本文關(guān)鍵詞：抗生素預(yù)處理后口服Der p2重組恥垢分枝桿菌調(diào)節(jié)免疫應(yīng)答的研究　出處：《第四軍醫(yī)大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 抗生素 腸道菌群 Der p2 重組恥垢分枝桿菌

【摘要】：背景 哮喘是一種全球范圍內(nèi)的發(fā)病率和死亡率逐漸上升的慢性疾病，嚴(yán)重威脅人類健康，降低患者生活質(zhì)量，給病人及社會(huì)造成了巨大的經(jīng)濟(jì)負(fù)擔(dān)。哮喘被認(rèn)為是具有一定遺傳學(xué)基礎(chǔ)的個(gè)體，受環(huán)境中過(guò)敏原反復(fù)刺激導(dǎo)致體內(nèi)免疫紊亂，以呼吸道慢性炎癥和一系列臨床癥狀為表現(xiàn)的一種病理狀態(tài)。Th2優(yōu)勢(shì)的Th1/Th2平衡紊亂是過(guò)敏性哮喘的免疫學(xué)本質(zhì)。而許多研究證明分枝桿菌采用多種免疫途徑均可調(diào)節(jié)Th1/Th2平衡。本研究小組前期構(gòu)建了Derp2-rBCG，經(jīng)口服接種免疫小鼠可誘導(dǎo)Derp2特異性的Th1優(yōu)勢(shì)免疫應(yīng)答。因分枝桿菌不是腸道固有菌，很難在腸道粘附定植，免疫效果不佳，而加大劑量則會(huì)引起腸道菌群失調(diào)和口咽部感染。研究發(fā)現(xiàn)腸道正常菌群能夠競(jìng)爭(zhēng)性抑制并排斥外來(lái)細(xì)菌的粘附和定植，起到一種生物屏障的作用。因此我們?cè)O(shè)想利用抗生素破壞這種生物屏障，從而提高分枝桿菌與腸道黏膜的親和力進(jìn)而增強(qiáng)其免疫調(diào)節(jié)作用。 目的 1.利用電穿孔技術(shù)，構(gòu)建以胞壁形式表達(dá)屋塵螨抗原Derp2的重組恥垢分枝桿菌疫苗pCW-Derp2-rMS。 2.給小鼠飲用低濃度抗生素溶液，觀察其對(duì)腸道細(xì)菌數(shù)的影響，探索使用抗生素減少腸道菌群又不引起腸道菌群失調(diào)的最佳用藥時(shí)間。 3.小鼠飲用抗生素溶液處理后，分別給予灌胃口服MS和pCW-Derp2-rMS免疫，比較兩者對(duì)Th細(xì)胞免疫應(yīng)答的調(diào)節(jié)。 實(shí)驗(yàn)方法和結(jié)果 1.Derp2-rMS的構(gòu)建及鑒定 采用電穿孔技術(shù)，將鑒定好的Derp2質(zhì)粒轉(zhuǎn)化到恥垢分枝桿菌(MS)，經(jīng)潮霉素抗性篩選rMS陽(yáng)性克隆，通過(guò)PCR特異性擴(kuò)增目的基因片段，鑒定 目的基因在rMS中的表達(dá)，證實(shí)Derp2質(zhì)粒成功轉(zhuǎn)化入MS，構(gòu)建了以胞壁形式表達(dá)外源蛋白的重組恥垢分枝桿菌（pCW-Derp2-rMS）。 2.口服抗生素對(duì)腸道菌群的影響 小鼠在SPF級(jí)別飼養(yǎng)環(huán)境下，分別給予10％蔗糖(Suc)溶液、終濃度2mg/ml替硝唑(Tin)溶液、終濃度5mg/ml氨芐青霉素(Amp)溶液飲用，連續(xù)7天。分別在第3、5、7天處死部分小鼠，無(wú)菌狀態(tài)下取小鼠回盲部腸內(nèi)容物，勻漿稀釋后涂LB瓊脂培養(yǎng)基和BHI瓊脂培養(yǎng)基平板(簡(jiǎn)易厭氧環(huán)境中)，培養(yǎng)并計(jì)數(shù)生長(zhǎng)的菌落數(shù)目（CFU）。結(jié)果顯示：Amp組小鼠腸內(nèi)容物中需氧菌數(shù)目隨著飲用氨芐青霉素溶液時(shí)間的增長(zhǎng)而逐漸減少（CFU3dCFU5dCFU7d），Amp組小鼠腸內(nèi)容物中需氧菌數(shù)量與同時(shí)間點(diǎn)Suc組、Tin組相比均有統(tǒng)計(jì)學(xué)差異（p㩳0.05）。Tin組小鼠腸內(nèi)容物中厭氧菌數(shù)目也隨著飲用替硝唑溶液時(shí)間的增長(zhǎng)而逐漸減少（CFU3dCFU5dCFU7d），Tin組小鼠腸內(nèi)容物中厭氧菌數(shù)量與同時(shí)間點(diǎn)Suc組、Amp組相比均有統(tǒng)計(jì)學(xué)差異（p㩳0.05）。飲用抗生素溶液7天后小鼠糞便變軟變稀增多，腸道菌群失調(diào)癥狀明顯，故考慮連續(xù)飲用抗生素溶液5天為宜。 3.抗生素預(yù)處理后口服MS/pCW-Derp2-rMS調(diào)節(jié)BALB/c小鼠Th細(xì)胞免疫應(yīng)答的研究 抗生素如前預(yù)處理小鼠后，每組分為3組，分別給予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周分別收集外周血血清和脾臟淋巴細(xì)胞培養(yǎng)上清，，用相應(yīng)ELISA試劑盒檢測(cè)其中IFN-γ、IL-2、IL-4水平。于8周時(shí)收集外周血和脾臟淋巴細(xì)胞，用流式細(xì)胞術(shù)檢測(cè)其中Th1/Th2的分布。結(jié)果表明：氨芐青霉素或替硝唑處理后可使Th1型細(xì)胞因子IFN-γ、IL-2水平下降，Th2型細(xì)胞因子IL-4水平輕微升高，使外周血和脾臟淋巴細(xì)胞中Th1型細(xì)胞所占比例下降，即可致Th1型免疫反應(yīng)呈下降趨勢(shì)。口服MS或pCW-Derp2-rMS可使Th1型細(xì)胞因子IFN-γ、IL-2水平升高（P㩳0.05），Th2型細(xì)胞因子IL-4水平輕微下降，使外周血和脾臟淋巴細(xì)胞中Th1型細(xì)胞所占比例升高，Th2型細(xì)胞所占比例下降，產(chǎn)生Th1優(yōu)勢(shì)的免疫應(yīng)答，且這種應(yīng)答是Derp2特異性的�？股仡A(yù)處理后接種pCW-Derp2-rMS與接種MS相比可使Th1型細(xì)胞因子IFN-γ、IL-2水平上升，外周血和脾臟淋巴細(xì)胞中Th1型細(xì)胞所占比例升高，即可使Th1型免疫反應(yīng)呈升高趨勢(shì)，且這種趨勢(shì)是Derp2特異性的。 結(jié)論： 1.小鼠飲用低濃度抗生素溶液，連續(xù)飲用5天為減少腸道菌群又不引起腸道菌群失調(diào)的最佳時(shí)間。 2.經(jīng)飲用抗生素溶液預(yù)處理的BALB/c小鼠分別給予MS和rMS口服免疫，觀察其對(duì)小鼠免疫應(yīng)答的影響。結(jié)果顯示氨芐青霉素和替硝唑可使Th1型免疫反應(yīng)呈下降趨勢(shì)；抗生素預(yù)處理后口服pCW-Derp2-rMS與口服MS相比可使Th1型免疫應(yīng)答呈上升趨勢(shì)，且這種趨勢(shì)是Derp2特異性的。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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