免疫抑制剂介导侵袭性白色念珠菌肠源性感染机制研究

发布时间：2019-02-23 11:56

【摘要】：目的：证实应用免疫制剂、广谱抗生素和(或)应激状态可致肠道菌群紊乱,导致原本少量的正常菌群(如白色念珠菌)及过路菌大量增殖,证实白色念珠菌血症的来源是肠道菌群。方法：本课题为动物实验,实验对象是SD大鼠,将80只清洁级SD大鼠随机分为4组,每组20只,分为第一组为免疫抑制组,第二组为免疫抑制+抗生素组；第三组免疫抑制+胰腺炎组；第四组为免疫抑制+胰腺炎+抗生素组。在以上个实验组中,免疫抑制模型的建立为在开始实验第1天按照200mg/kg予以腹腔注射免疫抑制剂环磷酰胺1次；抗生素的模型按照100mg/kg给予尾静脉注射头孢曲松注射液,每日一次给药,按照实验分组给予相应天数的抗生素注射；胰腺炎模型的建立为实验结束前1天开始禁食不禁水,于禁食12小时后给予20%L-精氨酸液腹腔注射,每次按2.0g/kg给药,共给药两次,中间间隔1小时。四组中又分为5天实验组和10天实验组,每小组各10只。其余饮食、饮水、鼠笼、日照时间等实验条件相同。分别于实验开始第5天、第10天,各取每组10只小鼠,离断颈椎后无菌操作环境下快速取心内血、肠内容物、空肠肠壁、结肠肠壁、肠系膜淋巴结及胰腺。取血、肠内容物及其他组织匀浆液做真菌培养,取空肠肠壁组、结肠肠壁及胰腺做病理镜检。对于真菌培养为阳性的标本再行初步念珠菌显色平板筛查,如果为白色念珠菌可应用25S rDNA编码区内存在一可转座I型内含子对白色念珠菌进行分型分析技术[19-20]。后进一步行抽提PCR扩增,电泳检测,切胶纯化测序,连接纯化,分析序列结果,达到菌株层面的鉴定,确定是否有真菌突破感染。结果：1.在实验第5天,免疫抑制+抗生素组的肠内容物真菌培养阳性结果与免疫抑制组对比有显著差异,表明实验第5天已存在菌群紊乱,真菌增殖。2.在实验第10天,免疫抑制+抗生素组及免疫抑制+抗生素+胰腺炎组动物的肠内容物真菌培养阳性结果与免疫抑制组对比有显著差异,表明实验第10天存在菌群紊乱,真菌增殖。3.免疫抑制+抗生素组与免疫抑制+抗生素+胰腺炎组动物的肠内容物真菌培养阳性结果比较,无论是在实验第5天还是在实验第10天,两者均无统计学差异,表明长时间应用抗生素的基础上,负荷应激因素,并不会加重菌群紊乱。4.在实验第5天,免疫抑制+抗生素组的心内血真菌培养阳性结果与免疫抑制组对比有显著差异,表明实验第5天已存在念珠菌血症；其他组间无显著统计学差异。5.在实验第5天及实验第10天,免疫抑制+抗生素组及免疫抑制+抗生素+胰腺炎组与免疫抑制组相比,从病理学及基因组学有明显突破肠道屏障证据,可造成侵袭性真菌感染。结论：免疫抑制条件下,长期应用广谱抗生素可造成肠道菌群紊乱,真菌增殖,随着抗生素应用时间的延长,负荷应激因素,可能加剧肠道菌群紊乱,从病理学及基因组学有明显突破肠道屏障证据,可造成侵袭性真菌感染,甚至白色念珠菌血症。
[Abstract]:Objective: To confirm that the application of immune preparation, broad-spectrum antibiotic and (or) normal condition can cause the intestinal flora to be disturbed, which results in a small number of normal bacteria (such as Candida albicans) and the proliferation of the passing bacteria, and it is confirmed that the source of the bacteremia is the intestinal flora. Methods: The subject was an animal experiment. The experimental subjects were SD rats, 80 clean-grade SD rats were randomly divided into 4 groups, each group was divided into the first group as the immunosuppression group, the second group was the immunosuppression group and the antibiotic group, and the third group was the group of immunosuppression + pancreatitis. The fourth group is an immunosuppression + pancreatitis + antibiotic group. In the above experimental group, the immunosuppression model was established to be injected with an immunosuppressant cyclophosphamide once at a dose of 200 mg/ kg on the first day of the start of the experiment; the model of the antibiotic was given to the tail in 100 mg/ kg for ceftriaxone injection, once a day, An antibiotic injection of the corresponding number of days was given in accordance with the experimental group; the establishment of the pancreatitis model began fasting at 1 day prior to the end of the experiment, and the 20% L-arginine liquid was administered intraperitoneally at a time of 12 hours of fasting, with a total of 2. 0g/ kg, and the intermediate interval was 1 hour. The four groups were divided into 5-day experimental group and 10-day experimental group. The rest of the diet, drinking water, rat cage, sunshine time and other experimental conditions were the same. The internal blood, intestinal contents, the intestinal wall of the jejunum, the intestinal wall, the mesenteric lymph node and the pancreas were quickly taken under the sterile operation environment after the test was started on the 5th and 10th day of the experiment. The blood, intestinal contents and other tissue homogenate were taken as the fungal culture, and the intestinal wall group, the intestinal wall of the colon and the pancreas were taken for pathological examination. In the case of Candida albicans, a transposable type I intron in the 25S rDNA coding region was used for the typing and analysis of Candida albicans[19-20]. and then the PCR amplification, the electrophoresis detection, the gel-cutting purification sequencing, the connection purification and the analysis sequence result are further performed, so that the identification of the strain level is achieved, and the fungal breakthrough infection is determined. Results: 1. In the fifth day of the experiment, the positive results of the fungal culture of the intestinal contents of the immunosuppression + antibiotic group were significantly different from that of the immunosuppression group, indicating that there was a bacterial population disorder and a fungus proliferation in the fifth day of the experiment. In the day 10 of the experiment, the positive results of the culture of the intestinal contents in the group of immunosuppression + antibiotic and the group of immunosuppression + antibiotic + pancreatitis were significantly different from that of the immunosuppression group, indicating that there was a bacterial population disorder and the growth of the fungus in the 10th day of the experiment. The immunosuppression + antibiotic group compared with the positive results of the intestinal contents of the immunosuppression + antibiotic + pancreatitis group animals, either on the day 5 of the experiment or on the day 10 of the experiment, there was no statistical difference between the two groups, indicating that on the basis of long-time application of antibiotics, The stress factor does not aggravate the bacterial population disorder. In the fifth day of the experiment, the positive results of the in-heart blood-fungus culture of the immunosuppression + antibiotic group were significantly different from that of the immunosuppression group, indicating that there was no significant statistical difference between the other groups in the fifth day of the experiment. In the day 5 and day 10 of the experiment, the immunosuppression + antibiotic group and the immunosuppression + antibiotic + pancreatitis group, compared with the immunosuppression group, had a clear breakthrough in the evidence of the intestinal barrier from the pathology and the genomics, which could result in invasive fungal infection. Conclusion: Under the condition of immunosuppression, the long-term application of broad-spectrum antibiotics can lead to the disorder of intestinal flora and the proliferation of the fungi. may result in invasive fungal infections, even white perbacteremia.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R519

【共引文献】