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標題: | 探討自體抗體在自體免疫水泡病的角色 Explore the roles of autoantibodies in the autoimmune bullous diseases |
作者: | Yung-Tsu Cho 卓雍哲 |
指導教授: | 張逸良(Yih-Leong Chang) 張逸良(Yih-Leong Chang | ntuhylc@gmail.com | ), |
關鍵字: | 自體免疫水泡病,天疱瘡,G型暨A型免疫球蛋白天疱瘡,類天疱瘡,自體抗體, autoimmune blistering disease,pemphigus,IgG/IgA pemphigus,pemphigoid,autoantibody, |
出版年 : | 2022 |
學位: | 博士 |
摘要: | 自體免疫性水泡病是一種因自體抗體所導致的皮膚或黏膜處會形成水泡或破皮的疾病,其發生率在台灣雖然不高,但對病患而言是一種嚴重影響生活及可能致死的疾病。自體免疫性水泡病可分成天疱瘡(pemphigus)及類天疱瘡(pemphigoid)兩大類型,其中又各自包含不同亞型的疾病。 天疱瘡的形成原因,目前主要的認知為病患體內的自體抗體結合至表皮細胞間的橋粒蛋白(desmosomal proteins),造成表皮細胞分離所致。診斷天疱瘡必須根據臨床表現、皮膚病理切片、皮膚組織直接免疫螢光染色、以及血清學檢測的結果才能確立,並判斷屬於天疱瘡中的何種亞型。天疱瘡中最主要的兩種亞型為落葉型天疱瘡(pemphigus foliaceus)以及尋常型天疱瘡(pemphigus vulgaris)。落葉型天疱瘡的病患體內具有抗第一型橋粒芯蛋白(anti-desmoglein 1)的G型免疫球蛋白(IgG)自體抗體,只會造成皮膚的病灶;尋常型天疱瘡的病患體內則是具有抗第三型橋粒芯蛋白(anti-desmoglein 3)的G型免疫球蛋白自體抗體,或是同時具有抗第一型以及抗第三型橋粒芯蛋白的自體抗體,導致出現以黏膜病灶為主的臨床表現(mucosa-predominant type pemphigus vulgaris),或是同時出現黏膜以及皮膚的病灶(mucocutaneous type pemphigus vulgaris)。 除了上述兩種主要的亞型之外,還有其他較為少見的天疱瘡亞型;其中相對少見且較少被探討的一種即為G型暨A型免疫球蛋白天疱瘡(IgG/IgA pemphigus)。G型暨A型免疫球蛋白天疱瘡的特點為病患同時存在有G型以及A型免疫球蛋白的抗橋粒芯蛋白自體抗體,但其臨床表現則是有較多的變化,且過去在僅有的少數報告文獻之中存在不少爭議之處;本論文的第一部分將以病歷回顧的方式收集天疱瘡病人的臨床資料、病理報告、血液檢查及免疫螢光檢查結果,並利用病患皮膚切片的剩餘檢體來進行免疫組織化學染色,藉由這些資訊來進一步了解G型暨A型免疫球蛋白天疱瘡的獨特表現。類天疱瘡的致病機轉與天疱瘡相似,不同之處在於類天疱瘡的自體抗體主要是作用在表皮與真皮間的基底細胞膜層(basement membrane zone)中的蛋白結構BP180和BP230。過去研究顯示這些自體抗體在結合其抗原之後,會造成補體系統的活化(complement system activation),進而吸引發炎細胞,如肥大細胞(mast cell)、嗜中性白血球(neutrophil)及嗜伊紅性白血球(eosinophil)的浸潤,這些細胞所釋放的發炎物質進一步破壞基底細胞膜層的結構造成水泡的形成;此外,有另外一些研究指出類天疱瘡的自體抗體可以直接作用在表皮細胞上,造成細胞表面的BP180蛋白發生內噬作用(internalization),於細胞內進一步被分解(degradation);這兩種機轉都可能貢獻在類天疱瘡水泡的形成。因此,在治療上若能使用藥物抑制自體抗體作用後產生的發炎反應,同時抑制自體抗體所導致的BP180內噬現象,預期將對疾病的控制有所助益。本論文的第二部分將用diacerein及berberine這兩種藥物來進行相關的測試,探究這兩種藥物是否可以減少自體抗體作用於表皮細胞後產生的發炎反應及是否可以抑制自體抗體所引起的BP180的內噬現象。 在論文第一部分的結果中,我們找到了22位G型暨A型免疫球蛋白天疱瘡的病例,發現了G型暨A型免疫球蛋白天疱瘡的一些獨特表現,臨床上病患呈現兩大類的皮膚表現,一類是以圓弧形或圓圈狀的紅斑合併水泡或破皮為主,另一類是表現與一般型的天疱瘡相似,主要是散在的水泡或破皮病灶,同時這些G型暨A型免疫球蛋白天疱瘡有40.9%的病患合併有口腔黏膜病灶的發生,在皮膚組織切片下比較特別的是G型暨A型免疫球蛋白天疱瘡會有表皮內的發炎細胞浸潤與聚集,其中有40.9%的病例可見噬中性白血球,31.8%的病例有噬伊紅性白血球;在直接免疫螢光染色的檢查中,100%的病例有G型免疫球蛋白以及A型免疫球蛋白的沉積,另外有高達81.8%的病例可見第三補體(C3)的沉積;這些表現的特點可以明顯與其他亞型的天疱瘡做區隔。同時在免疫組織化學染色的結果中,G型暨A型免疫球蛋白天疱瘡也在表皮層的部分表現出了較高的interleukin-8 (IL-8)和matrix metalloproteinase-9 (MMP-9),這兩者也反映出了A型免疫球蛋白、噬中性白血球及噬伊紅性白血球在G型暨A型免疫球蛋白天疱瘡致病機轉的可能角色。 在論文的第二部分中,我們使用純化過後的類天疱瘡病患的血清,來探討類天疱瘡自體抗體對表皮細胞的作用。我們利用HaCaT細胞來做測試,發現在加入類天疱瘡病患血清一起培養後,上清液中的發炎物質IL-6和IL-18會有上升的情形,可見自體抗體對HaCaT細胞具有一定刺激性的影響,然而這樣的影響在預先加入了治療藥物diacerein或berberine時會減輕;另一方面,我們使用免疫螢光染色來偵測HaCaT細胞在加入類天疱瘡病患血清後,其細胞表面BP180蛋白的變化情形,我們發現加入類天疱瘡病患血清後,HaCaT細胞表面的BP180會有內嗜現象,造成HaCaT細胞彼此間的分離,然而這個BP180的內嗜現象可以在預先加入了治療藥物diacerein或berberine時被抑制,且免疫螢光染色的結果顯示這種diacerein或berberine的抑制作用很可能是透過protein kinase C的影響。綜合這些結果,我們可以知道diacerein和berberine是具有治療類天疱瘡潛力的藥物。 總結來說,本論文統整了G型暨A型免疫球蛋白天疱瘡的特別表現,同時探討了G型免疫球蛋白、A型免疫球蛋白及相對應的發炎細胞在G型暨A型免疫球蛋白天疱瘡的可能角色;此外本論文展示了類天疱瘡自體抗體對表皮細胞的作用,包括了刺激發炎物質的產生及誘使BP180內嗜現象的發生,且發現了diacerein和berberine藥物有抑制這些作用的效果。期待本論文的結果可以對自體免疫水泡病病患的治療與疾病的瞭解提供更進一步的進展。 Autoimmune bullous diseases are a group of diseases affecting the skin and mucosal area which are caused by autoantibodies. The incidence of autoimmune bullous diseases is low in Taiwan. However, the diseases would impair the patients’ daily activities and may cause mortality. Autoimmune bullous diseases can be separated into two main types: pemphigus and pemphigoid. The pathomechanism of pemphigus is mainly resulted from the anti-desmoglein antibodies. These autoantibodies may lead to acantholysis. To make a diagnosis of pemphigus, the combinations of clinical presentation, histopathologic examination, direct immunofluorescence study, and the result of serology profile are required. The two main subtypes of pemphigus are pemphigus foliaceus (PF) and pemphigus vulgaris (PV). Patients with PF have anti-desmoglein 1 (anti-Dsg1) immunoglobulin G (IgG) antibodies, which cause lesions only located on the skin. Patients with PV have anti-desmoglein 3 (Dsg3) IgG antibodies or both anti-Dsg1 and anti-Dsg3 IgG antibodies, which lead to the formation of lesions mainly on the mucosal areas (mucosa-predominant type PV) or lesions on the both mucosal areas and skin (mucocutaneous type PV). In addition to the abovementioned two main types, there are several rare subtypes. One of them is IgG/IgA pemphigus. Patients with IgG/IgA pemphigus have both IgG and IgA anti-Dsg antibodies. The presentations of IgG/IgA pemphigus are less well-defined in the literature. The first part of this thesis focused on patients with IgG/IgA pemphigus, using a retrospective method to collect clinical data, histopathologic features, serology tests, and the results of immunofluorescence studies and immunohistochemical stains, and trying to better characterize these patients. The pathomechanism of pemphigoid is similar to that of pemphigus. The difference is that autoantibodies in pemphigoid mainly targeting to the basement membrane proteins, BP180 and BP230. Binding of these autoantibodies to theirs antigens would activate the complement system, recruiting inflammatory cells, such as mast cells, neutrophils and eosinophils. The inflammatory mediators released by these inflammatory cells would disrupt the structures of basement membrane, leading to the formation of blisters. On the other hand, some studies indicated that autoantibodies in pemphigoid can directly act on the epidermal cells, causing the internalization and degradation of BP180. This effect may also contribute to the formation of blisters in pemphigoid. Therefore, the compounds which can inhibit inflammatory responses and internalization of BP180 by autoantibodies are the candidate drugs to treat the disease. The second part of this thesis focused on the two potential drugs, berberine and diacerein, trying to examine the effects of these two drugs on inhibition of the autoantibodies-induced inflammation and internalization of BP180. In the result of the first part of thesis, we retrospectively identified 22 patients. These patients showed two types of skin presentations. One was annular or arciform erythemas with blisters or erosions (45.5%). Another was discrete erosions or blisters like those seen in the conventional pemphigus (54.5%). Around 40.9 % of the patients with IgG/IgA pemphigus had mucosal involvment. In histopathology, acantholysis (77.3%) and intra-epidermal aggregates of neutrophils (40.9%) and eosinophils (31.8%) could be identified and were the most characteristic features. In the direct immunofluorescence studies, all cases had both IgG and IgA depositions at the intercellular space, and 81.8% of the cases had C3 depositions at the intercellular space in their epidermis. Regarding to immunohistochemical stains, IgG/IgA pemphigus showed significantly more expressions of interleukin-8 and matrix metaloproteinase-9 in the epidermis comparing to those seen in the conventional pemphigus (P < 0.05). These features highlighted the potential roles of IgA autoantibody, neutrophils, and eosinophils in the pathomechanism of IgG/IgA pemphigus. In the second part of the thesis, we used purified sera from patients with pemphigoid to explore the effects of these autoantibodies on the keratinocytes. The levels of IL-6 and IL-18 in the culture supernatant were elevated when treating the cultured HaCaT cells with the purified patient sera. These effects would be reduced by pre-treatment with berberine or diacerein. On the other hand, the expression of BP180 on the surface of HaCaT cells was reduced and showed internalization when treating with the purified patient sera. This phenomenon would be inhibited by pre-treating with berberine or diacerein in a dose-dependent manner. We also identified that the effects of berberine and diacerein might be through the action on protein kinase C. As a result, berberine and diacerein have a therapeutic potential in treating patients with bullous pemphigoid. In summary, this thesis identified the characteristic presentation of IgG/IgA pemphigus, explored the potential roles of IgG, IgA, and inflammatory cells in the formation of IgG/IgA pemphigus. In addition, this thesis also demonstrated the effects of the autoantibodies in pemphigoid, including the provocation of inflammatory responses and induction of BP180 internalization. We also showed that berberine and diacerein may have therapeutic potentials by inhibiting these autoantibodies-induced effects. We hope the results of this thesis can provide a more detailed understanding of the autoimmune bullous diseases and can lead to a better treatment for these patients in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84906 |
DOI: | 10.6342/NTU202202739 |
全文授權: | 同意授權(限校園內公開) |
電子全文公開日期: | 2022-10-05 |
顯示於系所單位: | 病理學科所 |
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