隐球菌肉芽肿局部免疫微环境与肺隐球菌病结局的关系

The lung is the commonest route of entry for cryptococcal infection. Even when Cryptococci is engulfed by pulmonary macrophages, it can survive within them rather than being destroyed. Epidemiology data and clinical features indicate that this microorganism could cause latent infection in lungs. Reactivation of latent infection and disseminated disease could occur in patients who have received high-dose corticosteroids. Our previous study showed that genes relating to alternative activation of macrophages were highly up-regulated. Alternative activation of macrophages could lead to intracellular survival of pathogens, atypical granuloma formation, and fibrosis. In this study, pet-ct and Immunohistochemistry are applied to evalue the imaging changes and immune microenvironment of the pulmonary lesions. The aim of this study is to investigate the relationship among radiographic feautes, pulmonary immune microenvironment, structure of pulmonary granuloma, and fungi load. This study will explore radiographic and histopathologic features of pulmonary cryptococcosis to help predict the outcome of the infection. Meanwhile, the effect of glucocorticoid intervention in the progress of pulmonary cryptococcosis will also be assessed.

肺是隐球菌进入人体的主要门户器官，隐球菌可被肺泡巨噬细胞吞噬并能在细胞内存活。流行病学数据和肺部隐球菌病转归均提示肺是隐球菌潜伏感染的靶器官，而大剂量糖皮质激素使用是肺部隐球菌合并播散性感染的高危因素。前期我们发现隐球菌感染小鼠后，肺泡巨噬细胞替代激活途径相关基因表达显著增高。巨噬细胞替代激活与隐球菌胞内存活、肉芽肿结构和纤维化有密切关系。本课题中，我们通过PET-CT动态观察隐球菌感染后小鼠肺部影像学改变和病灶活动情况。对不同病灶进行病理和免疫组化观察，同时计算不同病灶菌荷量和中枢菌荷量，明确肺部病变影像学改变、免疫微环境、肉芽肿结构和病原菌生长、扩散之间的关系。将临床影像学和病理学与疾病预后相联系。同时，对慢性肺部感染小鼠进行激素干预，观察病情变化，明确激素作用条件下免疫微环境如何变化导致感染扩散。

我国的隐球菌病主要是由新生隐球菌格鲁比变种（Cryptococcus neoformasn var. grubii）引起的，在我国环境中分离的隐球菌菌株也以该变种为主，鸽粪中常可分离出该菌。目前，H99是临床和科研中常用的新生隐球菌格鲁比变种标准菌株。本研究发现，H99对Balb/c小鼠的致病力高于实验所用的新生隐球菌格鲁比变种的临床分离株和环境分离株（所有菌株均进行了MLST分析，鉴定为VNI型隐球菌）。新生隐球菌H99菌株按5×105对Babl/c小鼠进行呼吸道吸入感染造模，其引起的Babl/c小鼠的中位死亡期为18.5天，与其余临床分离株和环境分离株相比差异有统计学意义。部分临床分离株可在小鼠颅内分离出新生隐球菌菌株。动物实验中发现，临床分离株CH16和环境分离株T228-2及T298感染Babl/c小鼠后颅内未分离出隐球菌，说明这些菌株缺少对Babl/c小鼠的嗜中枢性。我们选取CH16新生隐球菌临床分离株进行小鼠肺部感染造模，6周雌性Babl/c小鼠感染后，可存活50天以上，在43天时进行小动物pet/ct检查，可见小鼠肺内多发高代谢病灶，大体解剖后可见散在分布的炎性肉芽肿样结构。通过菌荷量分析发现，炎性肉芽肿菌含量显著低于肺部胶样结节。该模型后期可用于隐球菌感染小鼠肺部炎症与嗜中枢性差异化分析，明确何种免疫反应参与了隐球菌小鼠模型的嗜中枢性。