PROJECT I 藉由離胺酸及半胱胺酸標定的二維差異電泳儀鑑定出在高糖環境下所引起之蛋白質體及分泌蛋白質體的表現量變化和巰基變化 PROJECT II 藉由離氨酸標定的二維差異電泳分析懷有正常胎兒以及唐氏症胎兒的母體胎盤的蛋白質體的表現量變化從中發現新穎性生物標誌

PROJECT I Effect of High Glucose on Expression Proteome, Redox-Proteome and Secreted Proteome in Cultured Retinal Pigmented Epithelium Cells Its Possible Relevance to Diabetic Retinopathy PROJECT II Placenta Proteome Analysis from Down Syndrome Pregnancies for Biomarker Discovery

陳又瑄(You-Hsuan Chen)

指導教授 ： 詹鴻霖

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2012年)

https://doi.org/10.6843/NTHU.2012.00408

摘要

PROJECT I 藉由離胺酸及半胱胺酸標定的二維差異電泳儀鑑定出在高糖環境下所引起之蛋白質體及分泌蛋白質體的表現量變化和巰基變化

在糖尿病患者中約有25%的病患會有糖尿病視網膜病變的發生，先前研究指出糖尿病視網膜病變其中主要的原因是由於高糖會使視網膜微血管功能減弱而使微血管中血液或組織液流到周圍空間近而導致視力不良甚至失明。除此之外，高糖會誘使一些蛋白行醣基化而導致蛋白失去功能並且發生醣氧化作用而使細胞產生活性氧化物質(Reactive oxygen species, ROS)。然而，目前對於高糖如何導致視網膜病變的詳細機制尚未明瞭。本研究中，利用正常的人類視網膜色素上皮細胞(ARPE-19)培養在不同糖濃度的培養液中，分別是5.5mM糖濃度培養液代表一般人平時正常血糖濃度, 25mM糖濃度培養液代表糖尿病患飯後兩小時的血糖濃度以及100mM 糖濃度培養液表示更不受控的糖尿病患者的血糖濃度，並用甘露醇調整各個培養液的滲透壓，之後再利用離胺酸及半胱胺酸標定的二維差異電泳結合基質輔助雷射脫附游離飛行是質譜儀(MALDI TOF-MS)鑑定出具有表現量差異且有巰基變化的蛋白質。結果顯示，56個蛋白質具有明顯表現量的差異而有33個蛋白質在半胱胺酸殘基上有巰基的變化。除此之外，分泌蛋白質有55個具有表現量的差異。在鑑定到的蛋白中，包含訊息傳遞、基因調節、和細胞中蛋白質傳遞相關的蛋白質在高糖環境下其表現量有明顯的改變。另外，在細胞代謝、細胞中蛋白質傳遞和細胞存活中相關的蛋白質具有明顯巰基的變化。而差異的分泌蛋白質主要功能在於維持細胞膜穩定和細胞黏附中的重要蛋白質(例如galectin-3-binding protein) 還有運輸相關的蛋白質(比如multidrug resistance-associated protein 1)，另外在這些蛋白中選取了MPP2,、haptoglobin and cathepsin D等感興趣的目標蛋白並且利用了第二型糖尿病有併發視網膜病變的病人血清和健康血清做更進一步的驗證。總結來說，本實驗結果顯示出完整的利用視網膜細胞為基礎的蛋白質體學分析並鑑定到許多細胞內經由高糖所導致而有明顯的蛋白質表現量和具有氧化還原變化的蛋白質，這些蛋白質會再更進一步利用臨床上的檢體做驗證期望能提供糖尿病視網膜病變一個預測和診斷更有力的目標。

PROJECT II 藉由離氨酸標定的二維差異電泳分析懷有正常胎兒以及唐氏症胎兒的母體胎盤的蛋白質體的表現量變化從中發現新穎性生物標誌

唐氏症是一種常見的基因異常疾病，其發生原因是在第二十一號染色體上發生缺陷，隨著孕婦年齡之遞增懷有唐氏症胎兒的可能性也隨之升高。侵入性檢測(比如羊水穿刺)檢出率高但是造成早產的機率遠比非侵入性母血檢測高出許多。因此，在此研究中，我們利用離氨酸標定的二維差異電泳結合基質輔助雷射脫附游離飛行式質譜儀 (MALDI-TOF-MS) 分析懷有正常胎兒及唐氏症胎兒的母體胎盤中的蛋白質並鑑定出具有表現量差異的蛋白質，希望從中找出新穎性的生物標誌而得以用於非侵入性的唐氏症檢測中。結果顯示，101個蛋白質具有明顯表現量的差異。在所有鑑定到的蛋白質當中以參與細胞骨架結構和調控 (例如 vimentin and Profilin-1) 的功能性上差異最多。我們的結果中也顯示出許多先前已被報導過的唐氏症標誌 (例如 serum amyloid P-component)。不過，我們也找到了一些新穎性標誌而且前從未被報導過的蛋白質 (例如 galectin-1, ataxin-3和SPRED2) 可作為唐氏症非侵入性檢測的目標蛋白質。總結來說，本研究利用了蛋白質體學的技術找到了有潛力的新穎性生物標誌，未來可對於唐氏症非侵入性檢測有更進一步的幫助。

關鍵字

糖尿病眼病變 ； 唐氏症

並列摘要

PROJECT I
Effect of High Glucose on Expression Proteome, Redox-Proteome and Secreted-Proteome in Cultured Retinal Pigmented Epithelium Cells Its Possible Relevance to Diabetic Retinopathy

Retinopathy has been observed in around 25% of diabetic patients. Previous research has suggested that diabetic retinopathy can cause poor vision and even blindness since high glucose has been evidenced to weaken retinal capillary leading to leakage of blood into the surrounding space. Besides, high glucose may induce glycosylation of proteins resulting in the loss of their biological functions as well as the generation of reactive oxygen species called glycoxidation. However, the detailed molecular mechanisms by which high glucose leads to diabetic retinopathy have not yet to be clarified. In the recent study, a model retinal pigmented epithelium cell line, ARPE-19, grown in mannitol-balanced 5.5 mM, 25 mM and 100 mM D-glucose culture media has been used as a model for proteomic analysis and the changes in protein expression and thiol reactivity were observed with lysine- and cysteine-labeling 2D-DIGE and MALDI-TOF mass spectrometry. Our proteomic analysis revealed that 56 identified proteins showed significant changes in protein expression, and 33 in thiol reactivity. Moreover, the results revealed that 55 identified proteins showed significant changes in protein expression. Many proteins that are known to be involved in signal transduction, gene regulation and transport were shown significant changes under high glucose conditions. In addition, the thiol-reactivity of proteins involving metabolism, transport and cells survival were altered by high glucose. Additionally, these secreted proteins mainly function in cytoskeleton-associated adhesion / junction (such as galectin-3-binding protein) and transport (multidrug resistance-associated protein 1). Additionally, the identified secreted markers including MPP2, haptoglobin and cathepsin D were further validated in plasma samples coming from type 2 diabetic patients with retinopathy and healthy donors.To sum up, we report a comprehensive retinal cell-based proteomic approach for the identification of potential expression and redox-associated retinal markers-induced by high glucose conditions. Furthermore, such proteins might further validate with clinical samples and provide as potential targets for the prognosis and diagnosis of diabetic retinopathy.
PROJECT II Placenta Proteome Analysis from Down Syndrome Pregnancies for Biomarker Discovery

Down syndrome is one of the most frequent chromosomal disorders, with prevalence approximately 1/500 to 1/800, depending on the maternal age distribution of the pregnant population. However, few reliable protein biomarkers have been used in diagnosis of this disease. Recent progresses in quantitative proteomics have offered opportunities to discover biomarkers for tracking the progression and for understanding the molecular mechanisms of Down syndrome. In present study, placental samples were analyzed by fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Totally, 101 proteins have been firmly identified representing 80 unique gene products. These proteins mainly functioned in cytoskeleton structure and regulation (such as vimentin and Profilin-1). Additionally, our quantitative proteomic approach has identified numerous previous reported Down syndrome markers such as serum amyloid P-component. On the contrary, we have presented several Down syndrome biomarkers including galectin-1, ataxin-3 and sprouty-related EVH1 domain-containing protein 2 (SPRED2) which have not been reported and may be associated with the progression and development of the disease. In summary, we report a comprehensive placenta-based proteomic approach for the identification of potential biomarkers for Down syndrome. The potential of utilizing these markers for prognosis and screen of Down syndrome warrants further investigations.

並列關鍵字

Diabetic Retinopathy ； Down Syndrome

參考文獻

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