【作者】 焦泽龙；

【导师】 李恒进； 邹先彪； 赵华；

【作者基本信息】 中国人民解放军军医进修学院 ， 皮肤病与性病学， 2011， 博士

【摘要】 背景随着新治疗技术如骨髓移植、器官移植和强化治疗的开展及广谱抗生素和免疫抑制药物的广泛应用,临床上发生念珠菌感染的现象越来越普遍,而抗真菌药物由于其毒副作用和易产生耐药的局限性,使得白念珠菌在免疫低下患者中的发病率依然居高不下。由此,为了探索免疫低下患者中预防白念珠菌感染的措施,有必要建立适当的免疫抑制后系统感染念珠菌的动物模型,来模拟免疫功能低下患者的情况。我们同时也研究了重组念珠菌天冬氨酰蛋白酶(screted aspartyl proteinase, Sap)预防接种对免疫功能低下时系统感染念珠菌的保护作用。第一部分目的建立免疫抑制小鼠系统感染白念珠菌模型,并观察其主要影响因素。方法1)以环磷酰胺(CTX)100mg/kg和200mg/kg两个剂量水平分别对6周龄BALB/c小鼠进行单次腹腔注射(各3组),48h后各对应组分别经尾静脉注射1×105、106、107/ml的白念珠菌,通过各组的死亡率来确定半数致死量。2)以50、100mg/kgCTX单次腹腔注射,观察外周血中白细胞、淋巴细胞计数被抑制的程度。3)分别采用4和8周龄小鼠来观察周龄对死亡率的影响。4)通过组织病理检查和肾组织真菌培养来验证模型。结果1)100mg/kgCTX+1×105/ml白念珠菌作用后,小鼠的死亡率接近50%。2)100mg/kgCTX单次腹腔注射后,外周血中白细胞、淋巴细胞计数下降超过30%。3)4周龄小鼠对模型建立条件的耐受性较差,死亡率高。4)组织病理检查和肾组织真菌培养结果表明模型小鼠肾脏中有菌丝生长或局灶性脓肿形成。结论1)成功建立了免疫抑制后系统感染白念珠菌的小鼠模型。2)模型建立条件下,白念珠菌对小鼠肾组织有高亲嗜性。第二部分目的观察重组Sap2蛋白免疫接种3次(间隔1周)对模型小鼠抗白念珠菌系统感染的保护作用。方法6周龄BALB/c小鼠随机分成3组：1)间隔1周,重组Sap2+弗氏完全佐剂(CFA)胫前肌注射3次,第3次免疫后1周时,按体重单次腹腔注射100mg/kgCTX,48h后经尾静脉注射1×105/ml的白念珠菌,剂量为0.01ml/g。2)间隔1周,CFA胫前肌注射3次,其余干预同第1组。3)间隔1周,PBS胫前肌注射3次,其余干预同第1组。分别以这3种分组方式饲养小鼠,并在不同时间点检测下列指标,探讨模型小鼠的免疫功能动态变化：1)白念珠菌感染后21天内各组小鼠的死亡率。2)间接ELISA法测定各组小鼠血清特异性IgG抗体水平。3)ELISA法测定小鼠血清中IL-2、IL-4、IFN-γ、TNF-α的含量。4)流式细胞仪分析小鼠脾淋巴细胞中的CD4+/CD8+T细胞比例。5)测定各组小鼠肾脏中的荷菌量。6)比较各组小鼠脾脏重量。7)对各组小鼠的肾、肝、肺组织进行组织病理检查。结果1)免疫组小鼠的死亡率显著低于对照组。2)免疫组小鼠血清中的特异性IgG抗体水平的显著高于对照组。3)念珠菌攻击后的第1、4、7天期间,IL-2、IL-4、IFN-γ知TNF-α在第4天时的测量值高于第1和第7天水平。其中TNF-α的测量值始终低于正常对照水平。4)念珠菌攻击后的第1、4、7天期间,各组小鼠的CD4+/CD8+比值呈现相似的变化,其中第4天时的测量值低于第1和第7天水平。5)免疫组小鼠的肾脏荷菌量显著低于对照组。6)免疫组小鼠的脾脏重量显著高于对照组。7)免疫组小鼠肾组织中检出菌丝和局灶性脓肿的发生率低于对照组。结论：1)重组Sap2蛋白间隔1周免疫接种3次对模型小鼠抗白念珠菌系统感染具有一定的保护作用。2)重组Sap2蛋白间隔1周免疫接种3次后会导致小鼠出现脾大。更多还原

【Abstract】 BackgroundWith such developments of novel treatment technologies as bone marrow transplantation, organ transplantation and intensive treatment and the wide use of broad spectrum antibiotics and immunosuppressors, the phenomenon of clinical infection of candida becomes more and more widespread, meanwhile the limitation of antifungal agents derived from the side effects and drug resistance makes prevalence of candida albicans high in the patients of low immunity. Thus, in order to explore the preventive measures against the infection of candida albicans in the low immune patients, it’s necessary to establish certain animal model of the infection of candida albicans after immunosuppression, which mimicking the conditions of the low immune patients, and followed by further investigation of the protection of vaccination for the systemic candida albicans infection in the low immune patients.Part oneObjectivesTo establish the immunosuppressive mouse model of systemic infection of candida albicans and observe the main influence factors.Methods1)6-week old BALB/c mice were injected intraperitoneally with two different dosages of cyclophosphamide, 100mg/kg and 200mg/kg respectively, then 48 hours later candida albicans were injected into the caudal veins at the following concentrations as 1×105、106、107/ml.the median lethal dose was defined by the mortality of each group.2)The inhibitory degrees of the white blood cell count,lymphocytes count in peripheral blood were observed after single intraperitoneal injection of cyclophosphamide with a dosage of 50 mg/kg orl00mg/kg.3)4- and 8-week old mice were used to observe the influence of week age on the mortality.4)The mouse model was validated by histopathological examination and fungal culture of renal tissues. Results1)The mortality of 6-week old balb/c mice was close to 50%,which were injected intraperitoneally with a single dose of 100mg/kg cyclophosphamide,followed by injection of candida albicans into caudal vein 48 hours later.2)The decreases of the white blood cells and lymphocytes in peripheral blood were more than 30% after single intraperitoneal injection of 100mg/kg cyclophosphamide.3)The mortality of 4-week old mice was high because of bad tolerability to all the conditions required for the mouse model.4)The results of histopathological examinations and cultures of fungi in renal tissue indicated that there were hypha growth and focal abcesse formation in the kidney of the mouse model.Conclusions1) The mouse model of systemic infection of candida albicans after immunosuppression was successfully established.2) High affinity of candida albicans to renal tissues was observed in the conditions of the mouse model.Part twoObjectivesTo observe the protection profile of recombinant Sap2 protein, with which the model mice were inoculated for 3 times (every 2 weeks), from systemic infection of candida albicans.Methods6-week old balb/c mice were randomized into three groups:1)recombinant Sap2 combined with complete Freund’s adjuvant(CFA) were injected intramuscularly for 3 times at one week interval in the tibial anterior area, then single dose of 100mg/kg cyclophosphamide was injected intraperitoneally according to body weight one week after the third inoculation,48 hours after which 1×105/ml candida albicans were injected into caudal vein at a dosage of 0.01ml/g.2) CFA were injected intramuscularly for 3 times at one week interval in the tibial anterior area at the same other interventions as group one.3)PBS were injected intramuscularly for 3 times at one week interval in the tibial anterior area at the same other interventions as group one. Mice were raised by the above three grouping ways and were determined at different time point the following indexes:1)mortality of each group within 21 days after infection of candida albicans.2)levels of specific IgG antibody in the sera of each group of mice determined by indirect ELISA.3)serous levels of IL-2、IL-4、IFN-γ、 TNF-αdetermined by ELISA.4)the ratio of CD4+/CD8+T cells in the spleen by flow cytometry.5)fungal quantity in the kidney of each group of mice.6)comparison of splenetic weight of each group of mice.7)histopathological examination of the kidney, liver and lung of each group of mice.Results1)Mortality of the inoculated mice group was significantly lower than that of control.2)The specific serous IgG antibody was significantly higher than that of control.3)At the test time point of day1/4/7 after candida albicans challenge, the serous levels of IL-2、IL-4、IFN-γand TNF-αat day4 were higher than that at dayl and day7.of note, the levels of TNF-αkept lower than that of normal control throughout the experiment.4) At the test time point of day1/4/7 after candida albicans challenge, the ration of CD4+/CD8+T cells in all groups presented with similar changes, of which the measured values at day4 were lower than that at dayl and day7. 5)The fungal quantities of inoculated mice were significantly lower that of control. 6)The splenic weight of the inoculated mice were significantly higher than that of control.7) The prevalence of hypha and focal abcesses in the kidney of inoculated mice was lower than that of control.Conclusions1)Three-time inoculation of these model mice (every 2 weeks) with recombinant Sap2 protein had certain protection against systemic infection of candida albicans.2) Three-time inoculation of the model mice (every 2 weeks) with recombinant Sap2 protein led to splenomegaly.更多还原