【摘要】：背景和目的煙曲霉是一類廣泛存在與自然環(huán)境中的腐生菌,在不同免疫狀態(tài)的宿主中可引起不同類型的曲霉病。在免疫抑制人群中常引起致命性的侵襲性肺曲霉病(Invasive Pulmonary Aspergillosis,IPA),盡管IPA的診斷方法和治療方式在不斷進展,但其發(fā)病率和死亡率仍較高。在過去幾十年中,全世界范圍內(nèi)陸續(xù)出現(xiàn)了三唑類抗真菌藥耐藥的曲霉菌株,這可能導(dǎo)致曲霉病治療選擇的減少以及治療的失敗,這亟需我們尋找新的治療方法。免疫功能正常的人群在吸入霉菌孢子后一般不會發(fā)病,表明宿主免疫功能在抵抗曲霉菌過程中發(fā)揮重要作用。已有研究提示肺泡表面活性蛋白D(Surfactant Protein D,SP-D)可通過增強機體免疫反應(yīng)來抵抗曲霉菌的入侵,對IPA模型小鼠起到一定的治療效果。本研究通過聯(lián)合應(yīng)用SP-D和伏立康唑與單獨應(yīng)用伏立康唑或SP-D對IPA小鼠治療效果的比較,并進一步探討SP-D增強伏立康唑治療效果可能存在的免疫機制。研究方法通過腹腔注射環(huán)磷酰胺建立免疫抑制小鼠模型,并以滴鼻方式感染臨床煙曲霉菌株成功建立并驗證BALB/c小鼠IPA模型。根據(jù)IPA小鼠在不同給藥濃度的伏立康唑及SP-D干預(yù)下15天生存率確定給藥劑量。在感染第0天、第1天和第2天,分別給予SP-D(10μg/d),伏立康唑(負荷劑量30 mg/kg/d,維持劑量15 mg/kg/d)及SP-D聯(lián)合伏立康唑治療。觀察小鼠第4天肺部的病理學變化、真菌負荷及15天生存率評估治療效果。進一步檢測小鼠第4天肺部IFN-γ、IL-4、IL-17及IL-23細胞因子水平變化及流式細胞術(shù)測脾臟內(nèi)TH1、TH2及TH17細胞的變化闡述可能發(fā)生的機制。結(jié)果PAS染色發(fā)現(xiàn)聯(lián)合治療組較單藥組和IPA模型組,肺組織中炎性細胞聚集明顯,孢子菌絲顯著減少;聯(lián)合給藥組較單藥組肺部真菌負荷顯著降低;聯(lián)合治療組較伏立康唑和模型組IFN-γ水平顯著增高(552.9±28.4 pg/ml,463±31.5pg/ml,297.9±16.1pg/ml,P0.01);IL-4水平顯著降低(83.8±10.9 pg/ml,129.3±15.2 pg/ml,200±16.5 pg/ml,P0.01);IL-17水平顯著降低(97.8±6.6pg/ml,164.9±21.6 pg/ml,203.9±50.2 pg/ml,P0.01);聯(lián)合治療組較伏立康唑組、SP-D組和IPA模型組,脾臟中TH1數(shù)量顯著增多(9.87±1.1%,5.65±0.41%,6.86±0.75%,4.2±0.21%,P0.01);TH2數(shù)量顯著減少(4.82±0.27%,7.01±0.58%,6.10±0.61%,8.64±0.18%,P0.01);TH17數(shù)量顯著減少(1.63±0.11%,2.23±0.25%,2.17±0.24%,3.02±0.24%,P0.01);15天生存率明顯延長(60%,40%,30%,10%)。結(jié)論表明聯(lián)合治療組通過平衡TH1、TH2和TH17之間的免疫反應(yīng)更好的發(fā)揮抗菌作用。因此SP-D可通過平衡不同TH細胞亞組之間的免疫反應(yīng)來增強伏立康唑?qū)PA小鼠的抗真菌治療作用,并顯著延長生存率,為臨床上治療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- 緯 in the combined treatment group was significantly higher than that in the voleconazole group and the model group (552.9 鹵28.4 PG / ml, 463 鹵31.5 pgml / ml, 297.9 鹵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 鹵10.9 pgml / ml 129.3 鹵15.2 PG / ml 200 鹵16.5 PG / ml P0.01) (97.8 鹵6.6 PG / ml, 164.9 鹵21.6 PG / ml, 203.9 鹵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 鹵1.1 鹵5.65 鹵0.41) and the number of TH2 decreased significantly (4.82 鹵0.277.01 鹵0.581.61 鹵0.18P0.01). The number of TH17 significantly decreased (1.63 鹵0.112.23 鹵2.23 鹵0.242.02 鹵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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R519

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