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研究生:方孟眉
研究生(外文):MENG-MEI FANG
論文名稱:Adalimumab (HUMIRA®)治療乾癬的相關基因的調控
論文名稱(外文):Regulation of psoriasis-related genes in patients treated with adalimumab (HUMIRA®)
指導教授:洪舜郁
指導教授(外文):SHUEN-IU HUNG
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:55
中文關鍵詞:乾癬基因藥物治療
外文關鍵詞:psoriasisadalimumabTNF-α
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乾癬是常見的自體免疫性疾病,除了在皮膚的病灶外,還會造成生活上許多不便。British Association of Dermatologists guidelines以Psoriasis Area Severity Index (PASI)評估乾癬的嚴重程度。近期研究證明不正常的T淋巴細胞活化和腫瘤壞死因子(TNF)-α在乾癬的致病機轉中扮演了重要的角色。Adalimumab (HUMIRA) ®是一種抗腫瘤壞死因子(TNF)-α的單株抗體,近期來被拿來做為治療中等到嚴重乾癬的用藥選擇。曾有臨床實驗顯示,到了末期48 %的病人的PASI index顯示病症可以改善達90 %甚至大於90 %。雖然adalimumab的療效看似很高,但是藉由此藥物調控的乾癬相關基因仍是未知。我們假設T淋巴球(包括Th1、Th2、Th17和 Treg)1 和其他相關訊號可能被adalimumab調控。首先,我們取得10個乾癬病人的血液和10個健康自願者的血液,接著分析乾癬相關基因的表現,包括由原型 T淋巴球活化的細胞介質:IL-6和IL-12;由Th1淋巴球活化的細胞介質:IL-1β、IFN-γ和TNF-α;由Th2淋巴球活化的細胞介質Th2:IL-4;由T淋巴球活化的細胞介質:IL-22; Treg 的細胞表面標的:FOXP3、CD4和CD25;其他`的血清中相較於健康人有意義地增加。然而,IL-4和IP-10在以adalimumab治療後的病人中和治療前比較後無有意義的血清濃度改變。此外,real-time PCR的結果顯示以adalimumab治療後的病人的FOXP3和治療前比較後有意義地增加基因表現。我們也利用流式細胞儀分選出病人PBMCs的CD4+CD45RBhiCD25- T細胞注入scid mice,並加以皮膚刺激,如在背部皮膚用膠帶反覆黏貼或塗抹DNCB,培養出乾癬小鼠。另外,我們拿取乾癬病人的血液,在不同的時間點分別給予不同劑量的adalimumab,再以流式細胞儀分析CD4+CD25+ T淋巴細胞的比例;並透過real-time PCR分析各樣基因表現,發現FOXP3也有顯著的改變。由此可知,當乾癬病人接受adalimumab的治療後,會影響調節性T淋巴球的表現,而有可能改變原本病情的嚴重程度。
Psoriasis is a common autoimmune disease of skin, which causes physical discomfort and disfigurement. Recent studies have demonstrated abnormal T cell proliferation and the tumor necrosis factor (TNF)-α play important roles in psoriasis. Adalimumab (HUMIRA®) is a fully humanized, anti-TNF monoclonal antibody, currently as a therapeutic choice for moderate to severe psoriasis. In the end of one recently clinical trial of adalimumab, a PASI (Psoriasis Area Severity Index) 90 (90% or greater improvement) response was achieved in 48% of patients. Although the response rate of adalimumab therapy seems high, the psoriasis-related gene(s) regulated by adalimumab remains unknown. We hypothesized that the gene expression of T cell (including Th1, Th2, Th17 and Treg)1 and other related molecules may be regulated by adalimumab therapy. Blood samples of 10 psoriatic patients with adalimumab treatment and 10 healthy volunteers were obtained. We analyzed the expression levels of psoriasis-related genes, including naïve T cell activating cytokines: IL-6 and IL-12; Th1 activating cytokines: IL-1β, IFN-γ and TNF-α; Th2 activating cytokines: IL-4; Th17 activating cytokines: IL-22; Treg surface marker: FOXP3, CD4 and CD25; and the other chemokines: IP-10/CXCL10, RANTES/CCL5 and IL-8/CXCL8. The results of ELISA and Bio-Plex showed that the serum levels of IL-4 significantly reduced in psoriatic patients, and IP-10 significantly increased in psoriatic patients when compared to healthy subjects. However, both of them didn’t show significantly difference after patients received the treatment of adalimumab. In addition, the results of real-time PCR showed that the gene expression of FOXP3 significantly increased in psoriatic patients after the treatment. This suggests that the adalimumab treatment in psoriasis changes the expression of some of the psoriatic-related genes which could be as biomarkers for therapeutic efficacy. Furthermore, we used flowcytometry to isolate the CD4+CD45RBhiCD25- T cells from PBMCs of psoriasis patients, and injected the cells into scid mice. To induce inflammatory reaction, we performed tapping and spread DNCB (Dinitrochlorobenzene) in the treated mice and observed the development of psoriasis-like lesions in the mice skin; in addition, we treated PBMCs of psoriatic patients with adalimumab and measured the percentage of CD4+CD25+ T cells by flowcytometry and analyzed the expression of psoriatic-related genes by real-time PCR. The gene expression of FOXP3 showed significant increase after the treatment, which was consistent with our previous in vivo experiment. Taken together, we suggested that the treatment of adalimumab of psoriatic patients may lead to the change of Treg related gene expression.
ABSTRACT..................................................1
中文摘要..................................................3
第一章 緒論............................................4
- 第一節 研究背景與動機................................4
一、乾癬的介紹........................................4
二、乾癬的致病機轉....................................4
三、乾癬的免疫調控因子................................5
四、乾癬的現行治療與評估..............................6
- 第二節 研究假說......................................9
- 第三節 研究目的與設計................................9
第二章 實驗流程.........................................11
第三章 材料與方法.......................................12
第四章 實驗結果.........................................15
一、比較乾癬病人與正常人的IL-1β、IL-4、IL-6、IL-22、
IFN-γ、TNF-α、 IP-10和RANTES血清中濃度...........15
二、分析白血球中的FOXP3、IL-8、IL-12、CD4和CD25在乾癬病
人以adalimumab治療前後的基因表現比較.............15
三、CD4+CD45RBhiCD25- T淋巴球注射入scid mice,再給予發
炎刺激產生類似乾癬的反應.........................16
四、體外培養(in vitro)病人的PBMCs並給予adalimumab,並分
析CD4+CD25+ T 淋巴球比例含量和FOXP3、IL-8、 IL-12和
CD4的基因表現....................................17
第五章 討論.............................................19

圖表
表一、動物實驗設計.......................................23
表二、統整圖四的各分析圖:各時間點中投予不同劑量的adalimumab
和TNF-α其CD4+CD25+ T淋巴球佔總量的比例.............24
圖一、IL-4、IP-10、IL-1β、IL-6、IL-22、IFN-γ、TNF-α和
RANTES在正常人與乾癬病人的血清中濃度比較...........25
圖二、FOXP3、IL-8、CD4、IL-12和CD25在乾癬病人以adalimumab
治療前後的基因表現比較.............................28
圖三、動物實驗結果.......................................31
圖四、體外培養(in vitro)病人的PBMCs分別給予adalimumab和
TNF-α,並利用流式細胞儀分析經過12小時後CD4+CD25+ T淋
巴球佔總量的比例...................................32
圖五、體外培養(in vitro)病人的PBMCs分別給予adalimumab和
TNF-α,並利用流式細胞儀分析經過24小時後CD4+CD25+ T
淋巴球佔總量的比例.................................35
圖六、取乾癬病人的白血球分析12小時和24小時FOXP3、IL-8、CD4
和IL-12的基因表現..................................38

附圖表
附圖一、以流式細胞儀分選CD4+CD45RBhiCD25- T 淋巴球.......41
附表一、Mean disease severity score......................42
附表二、以Bio-Plex human cytokine 27-plex panel檢測乾癬病人
和健康人的27個cytokine的數值,並比較乾癬病人以
adalimumab治療前後、健康人與乾癬病人治療前的差異(分
別顯示Mean和Standard Deviation)..................43
附表三、以Bio-Plex human cytokine 27-plex panel檢測乾癬病人
和健康人的27個cytokine的數值,並比較乾癬病人以
adalimumab治療前後、健康人與乾癬病人治療前的差異(分
別顯示P value)...................................45

參考文獻.................................................47
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