【作者】 卢鑫；

【导师】 施毅；

【作者基本信息】 南京大学 ， 内科学， 2012， 硕士

【摘要】 侵袭性肺曲霉病(invasive pulmonary aspergillosis, IPA)多发生于重度免疫缺陷人群,死亡率高。抗真菌药通过雾化吸入给药近来受到广泛关注。与系统用药相比,雾化给药少量药物即可达到肺部高药物浓度,并且因较少入血降低了药物系统毒性。本研究的目的是建立免疫抑制小鼠IPA模型,并评价雾化吸入卡泊芬净在预防和治疗IPA的疗效及安全性。主要分为以下三个部分：1.免疫抑制小鼠侵袭性肺曲霉病模型的建立目的：动物实验被广泛的应用于侵袭性肺曲霉病(IPA)诊断及治疗方面的研究,本文利用气管插管法建立免疫抑制小鼠IPA模型。方法：以环磷酰胺及地塞米松对小鼠进行免疫抑制处理后,气管插管气管内滴入烟曲霉孢子悬液,通过组织病理进行验证,并观察小鼠生存-时间曲线。同时运用平板菌落计数方法比较气管插管法与传统滴鼻法造模实际进入鼠肺的孢子量。结果：气管插管法可成功建立小鼠IPA模型。小鼠1周内死亡率55%,接种24小时后肺组织匀浆平板菌落计数显示气管插管法所造成的孢子入肺量为5.17±0.32CFU(1g)/g肺组织,而传统滴鼻法仅为3.82±0.49CFU(1g)/g肺组织。结论：气管插管法成功建立小鼠IPA模型,较滴鼻法更适于进行IPA相关研究。2.卡泊芬净雾化吸入给药防治侵袭性肺曲霉病药效学研究目的：通过动物实验对雾化吸入卡泊芬净对IPA的防治效果做初步探讨。方法：免疫抑制小鼠IPA模型,分别给予卡泊芬净腹腔注射及雾化用药,用药策略分为单纯治疗及预防+治疗,同时设置标准治疗——两性霉素B腹腔注射组及对照组,持续用药至感染后第7天。比较各组小鼠生存率及肺组织真菌负荷。结果：与标准治疗——两性霉素B腹腔注射相比,雾化吸入卡泊芬净作为单纯治疗性用药、或预防+治疗用药均能有效降低小鼠肺组织真菌负荷；但只有作为预防+治疗给药时才能有效提高小鼠生存率,且对IPA预防效果与腹注给药相当。结论：动物实验证明卡泊芬净雾化吸入给药是一种有效的预防IPA的给药方式。3.卡泊芬净雾化吸入给药局部安全性研究目的：观察连续多次雾化吸入卡泊芬净后小鼠肺组织病理学及局部炎症因子表达。方法：ICR小鼠分为三组,高剂量组每天雾化2小时,低剂量组每天雾化时间为1小时,设置生理盐水对照组,各组小鼠连续用药7天。各组小鼠行肺组织病理学检查并予以评分；提取肺组织RNA行荧光定量PCR,检测各组小鼠肺组织炎症因子表达。结果：雾化给药结束后各组肺组织病理评分无明显差异,荧光定量PCR检测显示雾化吸入卡泊芬净并未导致肺组织IL-1β、IL-6、TNF-α等炎症因子的活化。结论：动物实验证明卡泊芬净雾化吸入给药对肺组织刺激性小,安全性较好。综上所述,抗真菌药物雾化吸入用药在肺组织局部形成较高药物浓度,作为预防性用药时可显著增强抗真菌效果,且避免了高血药浓度导致的副作用。本研究利用动物实验观察了雾化卡泊芬净用于预防和治疗IPA疗效和安全性,与标准治疗药物——两性霉素B治疗相比,雾化卡泊芬净作为预防性用药时可提高小鼠生存率,并降低肺真菌负荷。雾化用药易于操作且安全性较好,有潜在的临床应用前景。更多还原

【Abstract】 Invasive aspergillosis is a significant cause of morbidity and mortality in heavily immunocompromised patients and is associated with significant hospital costs and therapy complications in those with multiple comorbidities. Targeted pulmonary delivery by aerosolization of antifungals has recently gained attention, The use of aerosols for targeting medication to the receptor sites in the lung has multiple advantages compared to systemic administration, including the use of less drug to provide the same therapeutic effect and the reduction in the likelihood of systemic side effects.Our objective was to establish the mice model of IPA and assess the Efficacy and safety of caspofungin inhalations for prevention and treatment of invasive pulmonary aspergillosis in mice.Three parts of this research are as following: 1. Establishment of animal model of invasive pulmonary aspergillosis in immunosuppressed miceObjective Animal experiments are widely used in the diagnosis and treatment of invasive pulmonary aspergillosis (IPA). We establish an experimental model of IPA in neutropenic ICR mice with method of tracheal intubation.Methods Female ICR mice was immunosuppressed with cyclosphamide and Dexamethasone and inoculated with1×107conodia of Aspergillus fumigates.Patholigic examination were evaluated to determine whether the IPA model was successfully constructed. survival curves was draw,Fungal burden analysis following24h inoculation to compare these two methods.Results IPA was confirmed in neutropenic ICR mice with method of tracheal intubation based on pathologic examination. Mortality of mice was55%within7d after inculation,Numbers of CFU from homogenized lung tissues of IPA mice were5.17±0.32and3.82±0.49(log/gram lung) with these two methods of tracheal intubation and intranasal challenge, respectively.Conclusion The IPA model in immunosuppressed ICR mice is constructed successfully,and tracheal intubation was better than intranasal challenge.2. Efficacy of caspofungin inhalations for prevention and treatment of invasive pulmonary aspergillosis in miceObjective To evaluated the effectiveness of aerosolizing caspofungin as prophylaxis and treatment against invasive pulmonary aspergillosis caused by Aspergillus fumigates in an established model.Methods Immunocompromised ICR mice received either caspofungin by intraperitoneal injection or by aerosolization.dosing began2days prior or after pulmonary incoculation with A. fumigates and continued until7days after pulmonary incoculation. Survival and pulmonary fungal burden were assessed.Results The use of aerosolization of caspofungin,either as a prophylactic or therapeutic agent, is effective in reducing pulmonary fungal burden of immunosuppressed animals with pulmonary aspergillosis. But only caspofungin inhalations for prevention, is effective in reducing mortality of immunosuppressed animals.Conclusion Aerosol administration of caspofungin, especially used as prophylactic,could be an additional approach to optimizing treatment of invasive pulmonary aspergillosis.3. safety of caspofungin inhalations in miceObjective To evaluated the histologic effects, immunogenic potential after servel inhalations of caspofungin.Methods ICR mice received caspofungin by aerosolization dosing for1h/day(LD group),2h/day(HD group), or saline control for up to7days. Coronal sections of the entire lung were stained,viewed by light microscopy, and the histopathologic inflammatory score was obtained for each lobe. Cytokine induction of IL-1β、IL-6、TNF-α was assessed by Real-time PCR.Results The lung histopathologic scores for HD group, LD group, and saline control on days7did not differ between groups. Real-time PCR analysis showed no cytokine induction of IL-1β、IL-6、TNF-α.Conclusion Aerosol administration of caspofungin for up to7days do not apper to be immunogic and do not cause changes in pulmonary histology. Aerosolization of caspofungin is a safe method of pulmonary delivery.In conclusion, Targeted airway delivery of antifungals as prophylaxis against invasive aspergillosis may lead to high lung drug concentrations while avoiding toxicities associated with systemically administered agents. We evaluated the effectiveness of aerosolizing caspofungin as prophylaxis and treatment against invasive pulmonary aspergillosis caused by Aspergillus fumigatus in an established murine model. Inhaled caspofungin significantly improved survival and limited the pulmonary fungal burden, as assessed by histopathology, compared to control and amphotericin B treatments. Inhalations were easy to administer and were well tolerated. This experience suggests The physiologic profile of the response to inhaled caspofungin is acceptable and is well tolerated. It merits further assessment in the treatment of pulmonary fungal disease.更多还原