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[目的]临床上各种疾病会导致肠黏膜屏障损伤并伴随大量的细菌易位发生。本实验拟使用发光细菌的小鼠活体成像技术,观察肠黏膜损伤状态下易位细菌的动态移位过程。[方法]构建重组荧光脂酶基因(Lux)的质粒pXen-1、pXen-18,转化大肠杆菌DH5a,筛选阳性细菌克隆,扩增培养后,定量(107)注射到肠黏膜屏障损伤模型小鼠肠腔不同部位内,利用生物发光活体成像技术进行细菌易位的病理生理观察研究。实验分为四组:小肠注射菌实验组和对照组、盲肠注射菌实验组和对照组。其中实验组为肠道缺血再灌注损伤,对照组无任何干预措施。[结果]发光细菌小鼠活体成像观察到,存在肠黏膜损伤的小鼠肠腔内的细菌数量及分布发生明显的改变,提示细菌易位在肠黏膜损伤时起着重要的病理生理作用。注入小肠内的发光细菌数量在试验过程中明显下降,提示小肠肠腔环境可以杀灭一定的细菌。[结论]生物发光活体成像技术,可以直观、可靠、敏感地观察肠道内细菌的存活状态,并可观察肠黏膜损伤状态下细菌易位的动态过程。该技术为肠黏膜屏障损伤细菌易位机制的研究提供了一个有力的工具。
[Objective] Clinically, various diseases lead to intestinal mucosal barrier damage accompanied by a large number of bacterial translocations. This experiment intends to use luminescent bacteria mouse live imaging technology to observe the dynamic shift of translocated bacteria under intestinal mucosal injury. [Method] The plasmids pXen-1 and pXen-18 of recombinant luciferase gene (Lux) were constructed and transformed into E. coli DH5a. The positive bacterial clones were screened and expanded. The cells were quantitatively injected into the intestinal mucosal barrier injury model mice Different parts of the intestine, the use of bioluminescence in vivo imaging of bacterial translocation pathophysiological study. The experiment was divided into four groups: small intestine injection bacteria experimental group and control group, cecal injection experimental group and control group. The experimental group was intestinal ischemia-reperfusion injury in the control group without any intervention. [Result] The results showed that the quantity and distribution of bacteria in intestinal lumen of mice with intestinal mucosal injury changed obviously, which indicated that bacterial translocation played an important pathophysiological role in intestinal mucosal injury. The number of luminescent bacteria injected into the small intestine significantly decreased during the test, suggesting that the intestinal lumen environment can kill a certain amount of bacteria. [Conclusion] Bioluminescence in vivo imaging technique can observe the live state of intestinal bacteria visually, reliably and sensitively, and observe the dynamic process of bacterial translocation under intestinal mucosa injury. This technique provides a powerful tool for studying the bacterial translocation mechanism of intestinal mucosal barrier injury.