经肠道播散诱导内源性感染小鼠模型的建立及评价
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摘要
目的建立经肠道播散诱导发生内源性感染的小鼠模型,为研究肠道微生态与内源性感染的相关机制提供可靠的实验模型。方法 24只ICR雌性小鼠随机分为模型组A、模型组B和对照组C。模型组A给予广谱抗生素溶液口服破坏肠道正常菌群后尾静脉注射5-氟尿嘧啶(5-FU)进行免疫抑制。在模型组A的基础上给予白假丝酵母菌灌胃引入机会性致病菌即为模型组B。对照组C同等方法给予生理盐水处理。实验过程中持续观察小鼠粪便菌群变化,平板计数法检测小鼠组织载菌量,HE染色观察小鼠肺、肝、盲肠和大肠组织病理变化,荧光定量PCR法观察小鼠肠道主要菌群定量变化。结果实验终点时模型组A小鼠组织器官均出现细菌感染,模型组B小鼠表现为细菌和真菌混合感染。两模型组小鼠肺和肝脏组织器官均表现为典型的炎症表现,而盲肠和大肠表现为黏膜炎症和屏障完整性被破坏。肠道菌群定量结果显示两模型组肠道主要菌群结构发生紊乱,肠道定植抗力下降,B/E值<1。结论在小鼠肠道菌群紊乱和免疫抑制的条件下,肠道致病菌或机会致病菌突破肠道黏膜屏障引起组织器官感染,该模型能够为从肠道微生态方面预防及控制内源性感染的研究提供可靠的模型基础。
Objective To establish a mouse model of endogenous infection induced by intestinal dissemination and provide a reliable experimental model for studies of the mechanism of intestinal microecology and endogenous infection.Methods Twenty-four female ICR mice were randomly divided into model group A,model group B,and control group C.The mice in group A were administered a broad spectrum antibiotic solution orally to disturb the balance of normal intestinal flora,and then 5-fluorouracil was injected into the tail vein for immunosuppression. The mice in group B were administered with Candida albicans by gavage after the same treatment as the group A. The control group C was administered with normal saline by the same method. Changes of fecal flora in the mice were continuously observed during the experiment. The amount of bacteria in mouse tissues was detected by the plate counting method. Pathological changes of the lung,liver,cecum,and large intestine were observed using HE staining. Quantitative changes of intestinal flora in the mice were observed by quantitative PCR. Results At the end of the experiment,bacterial infection occurred in the tissues and organs of the mice in group A,and mixed bacterial and fungal infections occurred in the group B. Lung and liver histology of the mice in both infection groups showed typical inflammatory manifestations,while the cecum and large intestine showed mucosal inflammation and disrupted barrier integrity. Quantitation of intestinal flora showed disruption of the main intestinal flora structure in the two model groups,and the ratio of the intestinal colonization resistance index was less than 1.Conclusions Under the conditions of intestinal flora disturbance and immune suppression,intestinal pathogenic bacteria or opportunistic pathogenic bacteria break through the intestinal mucosa barrier and cause tissue and organ infection in mice. This model can provide a reliable basis for the studies on prevention and control of endogenous infection from the perspective of intestinal microecology.
Objective To establish a mouse model of endogenous infection induced by intestinal dissemination and provide a reliable experimental model for studies of the mechanism of intestinal microecology and endogenous infection.Methods Twenty-four female ICR mice were randomly divided into model group A,model group B,and control group C.The mice in group A were administered a broad spectrum antibiotic solution orally to disturb the balance of normal intestinal flora,and then 5-fluorouracil was injected into the tail vein for immunosuppression. The mice in group B were administered with Candida albicans by gavage after the same treatment as the group A. The control group C was administered with normal saline by the same method. Changes of fecal flora in the mice were continuously observed during the experiment. The amount of bacteria in mouse tissues was detected by the plate counting method. Pathological changes of the lung,liver,cecum,and large intestine were observed using HE staining. Quantitative changes of intestinal flora in the mice were observed by quantitative PCR. Results At the end of the experiment,bacterial infection occurred in the tissues and organs of the mice in group A,and mixed bacterial and fungal infections occurred in the group B. Lung and liver histology of the mice in both infection groups showed typical inflammatory manifestations,while the cecum and large intestine showed mucosal inflammation and disrupted barrier integrity. Quantitation of intestinal flora showed disruption of the main intestinal flora structure in the two model groups,and the ratio of the intestinal colonization resistance index was less than 1.Conclusions Under the conditions of intestinal flora disturbance and immune suppression,intestinal pathogenic bacteria or opportunistic pathogenic bacteria break through the intestinal mucosa barrier and cause tissue and organ infection in mice. This model can provide a reliable basis for the studies on prevention and control of endogenous infection from the perspective of intestinal microecology.
引文
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[14] Clemons KV,Gonzalez GM,Singh G,et al. Development of an orogastrointestinal mucosal model of candidiasis with dissemination to visceral organs[J]. Antimicrob Agents Chemother,2006,50(8):2650-2567.
[15] Giro E,Levin AS,Basso M,et al. Seven-year trend analysis of nosocomial candidemia and antifungal(fluconazole and caspofungin)use in Intensive Care Units at a Brazilian University Hospital[J]. Med Mycol,2008,46(6):581-588.
[2]马园园,赛音其木格,张梅,等.血液肿瘤患者感染的实验室诊断进展[J].中华医院感染学杂志,2016,26(7):1677-1680.Ma YY, Sai YQMG, Zhang M, et al. Laboratory diagnosis progress of hematologic malignancies co-infection[J]. Chin J Nosocomiol,2016,26(7):1677-1680.
[3]张晓,张建超,李伟华.急诊肿瘤晚期感染患者的病原菌分布及炎性指标变化的意义分析[J].中华医院感染学杂志,2017,27(23):5348-5351.Zhang X,Zhang JC,Li WH. Distribution of pathogenic bacteria and the significance of inflammatory indexes changes in patients with advanced cancer complicated with infections in emergency department[J]. Chin J Nosocomiol,2017,27(23):5348-5351.
[4]吴玉娥,李航,陈梅玲,等.系统性念珠菌感染小鼠模型的制备[J].中国实验动物学报,2015(3):301-305.Wu Y,Li H,Chen ML,et al. Preparation of a murine model of systemic Candida albicans infection[J]. Acta Lab Anim Sci Sin,2015(3):301-305.
[5]罗银珠,潘金春,何丽芳,等.白假丝酵母菌经口感染ICR小鼠建立系统性感染模型[J].中国实验动物学报. 2016,24(6):591-595.Luo YZ,Pan JC,He LF,et al. Establishment of a ICR mouse model of systemic C. albicans infection induced by oral inoculation[J]. Acta Lab Anim Sci Sin,2016,24(6):591-595.
[6]陈丽,张伟丽,叶先飞,等.恶性血液病患者医院侵袭性真菌感染的危险因素分析[J].中国微生态学杂志,2015,27(4):417-420.Chen L,Zhang WL,Ye XF,et al. Risk factors of invasive fungal infections in patients with hematopoietic malignancies[J]. Chin J Microecol,2015,27(4):417-420.
[7]张秀荣.肠道菌群粪便涂片检查图谱[J].解放军医学杂志,2000,25(4):33-33.Zhang XR. Fecal smear examinations of intestinal flora[J]. Med J Chin PLA,2000,25(4):33-33.
[8] Deitch EA. Gut-origin sepsis:evolution of a concept[J].Surgeon,2012,10(6):350-356.
[9]朱迎钢,瞿介明.医院感染治疗中的一个难以决策的问题:定植还是感染?[J].中国呼吸与危重监护杂志,2011,10(5):421-423.Zhu YG, Qu JM. A difficult decision in hospital infection treatment:colonization or infection?[J] Chin J Respir Crit Care Med,2011,10(5):421-423.
[10] Clemons KV, Martinez M, Calderon L, et al. Efficacy of ravuconazole in treatment of systemic murine histoplasmosis[J].Antimicrob Agents Chemother,2002,46(3):922-924.
[11] Clemons KV,Stevens DA. Orogastrointestinal model of mucosal and disseminated candidiasis[J]. Methods Mol Biol,2012,845(845):557-567.
[12] Ashman RB, Vijayan D, Wells CA. IL-12 and related cytokines:function and regulatory implications in Candida albicans infection[J]. Clin Dev Immunol,2010,2011:686597.
[13] Kretschmar M,Felk A,Staib P,et al. Individual acid aspartic proteinases(Saps)1-6 of Candida albicans are not essential for invasion and colonization of the gastrointestinal tract in mice[J].Microb Pathog,2002,32(2):61-70.
[14] Clemons KV,Gonzalez GM,Singh G,et al. Development of an orogastrointestinal mucosal model of candidiasis with dissemination to visceral organs[J]. Antimicrob Agents Chemother,2006,50(8):2650-2567.
[15] Giro E,Levin AS,Basso M,et al. Seven-year trend analysis of nosocomial candidemia and antifungal(fluconazole and caspofungin)use in Intensive Care Units at a Brazilian University Hospital[J]. Med Mycol,2008,46(6):581-588.