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研究生:黃昭傑
研究生(外文):Chao-Chieh Huang
論文名稱:Thiazolidinediones對培養的人類葛瑞夫玆氏眼纖維母細胞玻尿酸合成之影響
論文名稱(外文):The Effect of Thiazolidinediones on Hyaluronan Synthesis in Cultured Human Orbital Fibroblast with Graves’ Disease
指導教授:王懷詩鄧錦泉
指導教授(外文):Hwai-Shi WangKam-Tsun Tang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:50
中文關鍵詞:葛瑞夫玆氏症葛瑞夫茲氏眼病變玻尿酸玻尿酸合成脢還氧化脢2脂肪分化油紅染色
外文關鍵詞:Graves’ diseaseGraves’ ophthalmopathyhyaluronic acidhyaluronic acid synthaseTZDsPPAR-γCOX-2HA ELISA
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葛瑞夫茲氏眼病變為伴隨葛瑞夫玆氏症常見的自體免疫疾病,其主要臨床症狀包括眼突、眼瞼外翻及結膜水腫。一般認為其主要原因為體內產生能辨識甲狀腺刺激荷爾蒙接受器的抗體,同時刺激了眼窩結締組織內纖維母細胞表面上的共同抗原-甲狀腺刺激荷爾蒙接受器,吸引免疫細胞浸潤,造成發炎的現象,使得眼窩內的纖維母細胞產生大量的葡萄糖胺聚醣。其中的主要成分為具有強吸水性的玻尿酸,進而造成眼窩內壓力上升,形成眼突的現象。Thiazolidinediones ( TZDs )為一種人工合成的PPAR-γ促效劑用於改善第二型糖尿病病人對胰島素的敏感度。2003年Kerry等人提出的病歷報告中指出一位同時患有葛瑞夫茲氏症及第二型糖尿病的病人,服用TZDs之後加劇了眼突的現象。另外有文獻指出環氧化脢2的基因表現以及前列腺素E2在葛瑞夫茲氏眼病變病人上有增加的情況。本實驗使用Q-PCR、HA ELISA偵測TZDs刺激下葛瑞夫玆氏眼病變眼窩纖維母細胞的玻尿酸合成脢和COX-2基因表現量以及玻尿酸的合成,並使用PPAR-γ及COX-2抑制劑嘗試找出可能路徑。最後利用油紅染色觀察TZDs對於脂肪分化的影響。本研究發現TZDs能夠透過活化PPAR-γ增加玻尿酸合成脢2及環氧化脢2 mRNA的表現。另外使用Meloxicam抑制環氧化脢2活性,發現能增加IL-1β刺激下玻尿酸合成脢2及環氧化脢2 mRNA表現。而在玻尿酸的合成方面,TZDs刺激下反而沒觀察到有增加的現象,但是發現抑制COX-2能增加IL-1β刺激下玻尿酸的合成。最後發現TZDs和IL-1β對於玻尿酸的合成以及脂肪分化都有加成的效果,這點能夠符合臨床觀察到的現象。透過以上結論,已初步了解TZDs在葛瑞夫茲氏眼病變病人身上造成影響的原因。
Graves’ ophthalmopathy is a common autoimmune disorder that display with Graves’ disease showing characteristic clinical feature such as proptosis,eyelid retraction and chemosis. It was thought that the pathogenic autoantibody activate TSH receptor shared by thyroid gland and orbital fibroblast were infiltrated by immunocyte and abundant of glycosaminoglycans was producted by fibroblasts. The major component is hydrophilic hyaluronaic acid that lead to the increase of orbital pressure and result in proptosis. Thiazolidinediones (TZDs), an synthetic agonist of PPAR-γ are used as insulin -sensitizing agent in patients type2 diabetes. Kerry et al. reported that a patient with Graves’ ophthalmopathy and type2 diabetes experienced exacerbating thyroid eye disease after he treated with TZDs. Some studies indicate that the gene level of COX-2 and the production of PGE2 are increased on patient with Graves’ ophthalmopathy. This study use Q-PCR to detect the mRNA expression of hyaluronan synthse and COX-2, HA ELISA to detect synthesis of hyalrunan under stimulated by TZDs than use PPAR-γ antagonist and COX-2 inhibitor to find out the possible pathway. Finally, I obtain the effect of TZDs to adipogensis by Oil Red O stain. Those result explained that TZDs can increase the mRNA expression of hyaluronan synthase2 and COX-2 by activing PPAR-γ. Another discovery is that the inhibition of COX-2 can increase the mRNA expression of hyaluronan synthase2 and COX-2.TZDs seem can’t stimulate the increase of hyaluronan but inhibition of COX-2 can increase production of hyalurona, like gene level. Finally, I found that TZDs and IL-1β have the synergistic effect on hyaluronan production and adipogensis. It is in accordance with clinical observation. By these studies,let us preliminary understand why TZDs affect patient with Graves’ ophthalmopathy.
壹、縮寫表 1
貳、中文摘要 3
参、英文摘要 5
肆、前言 7
一、葛瑞夫玆氏症眼病變 7
二、玻尿酸及玻尿酸合成脢 8
三、過氧化體增生活化受體(Peroxisome proliferators activated
- receptors) 8
四、環氧化脢2(cycloxygenase 2)與葛瑞夫茲氏眼病變 9
伍、研究材料及方法 11
一、藥品及試劑 11
二、儀器 12
三、引子表 13
四、葛瑞夫玆氏眼病變眼窩纖維母細胞原代培養及繼代 14
五、誘發脂肪分化 15
六、細胞總核糖核酸萃取 15
七、即時定量反轉錄聚合脢連鎖反應 16
八、玻尿酸合成分析 16
九、蛋白質濃度測量 17
十、油紅染色 17
十一、統計方法 18

陸、實驗結果
一、Thiazolidinediones及IL-1β對於葛瑞夫玆氏眼病變病人眼窩
纖維母細胞三種玻尿酸合成脢mRNA的影響 19
二、Thiazolidinediones及IL-1β對於葛瑞夫玆氏眼病變病人眼窩
纖維母細胞之COX-2 mRNA的影響 20
三、PPAR-γ抑制劑GW9662及COX-2抑制劑 Mexloxicam對於
受Thiazolidinediones刺激的葛瑞夫玆氏眼病變病人眼窩纖維
母細胞之玻尿酸合成脢mRNA的影響 20
四、PPAR-γ抑制劑GW9662及COX-2抑制劑 Mexloxicam對於
受Thiazolidinediones刺激的葛瑞夫玆氏眼病變病人眼窩纖維
母細胞之COX-2 mRNA的影響 21
五、PPAR-γ抑制劑GW9662及COX-2抑制劑 Mexloxicam對於
受Thiazolidinediones刺激的葛瑞夫玆氏眼病變病人眼窩纖維
母細胞之玻尿酸合成的影響 21
六、PPAR-γ抑制劑GW9662及COX-2抑制劑 Mexloxicam對於
受Thiazolidinediones刺激的葛瑞夫玆氏眼病變病人眼窩纖維
母細胞脂肪分化之影響 22
柒、討論 23
捌、參考文獻 26
玖、圖表 29
拾、附錄 44
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