幽门螺杆菌感染小鼠呼气试验模型建立及其影响因素分析

发布时间：2018-11-23 14:47

【摘要】：目的：通过建立幽门螺杆菌小鼠13C-UBT、14C-UBT模型，探究构建小鼠呼气模型的条件和方法，分析影响幽门螺杆菌小鼠呼气模型仪器、条件及生物因素，为研究幽门螺杆菌动物模型提供一种无创性实时检测方法，客观评价HP疫苗在小鼠模型中的免疫保护效果以及对H.pylori致病性的深入研究提供一种简单、快速、便宜而且可靠的诊断手段。方法：利用6~8周龄BABL/c小鼠构建小鼠呼气模型的条件方法：通过对小鼠饲喂不同13C-Urea、14C-Urea药物剂量，，间隔不同时间检测，建立13C-UBT、14C-UBT实验模型；饲喂不同H.pylori菌量，通过相同时间检测，探究13C-UBT在相同检测时间中饲喂不同菌量的小鼠的呼气关系；小鼠饲喂不同H.pylori菌量，不同时间检测，探究13C-UBT、14C-UBT中不同菌量和时间的相互关系。探究影响幽门螺杆菌小鼠呼气试验模型的生物因素，确定影响呼气试验的小鼠体内菌种的组织定位，分离培养并筛选影响呼气试验的小鼠体内尿素酶阳性细菌；对分离出的尿素酶阳性细菌进行飞行质谱鉴定及16srDNA-PCR扩增测序，两种方法确定菌株菌种；对分离出的尿素酶阳性细菌进行尿素酶活性进行定性定量的检测。结果：小鼠13C-UBT、14C-UBT同一呼气时间检测数值与小鼠饲喂药量成正相关；同一饲喂药量中随着饲喂药物后检测时间延长，衡量呼气的检测数值增大，一定时间后达到最大值，此后随时间的延长，检测数值减小。成功建立幽门螺杆菌小鼠13C-UBT、14C-UBT模型：13C-UBT模型中选用0.1mg-13C-Urea药量，给予小鼠300ml活动空间，饲喂药物后呼气10min，收集250ml气体进行检测；14C-UBT呼气中选用0.556KBq-14C-Urea药量，给予200ml活动空间，饲喂药物后呼气10min，收集150ml气体进行检测；饲喂小鼠不同菌量H.pylori进行相同呼气时间的检测，13C-Urea、14C-Urea呼气试验中呼气数值均随着H.pylori菌量的升高而增大；饲喂小鼠不同H.pylori菌量，13C-UBT、14C-UBT各曲线走势相同，低浓度的菌量受到小鼠体内因素的影响大，1×109CFU的H.pylori菌量有很好的增幅曲线和趋势。成功对小鼠呼气试验中参与细菌进行定位，参与影响呼气试验的细菌位于胃及小肠组织，筛选出155株尿素酶阳性细菌；利用飞行质谱及16srDNA-PCR扩增测序两种方法确定菌株菌种，大部分为大肠杆菌，剩余以侵肺巴斯德菌居多，也检测出缓慢葡萄球菌、流感嗜血杆菌、链球菌等细菌；通过对分离出的细菌进行尿素酶活性定性定量测定，分离出的细菌尿素酶活性远远小于H.pylori。结论：（1）成功建立H.pylori小鼠13C-UBT、14C-UBT模型，能够对小鼠进行无创性实时检测；（2）小鼠13C-UBT、14C-UBT模型中13C-Urea、14C-Urea饲喂药量、 H.pylori菌量、呼气检测时间均与检测数值相关。其中H.pylori低浓度菌量受小鼠体内因素的影响大，H.pylori菌量为1×109CFU受到的影响小；（3）虽分离出的影响呼气试验的细菌尿素酶活性小于H.pylori，但在进行小鼠呼气试验中仍要注意并尽量减少其对实验的影响。
[Abstract]:Objective: To study the conditions and methods of constructing the mouse exhale model by establishing the model of 13C-UBT and 14C-UBT in Helicobacter pylori, and to provide a non-invasive real-time detection method for the study of Helicobacter pylori animal model. Objective To evaluate the immune protective effect of the HP vaccine in the mouse model and to provide a simple, rapid, cheap and reliable diagnostic method for the in-depth study of the pathogenicity of the H. pylori. Methods: The method of constructing the exhale model of mice by using BABL/ c mice at 6 to 8 weeks was used to establish the experimental model of 13C-UBT and 14C-UBT by feeding different doses of 13C-Urea and 14C-Urea in mice. In the same time, the exhale relation of the mice fed with different bacteria was investigated in the same detection time. The mice were fed with different amounts of H. pylori and different time, and the amount and time of different bacteria in the 13C-UBT, 14C-UBT were explored. To explore the biological factors that affect the exhale test model of Helicobacter pylori, determine the tissue location of the strain in the mice that affect the breath test, isolate the culture, and screen the mice that affect the exhale test. and carrying out flight mass spectrum identification and 16srDNA-PCR amplification sequencing on the isolated urea-enzyme-positive bacteria, and determining the strain strains by two methods; and carrying out urease activity on the isolated urea-enzyme-positive bacteria, line characterization The results showed that the same exhale time of 13C-UBT and 14C-UBT in mice was positively correlated with that of the mice. the maximum value is reached, The detection value was decreased with the extension of time. The model of 13C-UBT and 14C-UBT was successfully established in the model of 13C-UBT and 14C-UBT in the model of 13C-UBT, and 300ml of the active space was given. After the drug was fed, it was exhale for 10min, and 250ml of the gas was collected for testing; and 0.556KBq-14C-Urea was used in the 14C-UBT exhale, and 200ml was given. The activity space, after the drug was fed for 10 minutes, collected 150ml of gas for testing; the same expiration time was detected by H. pylori in the feeding mice, and the exhale value in the 13C-Urea and 14C-Urea expired test increased with the increase of the amount of H. pylori, and the amount of H. pylori in the feeding mice was 13C-U. The curves of BT and 14C-UBT were the same, and the amount of the low-concentration bacteria was affected by the internal factors of the mice. There was a good growth curve and trend in the amount of Lori. The bacteria were located in the breath test of the mice successfully. The bacteria involved in the breath test were located in the stomach and small intestine, and 155 strains of urease-positive bacteria were screened out. Using the flight mass spectrometry and the 16srDNA-PCR amplification test, the bacteria were screened out. The strain is determined by two methods, most of which are E. coli, and the rest of the strain is the Pasteurella, and the bacteria such as the slow Staphylococci, the Haemophilus influenzae and the streptococcus are also detected; and the isolated bacteria are subjected to the qualitative and quantitative determination of the activity of the urease, and the strains are separated out. bacteria The activity of urease is much less than that of H. pylori. Conclusion: (1) H. pylori mouse 13C-UB was successfully established. T, 14C-UBT model, capable of non-invasive real-time detection of mice; (2) 13C-Urea, 14C-Urea feeding in mouse 13C-UBT, 14C-UBT model The amount of H. pylori and the detection time of H. pylori were related to the detection value. The amount of H. pylori in the mice was affected by the in vivo factors. (3) The activity of bacterial urease was less than that of H. py.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332

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