SP-D联合伏立康唑对免疫抑制小鼠侵袭性肺曲霉病治疗效果的研究

发布时间：2018-08-14 10:09

【摘要】：背景和目的烟曲霉是一类广泛存在与自然环境中的腐生菌,在不同免疫状态的宿主中可引起不同类型的曲霉病。在免疫抑制人群中常引起致命性的侵袭性肺曲霉病(Invasive Pulmonary Aspergillosis,IPA),尽管IPA的诊断方法和治疗方式在不断进展,但其发病率和死亡率仍较高。在过去几十年中,全世界范围内陆续出现了三唑类抗真菌药耐药的曲霉菌株,这可能导致曲霉病治疗选择的减少以及治疗的失败,这亟需我们寻找新的治疗方法。免疫功能正常的人群在吸入霉菌孢子后一般不会发病,表明宿主免疫功能在抵抗曲霉菌过程中发挥重要作用。已有研究提示肺泡表面活性蛋白D(Surfactant Protein D,SP-D)可通过增强机体免疫反应来抵抗曲霉菌的入侵,对IPA模型小鼠起到一定的治疗效果。本研究通过联合应用SP-D和伏立康唑与单独应用伏立康唑或SP-D对IPA小鼠治疗效果的比较,并进一步探讨SP-D增强伏立康唑治疗效果可能存在的免疫机制。研究方法通过腹腔注射环磷酰胺建立免疫抑制小鼠模型,并以滴鼻方式感染临床烟曲霉菌株成功建立并验证BALB/c小鼠IPA模型。根据IPA小鼠在不同给药浓度的伏立康唑及SP-D干预下15天生存率确定给药剂量。在感染第0天、第1天和第2天,分别给予SP-D(10μg/d),伏立康唑(负荷剂量30 mg/kg/d,维持剂量15 mg/kg/d)及SP-D联合伏立康唑治疗。观察小鼠第4天肺部的病理学变化、真菌负荷及15天生存率评估治疗效果。进一步检测小鼠第4天肺部IFN-γ、IL-4、IL-17及IL-23细胞因子水平变化及流式细胞术测脾脏内TH1、TH2及TH17细胞的变化阐述可能发生的机制。结果PAS染色发现联合治疗组较单药组和IPA模型组,肺组织中炎性细胞聚集明显,孢子菌丝显著减少;联合给药组较单药组肺部真菌负荷显著降低;联合治疗组较伏立康唑和模型组IFN-γ水平显著增高(552.9±28.4 pg/ml,463±31.5pg/ml,297.9±16.1pg/ml,P0.01);IL-4水平显著降低(83.8±10.9 pg/ml,129.3±15.2 pg/ml,200±16.5 pg/ml,P0.01);IL-17水平显著降低(97.8±6.6pg/ml,164.9±21.6 pg/ml,203.9±50.2 pg/ml,P0.01);联合治疗组较伏立康唑组、SP-D组和IPA模型组,脾脏中TH1数量显著增多(9.87±1.1%,5.65±0.41%,6.86±0.75%,4.2±0.21%,P0.01);TH2数量显著减少(4.82±0.27%,7.01±0.58%,6.10±0.61%,8.64±0.18%,P0.01);TH17数量显著减少(1.63±0.11%,2.23±0.25%,2.17±0.24%,3.02±0.24%,P0.01);15天生存率明显延长(60%,40%,30%,10%)。结论表明联合治疗组通过平衡TH1、TH2和TH17之间的免疫反应更好的发挥抗菌作用。因此SP-D可通过平衡不同TH细胞亚组之间的免疫反应来增强伏立康唑对IPA小鼠的抗真菌治疗作用,并显著延长生存率,为临床上治疗IPA提供了新的思路。
[Abstract]:Background and objective Aspergillus fumigatus is a kind of rot bacteria widely existing in natural environment, which can cause different types of aspergillosis in host with different immune states. (Invasive Pulmonary AspergillosisIPA is a fatal disease in immunosuppressive population. Although the diagnosis and treatment of IPA are progressing, the morbidity and mortality of IPA are still high. In the past few decades, triazole-resistant aspergillus strains have appeared all over the world, which may lead to the decrease of treatment options and the failure of treatment. The host immune function plays an important role in the resistance to Aspergillus sp. It has been suggested that the alveolar surfactant protein (D (Surfactant Protein DnSP-D) can resist the invasion of Aspergillus by enhancing the immune response of the body, and has a certain therapeutic effect on IPA model mice. In this study, the therapeutic effect of SP-D and voriconazole on IPA mice was compared with that of Volconazole or SP-D alone, and the possible immunological mechanism of SP-D enhanced Volconazole was discussed. Methods the immunosuppressive mice model was established by intraperitoneal injection of cyclophosphamide, and the IPA model of BALB/c mice was successfully established and verified by infecting clinical aspergillus fumigatus strain by nasal drip. The dosage was determined according to the 15-day survival rate of IPA mice treated with different concentrations of Volconazole and SP-D. On day 0, day 1 and day 2, SP-D (10 渭 g / d), Volconazole (load dose 30 mg / kg / d, maintenance dose 15 mg/kg/d) and SP-D combined with Volconazole were given respectively. The lung pathological changes, fungal load and 15-day survival rate of mice were observed on day 4. The changes of IL-17 and IL-23 cytokines in the lung of mice on the 4th day and the changes of TH2 and TH17 cells in the spleen by flow cytometry were analyzed. Results PAS staining showed that the inflammatory cells in the lung tissue of the combined treatment group were significantly lower than those in the single drug group and the IPA model group, and the spore mycelium was significantly decreased in the combined treatment group than in the single drug group, and the pulmonary fungal load in the combined treatment group was significantly lower than that in the single drug group. The level of IFN- 纬 in the combined treatment group was significantly higher than that in the voleconazole group and the model group (552.9 卤28.4 PG / ml, 463 卤31.5 pgml / ml, 297.9 卤16.1 pg / ml, P0.01), and the level of IL-4 in the combined treatment group was significantly lower than that in the voleconazole group and the IPA model group (83.8 卤10.9 pgml / ml 129.3 卤15.2 PG / ml 200 卤16.5 PG / ml P0.01) (97.8 卤6.6 PG / ml, 164.9 卤21.6 PG / ml, 203.9 卤50.2 pgml / ml P0.01), and was significantly lower in the combined treatment group than that in the Vollikone group and the IPA model group, and the level of IL-17 in the combined treatment group was significantly lower than that in the voleconazole group and the IPA model group. The number of TH1 in the spleen significantly increased (9.87 卤1.1 卤5.65 卤0.41) and the number of TH2 decreased significantly (4.82 卤0.277.01 卤0.581.61 卤0.18P0.01). The number of TH17 significantly decreased (1.63 卤0.112.23 卤2.23 卤0.242.02 卤0.24P0.01) and the 15-day survival rate was significantly prolonged (60404030P 0.01). Conclusion the results showed that the combined treatment group had better antimicrobial effect by balancing the immune reaction between TH1 TH2 and TH17. Therefore, SP-D can enhance the antifungal effect of Volconazole on IPA mice by balancing the immunoreaction between different th cell subgroups, and prolong the survival rate significantly, which provides a new idea for clinical treatment of IPA.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R519

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