在臨床上對疾病進程的診斷中，組織學的檢驗是很常見的。組織學的結果讓我們可以直接去觀察發炎部位的情況，是個可以讓我們發現疾病致病原因的工具之一。在本篇研究中，我們利用免疫組織化學染色方法配合較客觀的定量方式去觀察不同類型的T細胞浸潤在IPEX患者的腸道及腎臟檢體中的情況，以及利用甲苯異氰酸酯(TDI)造成小鼠肺部發炎模式中肺部T細胞分佈的情況。IPEX症候群是一種X染色體性聯遺傳的罕見疾病，常發生在幼小的嬰兒或男性孩童身上，產生的症狀包括免疫功能失調、多種內分泌器官病變和腸道病變，IPEX發生的原因目前已知與FOXP3基因突變有關。在本篇研究我們使用甲苯異氰酸酯來引起小鼠肺部發炎，甲苯異氰酸酯是目前常被用來製造聚氨酯等相關產品的化合物，因被發現會引起職業性氣喘而逐漸受到注目。要在組織切片上計算不同細胞類型所占的比例，並為了能得到更客觀的計量結果，我們利用TissueGnostics Cytometry儀器在高倍率條件下去掃描整個組織切片，配合以電腦為主的分析軟體，預計可以增進組織學定量上的正確性與客觀性。我們觀察了在免疫反應中對T細胞分化扮演相當重要角色的一些轉錄因子，包括T-bet、GATA-3、RORt和Foxp3。在對IPEX患者檢體的觀察中，我們發現在以免疫抑制藥物治療之前與Th2細胞分化相關的轉錄因子GATA-3表現的比例比控制組的患者來的高，猜測在IPEX症候群中Th2細胞可能是引起腸道和腎臟發炎症狀的主要細胞。臨床上使用的免疫抑制劑有效地降低了效應性T細胞在組織中的浸潤。在以甲苯異氰酸酯引起小鼠肺部發炎的模式中，我們發現Th17細胞是主要浸潤在肺部的細胞，與我們實驗室先前的研究結果發現與Th17相關的細胞激素IL-17在以甲苯異氰酸酯刺激的組別中較高是一致的。另外，與原生野生型小鼠比較之下，菸醯胺腺嘌呤二核酸磷酸氧化酶(NADPH oxidase)基因缺陷小鼠(Ncf1-/- mice) 在肺部浸潤的細胞中表現較低比例的效應性T細胞，肺部發炎程度也較低，顯示白血球的菸醯胺腺嘌呤二核酸磷酸氧化酶對於甲苯異氰酸酯引起的肺發炎反應是占有重要角色的。藉著利用以電腦分析為主，較客觀的組織學定量方法，我們可以直接觀察到不同疾病中免疫反應的變化，並在組織學的研究上提供了新的分析方法。

Histological examination is generally used in clinical diagnosis of pathogenic process. It provides a direct observation of local site inflammatory situations and is a tool to find out the cause of diseases. We used immunohistochemistry and objective quantification to detect the different types of T helper cells in the intestine and kidney biopsy blocks of IPEX patient and in the lung of Toluene diisocyanate(TDI)-challenged mice. IPEX (Immunodysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is a rare X-linked disease found in young infants and children, and the disease associated with FOXP3 mutation has been report. TDI, a compound widely used in the production of polyurethane foams and related products, is noted for inducing occupational asthma. TDI was used to induce airway inflammation in mouse model. For objective counting of the percentages of different cell types in tissues, we use TissueGnostics Cytometry to scan a large overview of slide with high magnification. Computer-assisted analysis enhanced the accuracy and objectiveness in histopathology results. We studied transcription factors critical for immune response including T-bet, GATA-3, RORt and Foxp3. The expression percentage of Th2-associated transcription factor GATA3 in IPEX patient were higher than control before treatment, suggesting Th2 cells were the main cells responsible for intestine and kidney inflammation in IPEX syndrome. Immunosupressive treatments effectively decreased the effect cells in the IPEX patient. In the TDI-induced lung inflammation mice model, we found Th17 cells were the dominant cells infiltrated in the lung, consistent with our previous studies that IL-17 level was increasing in TDI-challenged group. Moreover, NADPH oxidase deficiency (Ncf1-/-) mice show lower percentage of effector cells in infiltration cells than wild type mice, indicating the importance of leukocyte NADPH oxidase in the TDI-induced airway inflammation. By using objective quantification methods to analyze histochemical investigations, we become able to reveal certain critical changes in pathogenic immune response.

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