免疫抑制劑介導(dǎo)侵襲性白色念珠菌腸源性感染機(jī)制研究
發(fā)布時(shí)間：2019-02-23 11:56
【摘要】：目的：證實(shí)應(yīng)用免疫制劑、廣譜抗生素和(或)應(yīng)激狀態(tài)可致腸道菌群紊亂,導(dǎo)致原本少量的正常菌群(如白色念珠菌)及過路菌大量增殖,證實(shí)白色念珠菌血癥的來源是腸道菌群。方法：本課題為動物實(shí)驗(yàn),實(shí)驗(yàn)對象是SD大鼠,將80只清潔級SD大鼠隨機(jī)分為4組,每組20只,分為第一組為免疫抑制組,第二組為免疫抑制+抗生素組；第三組免疫抑制+胰腺炎組；第四組為免疫抑制+胰腺炎+抗生素組。在以上個(gè)實(shí)驗(yàn)組中,免疫抑制模型的建立為在開始實(shí)驗(yàn)第1天按照200mg/kg予以腹腔注射免疫抑制劑環(huán)磷酰胺1次；抗生素的模型按照100mg/kg給予尾靜脈注射頭孢曲松注射液,每日一次給藥,按照實(shí)驗(yàn)分組給予相應(yīng)天數(shù)的抗生素注射；胰腺炎模型的建立為實(shí)驗(yàn)結(jié)束前1天開始禁食不禁水,于禁食12小時(shí)后給予20%L-精氨酸液腹腔注射,每次按2.0g/kg給藥,共給藥兩次,中間間隔1小時(shí)。四組中又分為5天實(shí)驗(yàn)組和10天實(shí)驗(yàn)組,每小組各10只。其余飲食、飲水、鼠籠、日照時(shí)間等實(shí)驗(yàn)條件相同。分別于實(shí)驗(yàn)開始第5天、第10天,各取每組10只小鼠,離斷頸椎后無菌操作環(huán)境下快速取心內(nèi)血、腸內(nèi)容物、空腸腸壁、結(jié)腸腸壁、腸系膜淋巴結(jié)及胰腺。取血、腸內(nèi)容物及其他組織勻漿液做真菌培養(yǎng),取空腸腸壁組、結(jié)腸腸壁及胰腺做病理鏡檢。對于真菌培養(yǎng)為陽性的標(biāo)本再行初步念珠菌顯色平板篩查,如果為白色念珠菌可應(yīng)用25S rDNA編碼區(qū)內(nèi)存在一可轉(zhuǎn)座I型內(nèi)含子對白色念珠菌進(jìn)行分型分析技術(shù)[19-20]。后進(jìn)一步行抽提PCR擴(kuò)增,電泳檢測,切膠純化測序,連接純化,分析序列結(jié)果,達(dá)到菌株層面的鑒定,確定是否有真菌突破感染。結(jié)果：1.在實(shí)驗(yàn)第5天,免疫抑制+抗生素組的腸內(nèi)容物真菌培養(yǎng)陽性結(jié)果與免疫抑制組對比有顯著差異,表明實(shí)驗(yàn)第5天已存在菌群紊亂,真菌增殖。2.在實(shí)驗(yàn)第10天,免疫抑制+抗生素組及免疫抑制+抗生素+胰腺炎組動物的腸內(nèi)容物真菌培養(yǎng)陽性結(jié)果與免疫抑制組對比有顯著差異,表明實(shí)驗(yàn)第10天存在菌群紊亂,真菌增殖。3.免疫抑制+抗生素組與免疫抑制+抗生素+胰腺炎組動物的腸內(nèi)容物真菌培養(yǎng)陽性結(jié)果比較,無論是在實(shí)驗(yàn)第5天還是在實(shí)驗(yàn)第10天,兩者均無統(tǒng)計(jì)學(xué)差異,表明長時(shí)間應(yīng)用抗生素的基礎(chǔ)上,負(fù)荷應(yīng)激因素,并不會加重菌群紊亂。4.在實(shí)驗(yàn)第5天,免疫抑制+抗生素組的心內(nèi)血真菌培養(yǎng)陽性結(jié)果與免疫抑制組對比有顯著差異,表明實(shí)驗(yàn)第5天已存在念珠菌血癥；其他組間無顯著統(tǒng)計(jì)學(xué)差異。5.在實(shí)驗(yàn)第5天及實(shí)驗(yàn)第10天,免疫抑制+抗生素組及免疫抑制+抗生素+胰腺炎組與免疫抑制組相比,從病理學(xué)及基因組學(xué)有明顯突破腸道屏障證據(jù),可造成侵襲性真菌感染。結(jié)論：免疫抑制條件下,長期應(yīng)用廣譜抗生素可造成腸道菌群紊亂,真菌增殖,隨著抗生素應(yīng)用時(shí)間的延長,負(fù)荷應(yīng)激因素,可能加劇腸道菌群紊亂,從病理學(xué)及基因組學(xué)有明顯突破腸道屏障證據(jù),可造成侵襲性真菌感染,甚至白色念珠菌血癥。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R519
本文編號：2428797
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R519
【共引文獻(xiàn)】
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