然后以初筛浓度腹腔注射小鼠,构建布鲁菌感染小鼠模型;取60只SPF级BALB/c雌性小鼠随机平均分成6组,设为8mg/m L大黄素组、4mg/m L大黄素组、2mg/m L大黄素组、多西环素组、PBS组、空白组,进行灌胃处理(除空白组外)后,通过腹腔菌落计数、体重指数、肝脾指数及组织病理的变化评价大黄素对布鲁菌感染小鼠的保护效果。2、体外实验:取BALB/c小鼠分离骨髓细胞,加入MG-CSF诱导分化培养,用小鼠F4/80抗体、CD11b抗体标记细胞,流式检测骨髓细胞分化为巨噬细胞(MΦ)的比例;通过MTT法检测不同浓度大黄素对小鼠MΦ的存活率影响,同时以多西环素为对照,比较两种药物的IC50;用布鲁菌侵染小鼠巨噬细胞(MΦ和细菌比例为100:1)培养4 h,然后加入大黄素作用后,培养1 h、6 h、12 h、24 h、48 h,通过菌落计数法检测大黄素对MΦ内布鲁菌存活的影响,然后用q RT-PCR法检测大黄素对侵染布鲁菌的MΦ分泌TNF-α、IL-6、IFN-γ基因转录的影响,同时用ELISA法检测各组MΦ培养上清中TNF-α、IL-6、IFN-γ的含量。结果:1、体内实验结果:经过细菌生化、16SrDNA鉴定结果一致,均证实该实验菌株为羊布鲁菌;羊布鲁菌感染小鼠的初筛浓度为1×105CFU,并且该浓度布鲁菌感染小鼠模型构建成功;大黄素对布鲁菌感染小鼠模型的影响:8mg/m L和4mg/m L大黄素组腹腔载菌数量的lg CFU值分别为(4.023±0.199)(4.771±0.498),明显低于PBS组(5.161±0.501);2mg/m L、4mg/m L、8mg/m L大黄素组小鼠体重指数明显高于PBS组,而脾脏系数明显低于PBS组;8mg/m L大黄素组小鼠的肝脏系数明显低于PBS组,且与多西环素组无明显差异;与PBS组比较,2mg/m L、4mg/m L、8mg/m L大黄素组小鼠脾脏均有不同程度的改善,组织病理结构排列规则,白髓、红髓分界较清楚;8mg/m L大黄素组小鼠肝细胞分界清楚,排列正常。2、体外实验结果:培养第8天,骨髓细胞诱导分化为巨噬细胞的比例为91.28%;大黄素的IC50(608.4μg/m L)明显高于多西环素的IC50(225.5μg/m L);菌落计数结果:6 h、12 h、24 h、48 h大黄素组lg CFU值均显著低于空白组,大黄素组间比较,24 h大黄素组的lg CFU值最低,且低于多西环素组;q RT-PCR结果:24h大黄素组TNF-α、IL-6、IFN-γ表达水平比其他组高(1 h、6 h、12 h、48 h);相同时间点,大黄素组TNF-α基因表达水平均高于布鲁菌组。除1 h、12 h大黄素组外,其余各组IL-6表达比布鲁菌组高;6 h、12 h、24 h大黄素组比布鲁菌组的IFN-γ基因表达明显上调;12h大黄素组的IFN-γ基因表达(5.4370±0.458)最高,且与24 h大黄素组(5.3495±0.336)比较无明显统计学差异。ELISA结果:与布鲁菌组比较,6 h、12 h、24 h大黄素组TNF-α、IL-6、IFN-γ含量明显增加,24h大黄素组TNF-α、IL-6、IFN-γ含量最高;而12 h大黄素组IFN-γ含量(74.233±4.416)与24 h大黄素组(78.328±8.932)比较无统计学意义;结论:大黄素对感染布鲁菌小鼠具有保护小鼠,并呈浓度依赖性,其作用机制与增加TNF-α、IL-6、IFN-γ和小鼠巨噬细胞杀伤布鲁菌的能力有关系。
[Abstract]:Objective: To observe the protective effect of emodin on Brucella infection in mice, to observe the protective effect of the mono emodin to Brucella infection, in vitro culture of mouse macrophages, to study the regulation of emodin on the function of Brucella infection in mice, and to provide a theoretical and experimental basis for finding new drugs to treat brucellosis. Method: 1, in vivo experiment: VITEK Compact 2 automatic microbiological analysis system and 16SrDNA molecular biology method were used to re identify Brucella strains in laboratory. The mice were injected with different concentration of bacteria solution, the mice were infected with Brucella infected by SAT and spleen, then the mice were intraperitoneally injected with the initial screening concentration, and the mice model of Brucella infection was constructed. 60 SPF grade BALB/c female mice were randomly divided into 6 groups, including the 8mg/m L emodin group, the 4mg/m L emodin group, the 2mg/m L emodin group, the doxycycline group, the PBS group and the blank group. After the gastric colony count, the body mass index, the liver spleen index and the histopathology, the infection of Brucella infection was evaluated by the peritoneal colony count, body mass index, liver spleen index and histopathological changes. The protective effect of mice was.2 in vitro: in vitro, the bone marrow cells were isolated from BALB/c mice, and MG-CSF was added to induce differentiation and culture. The proportion of F4/80 antibody, CD11b antibody was labeled with CD11b, and the proportion of bone marrow cells differentiated into macrophage (M diameter) by flow cytometry; the survival rate of M diameter in mice was detected by MTT method, and doxycycline was also used. For comparison, the IC50 of two drugs was compared, and Brucella infected mice macrophages (M diameter and 100:1) to cultivate 4 h. Then after adding emodin, 1 h, 6 h, 12 h, 24 h, 48 h were cultured. The effect of emodin on the survival of M I was detected by colony counting method, and then q RT-PCR method was used to detect the infection of Brucella infected by emodin The effect of TNF- alpha, IL-6, IFN- gamma gene transcription was secreted, and the content of TNF- alpha, IL-6, IFN- gamma in the culture supernatant of each group was detected by ELISA. Results: 1, the results of the experiment in vivo showed that the experimental strain was consistent with the bacterial biochemistry, and all confirmed that the experimental strain was of Brucella. The initial concentration of the experimental strain was 1 x 105CFU, and the concentration was strong. The effect of emodin on the mice model of Brucella infection: the LG CFU value of 8mg/m L and 4mg/m L emodin group was (4.023 + 0.199) (4.771 + 0.498), obviously lower than that in PBS group (5.161 + 0.501), 2mg/m L, 4mg /m L, and the body mass index of the emodin group was significantly higher than that of the group. The spleen coefficient was significantly lower than that of the PBS group; the liver coefficient of the 8mg/m L emodin group was significantly lower than that of the PBS group, and there was no significant difference from that of the doxycycline group. Compared with the PBS group, the spleen of the mice of 2mg/m L, 4mg/m L and 8mg/m L emodin had different degrees of improvement, the pathological structure of the tissue, the white pulp and the red pulp demarcation were clear; The mice liver cells had a clear demarcation and arranged normal.2. The results of in vitro experiment: eighth days of culture, the proportion of bone marrow cells induced to macrophage was 91.28%, and the IC50 (608.4 mu g/m L) of emodin was significantly higher than that of IC50 (225.5 mu g/m L), and the colony count results: 6 h, 12 h, 24 h, and 48 h emodin group were significantly lower than the blank group, rhubarb group, and rhubarb. The LG CFU value of the 24 h emodin group was the lowest and lower than that of the multi cyclin group. Q RT-PCR results: the expression level of TNF- alpha, IL-6, IFN- gamma in the 24h emodin group was higher than that of the other groups (1 h, 6 h, 12 h, 48). Brucella group was high, 6 h, 12 h, and 24 h emodin group IFN- Y gene expression was obviously up-regulated, 12h emodin group IFN- gamma expression (5.4370 + 0.458) was the highest, and 24 h emodin group (5.3495 + 0.336) had no significant statistical difference.ELISA results: compared with the Brucella group, 6 h, 12 h, 24 h emodin group TNF- alpha, 24 h emodin content content The content of 24h emodin group TNF- alpha, IL-6, IFN- gamma is the highest, but the content of IFN- gamma in 12 h emodin group (74.233 + 4.416) and 24 h emodin group (78.328 + 8.932) has no statistical significance. Conclusion: emodin has a protective effect on mice infected with Brucella, and is dependent on the concentration of TNF- a, IL-6, IFN- gamma and mice. The ability of phagocytosis to kill Brucella is related.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R516.7

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