抗生素预处理后口服Der p2重组耻垢分枝杆菌调节免疫应答的研究

发布时间：2018-01-14 04:33

本文关键词：抗生素预处理后口服Der p2重组耻垢分枝杆菌调节免疫应答的研究　出处：《第四军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景哮喘是一种全球范围内的发病率和死亡率逐渐上升的慢性疾病，严重威胁人类健康，降低患者生活质量，给病人及社会造成了巨大的经济负担。哮喘被认为是具有一定遗传学基础的个体，受环境中过敏原反复刺激导致体内免疫紊乱，以呼吸道慢性炎症和一系列临床症状为表现的一种病理状态。Th2优势的Th1/Th2平衡紊乱是过敏性哮喘的免疫学本质。而许多研究证明分枝杆菌采用多种免疫途径均可调节Th1/Th2平衡。本研究小组前期构建了Derp2-rBCG，经口服接种免疫小鼠可诱导Derp2特异性的Th1优势免疫应答。因分枝杆菌不是肠道固有菌，很难在肠道粘附定植，免疫效果不佳，而加大剂量则会引起肠道菌群失调和口咽部感染。研究发现肠道正常菌群能够竞争性抑制并排斥外来细菌的粘附和定植，起到一种生物屏障的作用。因此我们设想利用抗生素破坏这种生物屏障，从而提高分枝杆菌与肠道黏膜的亲和力进而增强其免疫调节作用。目的 1.利用电穿孔技术，构建以胞壁形式表达屋尘螨抗原Derp2的重组耻垢分枝杆菌疫苗pCW-Derp2-rMS。 2.给小鼠饮用低浓度抗生素溶液，观察其对肠道细菌数的影响，探索使用抗生素减少肠道菌群又不引起肠道菌群失调的最佳用药时间。 3.小鼠饮用抗生素溶液处理后，分别给予灌胃口服MS和pCW-Derp2-rMS免疫，比较两者对Th细胞免疫应答的调节。实验方法和结果 1.Derp2-rMS的构建及鉴定采用电穿孔技术，将鉴定好的Derp2质粒转化到耻垢分枝杆菌(MS)，经潮霉素抗性筛选rMS阳性克隆，通过PCR特异性扩增目的基因片段，鉴定目的基因在rMS中的表达，证实Derp2质粒成功转化入MS，构建了以胞壁形式表达外源蛋白的重组耻垢分枝杆菌（pCW-Derp2-rMS）。 2.口服抗生素对肠道菌群的影响小鼠在SPF级别饲养环境下，分别给予10％蔗糖(Suc)溶液、终浓度2mg/ml替硝唑(Tin)溶液、终浓度5mg/ml氨苄青霉素(Amp)溶液饮用，连续7天。分别在第3、5、7天处死部分小鼠，无菌状态下取小鼠回盲部肠内容物，匀浆稀释后涂LB琼脂培养基和BHI琼脂培养基平板(简易厌氧环境中)，培养并计数生长的菌落数目（CFU）。结果显示：Amp组小鼠肠内容物中需氧菌数目随着饮用氨苄青霉素溶液时间的增长而逐渐减少（CFU3dCFU5dCFU7d），Amp组小鼠肠内容物中需氧菌数量与同时间点Suc组、Tin组相比均有统计学差异（p㩳0.05）。Tin组小鼠肠内容物中厌氧菌数目也随着饮用替硝唑溶液时间的增长而逐渐减少（CFU3dCFU5dCFU7d），Tin组小鼠肠内容物中厌氧菌数量与同时间点Suc组、Amp组相比均有统计学差异（p㩳0.05）。饮用抗生素溶液7天后小鼠粪便变软变稀增多，肠道菌群失调症状明显，故考虑连续饮用抗生素溶液5天为宜。 3.抗生素预处理后口服MS/pCW-Derp2-rMS调节BALB/c小鼠Th细胞免疫应答的研究抗生素如前预处理小鼠后，每组分为3组，分别给予100μlMS、pCW-Derp2-rMS(1×1010CFU/ml)和甘油灌胃免疫小鼠。于末次免疫后4周、8周分别收集外周血血清和脾脏淋巴细胞培养上清，，用相应ELISA试剂盒检测其中IFN-γ、IL-2、IL-4水平。于8周时收集外周血和脾脏淋巴细胞，用流式细胞术检测其中Th1/Th2的分布。结果表明：氨苄青霉素或替硝唑处理后可使Th1型细胞因子IFN-γ、IL-2水平下降，Th2型细胞因子IL-4水平轻微升高，使外周血和脾脏淋巴细胞中Th1型细胞所占比例下降，即可致Th1型免疫反应呈下降趋势。口服MS或pCW-Derp2-rMS可使Th1型细胞因子IFN-γ、IL-2水平升高（P㩳0.05），Th2型细胞因子IL-4水平轻微下降，使外周血和脾脏淋巴细胞中Th1型细胞所占比例升高，Th2型细胞所占比例下降，产生Th1优势的免疫应答，且这种应答是Derp2特异性的。抗生素预处理后接种pCW-Derp2-rMS与接种MS相比可使Th1型细胞因子IFN-γ、IL-2水平上升，外周血和脾脏淋巴细胞中Th1型细胞所占比例升高，即可使Th1型免疫反应呈升高趋势，且这种趋势是Derp2特异性的。结论： 1.小鼠饮用低浓度抗生素溶液，连续饮用5天为减少肠道菌群又不引起肠道菌群失调的最佳时间。 2.经饮用抗生素溶液预处理的BALB/c小鼠分别给予MS和rMS口服免疫，观察其对小鼠免疫应答的影响。结果显示氨苄青霉素和替硝唑可使Th1型免疫反应呈下降趋势；抗生素预处理后口服pCW-Derp2-rMS与口服MS相比可使Th1型免疫应答呈上升趋势，且这种趋势是Derp2特异性的。
[Abstract]:background
Asthma is a chronic disease morbidity and mortality in a global scope gradually increased, a serious threat to human health, reduce the quality of life of patients, resulting in a huge economic burden to patients and society. Asthma is thought to have some genetic basis of the individual, affected by environmental allergens in repeated stimulation resulted in the immune disorders, Th1/Th2 balance disorder of respiratory tract chronic inflammation and a series of clinical symptoms as a pathological state of.Th2 is the essence of immunological advantage of allergic asthma. Many studies have shown that Mycobacterium using a variety of immune pathway can regulate the balance of Th1/Th2. The research group pre constructed Derp2-rBCG, Th1 immune response after oral immunization of mice induced by Derp2. Specific for Mycobacterium not gut commensal bacteria, it is difficult in the intestinal colonization, the immune effect is poor, which will lead to increase the dose Since the dysbacteriosis and oropharyngeal bacteria infection. The study found that the adhesion and colonization of normal intestinal flora can competitively inhibit and exclude foreign bacteria, as a biological barrier. Therefore we envision the use of antibiotics to destroy this biological barrier, so as to improve the mycobacterial and intestinal mucosal vaccines can enhance the immune function.
objective
1. using electroporation technique to construct recombinant Mycobacterium tuberculosis vaccine pCW-Derp2-rMS. with cell wall form of house dust mite antigen Derp2
2., mice were treated with low concentration of antibiotic solution, and their effects on the number of intestinal bacteria were observed. The best time to use antibiotics to reduce the intestinal flora and not cause intestinal flora imbalance was explored.
After the 3. mice were treated with antibiotic solution, MS and pCW-Derp2-rMS immunization were administered orally to the stomach, and the immunological response of the two to Th cells was compared.
Experimental methods and results
Construction and identification of 1.Derp2-rMS
Electroporation technology was used to transform the identified Derp2 plasmid into Mycobacterium MS. RMS positive clones were screened by hygromycin resistance, and the target gene fragments were amplified by PCR.
The expression of the target gene in rMS confirmed that Derp2 plasmid was successfully transformed into MS, and a recombinant Mycobacterium tuberculosis (pCW-Derp2-rMS) expressing foreign protein in cell wall form was constructed.
2. effect of oral antibiotics on intestinal microflora
The mice at the SPF level and breeding environment, were treated with 10% sucrose (Suc) solution, the final concentration of 2mg/ml tinidazole (Tin) solution, 5mg/ml final concentration of ampicillin (Amp) solution for drinking, for 7 consecutive days. Some of the mice were sacrificed at day 3,5,7 respectively in mice under sterile conditions, ileocecus intestinal contents, homogenate dilution after coated with LB agar and BHI agar medium (simple anaerobic environment), and the number of colony counting culture growth (CFU). The results showed that the number of aerobic bacteria in Amp mice intestinal contents decreased with increasing time of drinking ampicillin solution (CFU3dCFU5dCFU7d), the number of aerobic bacteria in Amp mice intestinal contents and at the same time point of Suc group, there were statistic differences in Tin group (P? 0.05) the number of anaerobic bacteria in.Tin mice intestinal contents in drinking with tinidazole solution growth of time gradually decreased (CFU3dCFU5dCFU7d), Tin group The number of anaerobes in intestinal contents of mice was statistically different from that in group Suc and group Amp (P? 0.05). After 7 days, the feces became soft and thinner, and the symptoms of intestinal flora imbalance were obvious. Therefore, it is advisable to take 5 days of continuous antibiotic solution.
Study on Th cell immune response in BALB/c mice by oral MS/pCW-Derp2-rMS after pretreatment with 3. antibiotics
Antibiotics such as pretreatment of mice in each group were divided into 3 groups, were given 100 lMS, pCW-Derp2-rMS (1 x 1010CFU/ml) and glycerol intragastric immunization mice. In 4 weeks after the final immunization, 8 weeks were collected from peripheral blood serum and spleen lymphocyte culture supernatant, with the corresponding ELISA kit with IFN- gamma, IL-2, IL-4 level. At week 8 collected peripheral blood and spleen lymphocytes were detected by flow cytometry and Th1/Th2 distribution. The results showed that: ampicillin or tinidazole after treatment can make Th1 type cytokines IFN-, IL-2 decreased, Th2 type cytokine IL-4 levels increased slightly, which accounted for type Th1 cells in peripheral blood and spleen lymphocyte ratio decreased, decreased Th1 type immune response can be induced by oral administration of MS or pCW-Derp2-rMS. The Th1 type cytokines IFN-, elevated levels of IL-2 (P? 0.05), Th2 type cytokines IL-4 level decreased slightly, the peripheral Th1 increased the proportion of cells in blood and spleen lymphocytes, Th2 cell proportion, immune responses generated Th1 advantage, and this response is Derp2 specific. Antibiotics after pretreatment with pCW-Derp2-rMS and MS inoculation compared to make Th1 cytokines IFN-, IL-2 levels increased, Th1 cells the proportion of increase in peripheral blood and spleen lymphocytes, the immune response to Th1 is increasing, and this trend is Derp2 specific.
Conclusion:
1. mice drinking low concentration of antibiotic solution for 5 days is the best time to reduce intestinal flora and do not cause intestinal flora imbalance.
2. pretreated drinking antibiotic solution BALB/c mice were given MS and rMS oral immunization, and observe its effects on the immune response in mice. The results showed that ampicillin and Tinidazole can make the immune response to Th1 decreased; compared with antibiotics after pretreatment of oral pCW-Derp2-rMS and oral administration of MS can make the Th1 type immune response showed an upward trend, and this trend is Derp2 specific.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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