本文選題：大黃素 + 布魯菌��； 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:通過構(gòu)建小鼠感染布魯菌模型,觀察單體大黃素對布魯菌感染小鼠的保護(hù)效果,體外培養(yǎng)小鼠巨噬細(xì)胞,研究大黃素對布魯菌侵染小鼠巨噬細(xì)胞功能的調(diào)節(jié),為尋找新的藥物治療布魯菌病提供理論和實(shí)驗(yàn)依據(jù)。方法:1、體內(nèi)實(shí)驗(yàn):采用VITEK Compact 2全自動(dòng)微生物分析系統(tǒng)、16SrDNA分子生物學(xué)方法,對實(shí)驗(yàn)室保存布魯菌株進(jìn)行再次鑒定;以不同濃度菌液注射小鼠,通過SAT和脾臟載菌初篩布魯菌感染小鼠的濃度,然后以初篩濃度腹腔注射小鼠,構(gòu)建布魯菌感染小鼠模型;取60只SPF級BALB/c雌性小鼠隨機(jī)平均分成6組,設(shè)為8mg/m L大黃素組、4mg/m L大黃素組、2mg/m L大黃素組、多西環(huán)素組、PBS組、空白組,進(jìn)行灌胃處理(除空白組外)后,通過腹腔菌落計(jì)數(shù)、體重指數(shù)、肝脾指數(shù)及組織病理的變化評價(jià)大黃素對布魯菌感染小鼠的保護(hù)效果。2、體外實(shí)驗(yàn):取BALB/c小鼠分離骨髓細(xì)胞,加入MG-CSF誘導(dǎo)分化培養(yǎng),用小鼠F4/80抗體、CD11b抗體標(biāo)記細(xì)胞,流式檢測骨髓細(xì)胞分化為巨噬細(xì)胞(MΦ)的比例;通過MTT法檢測不同濃度大黃素對小鼠MΦ的存活率影響,同時(shí)以多西環(huán)素為對照,比較兩種藥物的IC50;用布魯菌侵染小鼠巨噬細(xì)胞(MΦ和細(xì)菌比例為100:1)培養(yǎng)4 h,然后加入大黃素作用后,培養(yǎng)1 h、6 h、12 h、24 h、48 h,通過菌落計(jì)數(shù)法檢測大黃素對MΦ內(nèi)布魯菌存活的影響,然后用q RT-PCR法檢測大黃素對侵染布魯菌的MΦ分泌TNF-α、IL-6、IFN-γ基因轉(zhuǎn)錄的影響,同時(shí)用ELISA法檢測各組MΦ培養(yǎng)上清中TNF-α、IL-6、IFN-γ的含量。結(jié)果:1、體內(nèi)實(shí)驗(yàn)結(jié)果:經(jīng)過細(xì)菌生化、16SrDNA鑒定結(jié)果一致,均證實(shí)該實(shí)驗(yàn)菌株為羊布魯菌;羊布魯菌感染小鼠的初篩濃度為1×105CFU,并且該濃度布魯菌感染小鼠模型構(gòu)建成功;大黃素對布魯菌感染小鼠模型的影響:8mg/m L和4mg/m L大黃素組腹腔載菌數(shù)量的lg CFU值分別為(4.023±0.199)(4.771±0.498),明顯低于PBS組(5.161±0.501);2mg/m L、4mg/m L、8mg/m L大黃素組小鼠體重指數(shù)明顯高于PBS組,而脾臟系數(shù)明顯低于PBS組;8mg/m L大黃素組小鼠的肝臟系數(shù)明顯低于PBS組,且與多西環(huán)素組無明顯差異;與PBS組比較,2mg/m L、4mg/m L、8mg/m L大黃素組小鼠脾臟均有不同程度的改善,組織病理結(jié)構(gòu)排列規(guī)則,白髓、紅髓分界較清楚;8mg/m L大黃素組小鼠肝細(xì)胞分界清楚,排列正常。2、體外實(shí)驗(yàn)結(jié)果:培養(yǎng)第8天,骨髓細(xì)胞誘導(dǎo)分化為巨噬細(xì)胞的比例為91.28%;大黃素的IC50(608.4μg/m L)明顯高于多西環(huán)素的IC50(225.5μg/m L);菌落計(jì)數(shù)結(jié)果:6 h、12 h、24 h、48 h大黃素組lg CFU值均顯著低于空白組,大黃素組間比較,24 h大黃素組的lg CFU值最低,且低于多西環(huán)素組;q RT-PCR結(jié)果:24h大黃素組TNF-α、IL-6、IFN-γ表達(dá)水平比其他組高(1 h、6 h、12 h、48 h);相同時(shí)間點(diǎn),大黃素組TNF-α基因表達(dá)水平均高于布魯菌組。除1 h、12 h大黃素組外,其余各組IL-6表達(dá)比布魯菌組高;6 h、12 h、24 h大黃素組比布魯菌組的IFN-γ基因表達(dá)明顯上調(diào);12h大黃素組的IFN-γ基因表達(dá)(5.4370±0.458)最高,且與24 h大黃素組(5.3495±0.336)比較無明顯統(tǒng)計(jì)學(xué)差異。ELISA結(jié)果:與布魯菌組比較,6 h、12 h、24 h大黃素組TNF-α、IL-6、IFN-γ含量明顯增加,24h大黃素組TNF-α、IL-6、IFN-γ含量最高;而12 h大黃素組IFN-γ含量(74.233±4.416)與24 h大黃素組(78.328±8.932)比較無統(tǒng)計(jì)學(xué)意義;結(jié)論:大黃素對感染布魯菌小鼠具有保護(hù)小鼠,并呈濃度依賴性,其作用機(jī)制與增加TNF-α、IL-6、IFN-γ和小鼠巨噬細(xì)胞殺傷布魯菌的能力有關(guān)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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R516.7

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