【作者】 王乐；

【导师】 刘维达；

【作者基本信息】 北京协和医学院 ， 皮肤病与性病学， 2014， 博士

【摘要】 第一章小鼠口腔和阴道双部位念珠菌病模型的构建及其应用第一节小鼠口腔和阴道双部位白念珠菌感染模型的构建目的探索构建小鼠口腔和阴道双部位白念珠菌感染模型的方法。方法设置单独泼尼松龙处理组、单独雌激素处理组以及联合用药组,通过在接种后4天和7天的临床表现、真菌直接镜检、组织真菌载量测定以及组织病理学检查比较三种不同方法的建模效果,选择其中的最佳方案。结果联合用药组小鼠在接种后第4天舌背粘膜表面出现白斑,阴道口红斑、有白色分泌物,双部位真菌镜检均见大量菌丝,双部位可分离较高的真菌载量,可达105～106cfu/g,组织病理学显示粘膜表面有较多菌丝覆盖,部分侵入上皮引起上皮结构破坏以及炎症细胞浸润。接种后第7天上述表现较接种后第4天有所消退。单独使用雌激素或泼尼松龙组的造模效果不及联合用药组。结论(1)泼尼松龙联合雌激素可成功构建小鼠口腔和阴道双部位念珠菌病感染模型；(2)泼尼松龙和雌激素联合用药的造模效果优于单独使用泼尼松龙或雌激素的方法。第二节泼尼松龙联合雌激素构建小鼠口腔和阴道双部位白念珠菌感染模型的动态研究目的观察泼尼松龙联合雌激素构建小鼠口腔和阴道双部位白念珠菌感染模型在不同时间的感染变化情况方法采用泼尼松龙联合雌激素的方法,对接种后1、3、5和7天进行连续观察,观察指标选取小鼠体重变化、口腔临床表现、双部位局部真菌载量以及组织病理学。结果接种后第1天开始,小鼠体重即呈现逐渐下降趋势,在第1-3天下降最为明显。舌背粘膜在接种后第2-3天开始出现白斑,第3-5天最为明显,白斑平均面积接近舌背面积的90%。双部位的菌量自接种后第1天开始上升,第3-5天最高(105～106cfu/g),之后下降。病理学检查显示接种后第1天舌背和阴道管腔中有酵母细胞,少见菌丝侵入上皮,第3-5天舌背可见有较厚菌丝团覆盖,菌丝较长并侵入上皮组织,伴随上皮结构对破坏和炎症细胞浸润。第7天时双部位菌丝明显减少,对上皮的破坏减轻,上皮开始恢复正常。结论泼尼松龙联合雌激素构建的小鼠双部位白念珠菌感染模型在接种后第3-5天最为明显,此后则渐恢复正常。第三节不同糖皮质激素联合雌激素构建不同品系小鼠的口腔和阴道双部位白念珠菌感染模型目的比较不同糖皮质激素联合雌激素构建不同品系小鼠口腔和阴道双部位白念珠菌感染模型的效果差异。方法设置泼尼松龙联合雌激素处理组、可的松联合雌激素处理组以及地塞米松联合雌激素组,通过对两种品系的小鼠接种后不同时间的体重变化以及3天时的组织真菌载量比较三种不同方法的建模效果。结果泼尼松龙联合雌激素组ICR小鼠的体重在接种后1天持续下降至7天,而可的松和地塞米松联合雌激素处理组在第3-5天时体重稳定并有轻度回升。泼尼松龙联合雌激素组真菌载量最高,在此条件下ICR小鼠感染ATCC62342(口腔1.05×106cfu/g,阴道5.50×105cfu/g)后的真菌载量较SC5314高(口腔4.79×10Scfu/g,阴道4.07×105cfu/g), BALB/c小鼠感染ATCC62342(口腔1.92x106cfu/g,阴道1.33×106cfu/g)后的真菌载量亦较SC5314高(口腔7.57×105cfu/g,阴道2.58×105cfu/g)。结论(1)泼尼松龙较可的松和地塞米松造模效果更佳：(2)白念珠菌／TCC62342较SC5314造模效果更好；(3)ICR小鼠和BALB/c小鼠均能成功构建模型,后者真菌载量略高。第四节环磷酰胺和糖皮质激素联合雌激素构建小鼠口腔和阴道双部位白念珠菌感染模型目的比较环磷酰胺和糖皮质激素联合雌激素构建小鼠口腔和阴道双部位白念珠菌感染模型的效果差异。方法设置泼尼松龙联合雌激素处理组、环磷酰胺联合雌激素处理组以及泼尼松龙和环磷酰胺联合雌激素用药组,通过对两种品系的小鼠接种后3天时的体重变化和组织真菌载量比较三种不同方法的建模效果。结果两种品系的小鼠在接种白念珠菌后第3天,体重均出现明显下降(低于接种当天体重的90%),其中BALB/c小鼠下降更为明显,联合用药组小鼠体重甚至不足接种当天的75%。在不同的处理因素中,联合用药组在不同品系小鼠和不同菌株条件下,体重下降均最明显。真菌载量方面,泼尼松龙联合雌激素组、泼尼松龙联合环磷酰胺以及雌激素组真菌载量最高,但后者在接种3天以后小鼠的死亡率较高。结论(1)泼尼松龙较环磷酰胺造模效果更佳；(2)泼尼松龙联合环磷酰胺以及雌激素造模效果亦较好,但该条件下小鼠死亡率较高。第五节小鼠口腔和阴道双部位感染白念珠菌模型的应用初探目的评价小鼠口腔和阴道双部位白念珠菌感染模型在药效学以及免疫学研究方面的应用。方法首先选用ICR小鼠通过泼尼松龙联合雌激素构建双部位感染模型。(1)通过在小鼠饮水中添加一定浓度的氟康唑进行治疗,在接种后第3天和第5天测定双部位的真菌载量,评价治疗效果；(2)在接种后不同时间点采集小鼠的口腔和阴道组织,匀浆后使用Elisa方法测定组织中IL-17和IL-23的含量,观察其感染过程中的动态表达水平。结果(1)接种SC5314的ICR小鼠和BALB/c小鼠经氟康唑治疗3天即出现菌量明显下降,至第5天可完全清除真菌；而接种ATCC62342的两个品系小鼠经氟康唑治疗无效,接种后的菌量变化与未治疗组无差异；(2)在感染建立后,口腔组织中IL-17的表达水平开始升高,以第3～-5天达到高峰,而阴道组织中的IL-17仅在接种后1天升高,此后即恢复正常水平；口腔和阴道双部位中的IL-23含量自感染开始即明显升高,且维持较高的表达水平可达接种后2周。结论本实验构建的小鼠口腔和阴道双部位白念珠菌感染模型可用于药效学及宿主-真菌相互作用等免疫学研究。第二章不同感染部位来源的白念珠菌毒力相关性状研究第一节不同感染部位来源的白念珠菌胞外水解酶毒力研究目的探索不同部位来源白念珠菌胞外水解酶的差异。方法收集64株来自于血液、阴道、口腔和支气管肺泡灌洗的白念珠菌菌株。菌株在YEPD培养基中37℃培养48h。制备1×107CFU/mL的白念珠菌菌悬液,分别取51μl点滴于卵黄培养基、胎牛白蛋白培养基、吐温-80培养基、溶血素培养基表面。前三种常态37℃培养,最后一种培养基在5%C02环境下培养。分别于第5天、5天、10天、2天观察酶活力。结果口腔、阴道来源的白念珠菌产生蛋白酶的能力强于血液来源。血液、口腔来源的白念珠菌产生磷脂酶的能力最强,阴道株最弱。白念珠菌血液株分泌酯酶的能力最强,阴道来源分泌最低。口腔、阴道来源的白念珠菌溶血素分泌能力强于血液来源株。结论不同部位来源的白念珠菌在产生毒力因子蛋白酶、磷脂酶、酯酶和溶血素方面存在着一定的差异。第二节不同感染部位来源的白念珠菌药物敏感性研究目的探索不同部位来源白念珠菌对抗真菌药物敏感性的差异性。方法收集分别来自于血液、阴道、口腔和支气管肺泡灌洗的白念珠菌菌株,参照CLSI M27-A2方案对上述菌株进行药物敏感性实验。实验药物包括两性霉素B、氟康唑、伊曲康唑、伏立康唑、特比萘芬、卡泊芬净和米卡芬净。结果两性霉素B对血液、口腔、阴道、支气管肺泡灌洗液来源的64株白念珠菌的MIC50、MIC90分别为0.25μg/ml、0.5μg/ml,0.5μg/ml、1.0μg/ml,0.25μg/ml、0.5μg/ml,0.25μg/ml、0.5μg/ml。伏立康唑对血液、口腔、阴道、支气管肺泡灌洗液来源的白念珠菌的MIC50、MIC90分别为0.03μg/ml、0.03μg/ml,0.125μg/ml、1μg/ml,0.03μg/ml、0.03μg/ml,0.03μg/ml、0.03μg/ml。伊曲康唑对血液、口腔、阴道、支气管肺泡灌洗液来源的白念珠菌的MIC50、MIC90分别为0.03μg/ml、0.03μg/ml,0.06μg/ml、0.125μg/ml,0.06μg/ml、2μg/ml。氟康唑对血液、口腔、阴道、支气管肺泡灌洗液来源的白念珠菌的MIC50、MIC90分别为0.25μg/ml、0.25μg/ml,0.5μg/ml、32μg/ml,1μg/ml、8μg/ml,0.25μg/ml、0.25μg/ml。特比萘芬对血液、口腔、阴道、支气管肺泡灌洗液来源的白念珠菌的MIC50、MIC90分别为0.125μg/ml、0.5μg/ml,0.03μg/ml、0.125μg/ml,0.125μg/ml、4μg/ml,0.03μg/ml、0.125μg/ml。卡泊芬净对血液、口腔、阴道、支气管肺泡灌洗液等来源的64株白念珠菌的MIC50、MIC90分别为0.03μg/ml、0.125μg/ml,0.03μg/ml、0.03μg/ml,0.03μg/ml、0.06μg/ml,0.03μg/ml、0.125μg/ml。米卡芬净对血液、口腔、阴道、支气管肺泡灌洗液等来源的64株白念珠菌的MIC50、MIC90分别为0.03μg/ml、0.03μg/ml,0.03μg/ml、0.03μg/ml,0.03μg/ml、0.03μg/ml,0.03μg/ml、0.03μg/ml。结论不同抗真菌药物对不同部位来源菌株的药物敏感性存在着一定的差异,口腔、阴道来源的白念珠菌相对血液、肺泡灌洗液来源的白念珠菌出现耐药的频率更高。更多还原

【Abstract】 Chapter Ⅰ Establishment and application of a mouse model of candidiasis with concurrent oral and vaginal mucosal infectionSection Ⅰ Establishment of a mouse model of candidiasis with concurrent oral and vaginal mucosal infectionObjective To establish a mouse model of candidiasis with concurrent oral and vaginal mucosal infection. Methods Three groups were set as prednisolone alone, estrogen alone and combinational scheme. These groups were compared and the best one was selected based on clinical manifestation, fungal direct KOH examination, tissue fungal burden and histopathological analysis. Results Mice from the combination group developed oral thrush and vaginal candidiasis at day4(relative to day of inoculation (Day0)). Direct fungal examination showed plenty of hyphae and fungal burden of oral and vaginal tissue reached a high level of105～106cfu/g. Histopathological examination also revealed mucosal epithelium was covered with thick pseudomembranous substance mainly consisting of hyphae accompanied by deep epithelial penetration by hyphae and PMN infiltration. However, these characterizations had a remission at day7. Outcomes from groups of prednisolone alone and estrogen alone were not as good as the combination group. Conclusion (1) Prednisolone combined with estrogen could successfully establish a mouse model of candidiasis with concurrent oral and vaginal mucosal infection;(2) The combinational scheme was superior than single treatment scheme.Section Ⅱ Dynamic investigation of mouse model with concurrent oral and vaginal mucosal candida albicans infection.Objective To observe the changes of mouse model with concurrent oral and vaginal mucosal candida albicans infection at different time points. Methods Establishment the mouse model with concurrent oral and vaginal mucosal candida albicans infection using the combinational scheme and observe the changes such as weight, clinical manifestations, tissue fungal burden and histopathological examination at day1,3,5and7. Results The weight of mice gradually descended after inoculation, especially from day1to3. Pseudomembranous lesions appeared on dorsal mucosa of tongue at day2-3and had a more prominent manifestation between day3and day5that covering nearly90%of the whole mucosal surface. Tissue fungal burden had a upward tendency from day1and reached a maximum between day3and5(about105～106cfu/g), while the values descended after day5. Histopathological examination showed yeast cells existing on the mucosal surface of tongue and in the vaginal lumen with rare hyphae invading epithelium. Then thick layer of hyphae had formed on the mucosal surface since day3and hyphae invaded deeply into the local tissue accompanied by destruction of epithelium and inflammatory infiltration. The epithelium of both anatomical tissues began to recover after day5and had a prominent recession at day7. Conclusion The mouse model with concurrent oral and vaginal mucosal candida albicans infection had the most typical manifestation from day3to5after infection using the method of combinational scheme.Section Ⅲ Establishment of a mouse model of candidiasis with concurrent oral and vaginal mucosal infection using different glucocorticoids with estrogen.Objective To explore the best regimen among different glucocorticoids to establish mouse model with concurrent oral and vaginal mucosal candida albicans infection. Methods Two kinds of mice were used and separately divided into three groups of prednisolone with estrogen, cortisone with estrogen and dexamethasone with estrogen. The best one was chosen mainly based on results of weight loss and fugal burden at day3. Results The weight had a persistent loss from day1to7during group of ICR mice treated with prednisolone and estrogen, while the weight loss of the other two groups stopped and rebounded from day3-5. Both kinds of mice treated with prednisolone and estrogen had the highest fungal burden and the results of ICR mice were ATCC62342(oral,1.05×106cfu/g; vagina,5.50×105cfu/g) Vs SC5314(oral,4.79×105cfu/g; vagina 4.07×105cfu/g). As to BALB/c mice, the outcomes were ATCC62342(oral,1.92×106cfu/g; vagina,1.33×106cfu/g) Vs SC5314(oral,7.57×105cfu/g; vagina,2.58x105cfu/g). Conclusion (1) Prednisolone was most suitable for model establishment;(2) ATCC62342had a more favorable result than SC5314;(3) Both ICR mice and BALB/c mice were suitable for model establishment.Section IV Establishment of a mouse model of candidiasis with concurrent oral and vaginal mucosal infection using cyclophosphamide and (or) prednisolone combined with estrogen.Objective To explore the best regimen using cyclophosphamide (CTX) and (or) prednisolone combined with estrogen to establish mouse model with concurrent oral and vaginal mucosal candida albicans infection. Methods Two kinds of mice were used and separately divided into three groups of prednisolone with estrogen, CTX with estrogen and CTX combined with prednisolone and estrogen. The best one was chosen mainly based on results of weight loss and fugal burden at day3. Results The weight had a prominent loss at day3among all groups of mice, especially during BALB/c mice. All mice had a weight loss than10%refer to the weight before infection and BALB/c mice treated with the combinational scheme were75percent of initial weight. The effects of combinational scheme were the most striking in two strains of mice and candida albicans. As to fungal burden, prednisolone combined with estrogen and the combinational scheme had the most values, while the latter also had a high mortality of mice. Conclusion (1) Prednisolone was more suitable for model establishment than CTX;(2) The scheme of prednisolone combined with CTX and estrogen had a prominent fatal effect on mice though the fungal burden was synchronously favorable.Section V Application of the mouse model of candidiasis with concurrent oral and vaginal mucosal infection using prednisolone with estrogen. Objective To explore the probable applications of mouse model with concurrent oral and vaginal mucosal candida albicans infection. Methods ICR mice were first used to establish the model under the treatment of prednisolone with estrogen. Then (1) Fluconazole was used for the therapeutic assessment to candida albicans infection;(2) Oral and vaginal tissue were resected and the following homogenates were examined for the dynamic changes of IL-17and IL-23using the method of Elisa. Results (1) Both strains of mice infected by SC5314had a prominently declined fungal burden at day3through fluconazole therapy and candida albicans could be totally cleared out at day5. On the contrary, mice infected by ATCC62342failed to reduce the fungal burden throughout one week;(2) IL-17in oral tissue began to rise after infection and reached maximum between day3and5, while IL-17in vaginal tissue have a elevated value only at day1and then descend to normal level. IL-23from both sites had a prominent elevation throughout the total observation time. Conclusion The mouse model with concurrent oral and vaginal mucosal infection was suitable for research of in vivo pharmacodynamics and mucosal immunology.Chapter II Virulence comparison of candida albicans isolated from different human candidiasis Section I Evaluation of extracellular enzymatic activities of candida albicans isolated from different human candidiasisObjective To investigate the enzymatic activities of64candida albicans isolated from different human candidiasis. Methods The egg yolk agar, bovine serum albumin agar, tween-80agar and blood agar were used to detect phopholipase, proteinase, esterase and haemolytic activities, respectively. Results (1) Phospholipase activities:strains from blood and oral mucosa showed most powerful, while vaginal mucosa the weakest;(2) Proteinase activities:strains from oral and vaginal mucosa revealed stronger proteinase activities than blood strains;(3) Esterase activities:strains isolated blood showed strongest activity than strains from other sources;(4) Haemolytic activities:isolations from oral and vaginal mucosa revealed stronger proteinase activities than blood isolations Conclusion Candida albicans from different origins had great variations in the activities of extracellular enzymes.Section II Antifungal susceptibility profiling of candida albicans isolated from different human candidiasisObjective To profile the susceptibility of candida albicans isolated from different human candidiasis to seven commonly used antifungal agents. Methods The antifungal susceptibility tests were performed according to the CLSI-A2protocol. The tested antifungal agents included amphotericin B, fluconazole, itraconazole, voriconazole, terbinafine, caspofungin and micafungin. Results All isolates of candida albicans were susceptible to amphotericin B, caspofungin and micafungin. Five strains resisted to fluconazole (1isolated from blood,1isolated from oral and3isolated from vagina); Six strains resisted to itraconazole (2isolated from blood,2isolated from oral and2isolated from vagina); Two strains resisted to voriconazole (1isolated from blood and1isolated from oral); Nine strains resisted to terbinafine (2isolated from blood,5isolated from oral and2isolated from vagina). Conclusion Candida albicans had variable susceptibility to different antifungal agents. There are also differences of in vitro antifungal susceptibility among candida albicans isolated from different origin to fluconazole, itraconazole, voriconazole and terbinafine. Strains of candida albicans from superficial candidiasis seemed more likely resisting to antifungal agents than strains from invasive candidiasis.更多还原