肠道屏障在肠源性侵袭性真菌病中的作用

发布时间：2018-05-15 07:14

本文选题：侵袭性真菌病 + 真菌　；参考：《复旦大学》2013年硕士论文

【摘要】：目的：证实应用广谱抗生素和(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,证实急性应激状态下肠道屏障在侵袭性真菌病发病过程中的作用。方法：本课题为动物实验,实验对象是Wistar大鼠,将60只大鼠分为三组,雌雄不记,每组20只,第一组为空白对照组；第二组为抗生素组,每日静脉给予头孢曲松(Ceftriaxone, CTRX)100mg/kg一次[16][17]第三组为抗生素+胰腺炎组,腹腔注射20%精氨酸(L-Arginine,L-ARG)1.25mg/kg,两次,中间间隔一小时,造成急性出血性胰腺炎模型,同样给予头孢曲松,剂量及方法同第二组。其余饮食、饮水、鼠笼等实验条件相同。分别于实验开始第7天、第14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、门静脉血、肠内容物(Intestinal Content,IC)、肠壁及肠系膜淋巴结。取血、组织匀浆(Tissue Homogenate,TH)及肠内容物做真菌培养,取肠壁组织及胰腺做病理镜检。若同一实验动物有两个或两个以上样本培养结果阳性,则分离真菌,应用18S rDNA内转录间隔区(Internal Transcribed Spacer, ITS)序列分析技术,抽提基因组DNA,电泳检测,PCR扩增,电泳检测,切胶纯化测序,拼接序列,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7天,抗生素组及抗生素+胰腺炎组动物的肠内容物真菌培养阳性结果与空白组对比有显著差异,表明实验第7天已存在菌群紊乱,真菌增殖。 2.在实验第14天,抗生素组及抗生素+胰腺炎组动物的肠内容物真菌培养阳性结果与空白组对比有显著差异,表明实验第14天仍存在菌群紊乱,真菌增殖。 3.抗生素组与抗生素+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7天还是在实验第14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7天及实验第14天,抗生素组及抗生素+胰腺炎组与空白组相比,无明显突破肠道屏障证据,未造成侵袭性真菌感染。 5.肠道屏障的免疫功能在其防止侵袭性真菌病的发生过程中有重要作用。结论：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,并不会加剧菌群紊乱；在真菌成为肠道优势菌群的情况下,肠道固有屏障的保护作用,仍可保证急性应激情况下,真菌不能突破肠道屏障,造成侵袭性真菌病,肠道屏障的免疫功能在其中可能起重要作用。
[Abstract]:Objective: It was proved that the use of broad-spectrum antibiotics and / or stress could lead to intestinal flora disorder, resulting in the proliferation of a small number of normal flora (E. g., Candida albicans) and passageway bacteria. The role of intestinal barrier in the pathogenesis of invasive mycosis was confirmed under acute stress. Methods: This subject is an animal experiment, the experimental object is Wistar rats, 60 rats are divided into three groups, male and female, 20 rats in each group, the first group is a blank control group, the second group is an antibiotic group, Ceftriaxone was given intravenously daily and CTRX)100mg/kg was given once [16] [17] the third group was treated with antibiotic pancreatitis, 20% arginine L-Arginine L-ARGN 1.25mg / kg intraperitoneally, twice, with an interval of one hour, to make the model of acute hemorrhagic pancreatitis, and also to give ceftriaxone, the same dose of ceftriaxone. The dose and method were the same as those in the second group. The rest of the diet, drinking water, squirrel cage and other experimental conditions are the same. On the 7th and 14th day of the experiment, 10 mice in each group were taken from each group. The blood of heart, portal vein, intestinal contents, intestinal wall and mesenteric lymph nodes were collected quickly in aseptic environment after cervical amputation. Blood, tissue homogenate, tissue homogenate (THH) and intestinal contents were used for fungal culture, and intestinal wall tissue and pancreas were taken for histopathological examination. If two or more samples of the same laboratory animals were positive, fungi were isolated. Genomic DNAs were extracted by internal Transcribed Spacer, ITS) sequence analysis technique of 18s rDNA internal transcribed spacer, and then amplified by electrophoresis, purified and sequenced by gumming. Splicing the sequence and analyzing the sequence results to identify the strain level to determine whether there is a fungal breakthrough infection. Results: 1. On the 7th day of the experiment, the positive results of fungal culture of intestinal contents in the antibiotic group and the antibiotic pancreatitis group were significantly different from those in the blank group, indicating that on the 7th day of the experiment, there was a disturbance of the flora and the proliferation of the fungi. 2. On the 14th day of the experiment, the positive results of fungal culture of intestinal contents in the antibiotic group and the antibiotic pancreatitis group were significantly different from those in the blank group, indicating that on the 14th day of the experiment, there was still a disturbance of the flora and the proliferation of the fungi. 3. There was no significant difference between the antibiotic group and the antibiotic pancreatitis group in the culture of intestinal contents fungi on the 7th and 14th day, indicating that the antibiotics were used for a long time. Load stress factors will not aggravate the disturbance of flora. 4. On the 7th day and 14th day, there was no evidence of breakthrough of intestinal barrier and no invasive fungal infection in the antibiotic group and the antibiotic pancreatitis group compared with the blank group. 5. The immune function of intestinal barrier plays an important role in the prevention of invasive mycosis. Conclusion: Long-term use of broad-spectrum antibiotics can cause intestinal microflora disorder and fungal proliferation. With the prolongation of antibiotic application time, load stress factors will not aggravate the flora disorder. The protective effect of intestinal inherent barrier can still ensure that under acute stress, fungi can not break through intestinal barrier, causing invasive mycosis, in which the immune function of intestinal barrier may play an important role.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R519

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