慢性發炎藉由多種致病作用被認為會影響腎臟衰期病人的死亡率和罹病率，像是對於氧化壓力、加速老化、內皮細胞功能不完全和動脈粥狀硬化、營養不良貧血、透析相關澱粉樣變症、白血球減少、免疫系統缺失等疾病皆有相關性。血液透析廣泛用於治療腎臟衰竭病人，用來移除人體中的廢物，像是尿素和血清肌酸酐。然而，目前已知血液透析的過程中會產生明顯的發炎現象和氧化壓力。血液透析中的氧化壓力來自於透析管中的滲透壓和氧化環境。氧化壓力會修飾蛋白質導致血液透析病人產生短期或是長期的腎臟相關併發疾病。本次實驗中我們先鑑定16 位病人透析管中的氧化蛋白。利用 Oxyblot TM assay 去判定蛋白氧化的程度。之後氧化蛋白會利用LC-MS/MS 去鑑定身份。有多種的血清蛋白偵測出來會被氧化，包括albumin、 immunoglobin、 apoA 、beta-globin 和hemoglobin 等蛋白質。之前的報導已經證實在透析病人的血清蛋白中 albumin 容易受到 ROS 攻擊，而且會增加 albumin 中carbonyl groups 的程度。之後我們去測試發炎的反應，血液透析病人的氧化蛋白會讓 HEK293T 和 HEK293 細胞株誘發前發炎因子 TNF-α 產生。這些結果顯示出血液透析會誘導蛋白氧化作用，所導致的發炎反應可能會對末期腎病患者導致其他併發症。

關鍵字：血液透析、氧化壓力、腎臟衰竭、TNF-α、發炎反應

Chronic inflammation is considered strongly influence the morbidity and mortality of patients with end-stage renal disease (ESRD) through its multiple pathogenic roles, in association with oxidative stress, accelerated aging, endothelial dysfunction and atherosclerosis, malnutrition, dialysis-related amyloidosis, anaemia, and immune dysfunction . Hemodialysis （HD） is widely used for kidney failure patients,it is a method for removing waste products such as creatinine and urea. However, at present it is well known the course of hemodialysis can create obvious inflammation condition and oxidation pressure. The oxidation stress of HD can arise from the osmosis pressure and oxidative environment of dialysis tube. The oxidative stresses will finally modify proteins which turn out initiate the short term and long term complications related to renal diseases of HD patients. We identified oxidated proteins in the hemodialysis tube of 16 HD patients. The protein oxidation level was determined by Oxyblot assay. The oxidation proteins were further identified by LC/MS detection. Many serum proteins were detected to be oxidized including albumin, apoA, immunoglobin,beta-globin, hemoglobin, etc. It has been well documented that albumin is quite vulnerable to ROS and elevated levels of carbonyl groups of albumin have been reported in plasma of dialysis patients. Inflammatory effects were further tested. The oxidation proteins of HD patients induce pro-inflammatory factor TNF-alpha expression of HEK293T and HEK293 cells. These results indicate HD induce protein oxidation, and inflammatory response which may responsible for complications of End-stage kidney disease (ESRD).

Keywords：Hemodialysis, oxidative stress, ESRD, HEK293, inflammatory

Contents
中文摘要............................................................................... iv
英文摘要................................................................................ v
英文縮寫表 .......................................................................... vi

壹、序言（Introduction）.................................................. 1
一﹒血液透析與腎臟衰竭疾病............................................1
1.1 腎臟的基本功能 ............................................................1
1.2 腎臟的基本構造與解剖圖 ........................................... 2
1.2.1 腎上腺髓質與皮質部 ................................................ 3
1.2.2 腎臟荷爾蒙的組成及基本作用 ................................ 3
1.3 偵測腎功能依據 ............................................................ 3
1.3.1 尿素氮（BUN） ........................................................ 4
1.3.2 肌酸酐（Creatinine） ............................................. 4
1.3.3 肌酸酐清除率（Creatinine Clearance） ........... 4
1.4 慢性腎臟疾病分期 ........................................................ 5
1.5 腎臟衰竭 .........................................................................6
1.5.1 急性腎衰竭（ARF） ................................................. 6
1.5.2 慢性腎衰竭（CRF） ............................... .................6
1.6 慢性腎衰竭的治療 .................................. ......................6
1.6.1 血液透析（Hemodialysis） ................................... 6
1.6.2 腹膜透析（Peritoneal dialysis） ........................... 7
1.6.3 腎移植（Transplantation） .................................... 7
1.7 慢性腎衰竭之病因 ........................................................ 8
1.7.1 原發性腎疾（Glomerulonephritis） ..................... 8
1.7.2 先天性和遺傳性腎病..................................................8
1.7.3 高血壓腎病 (Hypertensive kidney disease) ....... 9
1.7.4 糖尿病腎病（Diabetic kidney disease ） ........... 9
1.8 血液透析與氧化壓力 ............................. ..................10


貳、實驗目的..................................................................... 12

參、材料與方法（Materials and Method）.................. 13
一﹒血液透析病人之血液與透析管取樣......................... 13
1.檢體來源.......................................................................... 13
2.血液取樣與蛋白質萃取...................................................13
3.白血球萃取.......................................................................13
二﹒透析管吸附蛋白萃取................................................. 13
1.蛋白質之萃取與濃縮.......................................................13
2.TCA/Acetone 沈澱 ........................................................ 14
3.蛋白質定量...................................................................... 14
三﹒細胞培養..................................................................... 15
3.1 實驗材料 ..................................................................... 15
3.2 實驗方法 ..................................................................... 15
3.2.1 細胞培養液的配置 ................................................ 15
3.2.2 解凍細胞 .................................................................. 16
3.2.3 繼代培養 .................................................................. 16
3.2.4 冷凍細胞 ................................................................ ..16
3.2.5 細胞計數 .................................................................. 17
3.2.6 腎臟胚胎細胞株 HEK293、HEK293T 的培養與處理 .... 17
四﹒氧化蛋白測定......................................................... ....18
五﹒TNF-α ELISA 測發炎反應 ..................................... ..19
六﹒MTT 抗癌活性測試 ................................. ..................20
七﹒細胞蛋白質萃取和濃度分析.......................... ...........20
7.1 聚丙烯硫胺膠體電泳（SDS-PAGE） ................... ..21
7.2 銀離子染色（Silver stain） ..................... ..............21
7.3 Comassie Brilliant Blue G-250 ..............................22
7.4 Digestion/Extraction ............................ ....................22
八﹒西方墨點電泳法（Western Blotting）.................. .22
九﹒氧化蛋白配置...................................... .......................24

肆、實驗結果（Results）............................... ................25
一﹒血液透析病人之檢體分析結果........................ .........25
二﹒透析管蛋白氧化程度與 CRP 的結果................... ...25
三﹒透析管蛋白和血清蛋白氧化程度的比較................ .25
四﹒白蛋白對於腎臟細胞 TNF-α的影響 ...................... .26
五﹒透析管吸附蛋白對腎臟細胞株的發炎反應........... 26
六﹒一維電泳膠片之分析結果........................................ 27
七﹒LC MS/MS 質譜分析與蛋白質資料庫比對 ............ 27
八﹒氧化蛋白與細胞發炎反應的結果............................. 27
九﹒氧化蛋白對 COX-2 的影響....................................... 28
十﹒氧化蛋白使 HEK293 細胞株增生快速.................... 28

伍、實驗討論（Discussion）........................................ 29
一﹒透析管蛋白氧化程度與發炎反應相關性討論......... 29
二﹒病人血清與透析管結合蛋白之氧化程度實驗討論. 30
三﹒透析管蛋白與氧化蛋白誘導細胞發炎反應實驗討論31
四﹒氧化蛋白導致腎臟細胞增生實驗討論..................... 32

陸、結論（Conclusion）................................................ 35

柒、圖與表（Figures & Tables）................................. 36
表一﹒血液透析病患基本資料與相關臨床數據表.........36
表二﹒LC MS/MS 鑑定透析管蛋白覽表 ........................ 37
圖一﹒16 位病人透析管吸附蛋白氧化程度和 CRP 值 ....... 39
圖二﹒比較病人血清蛋白和透析管蛋白氧化程度........ 40
圖三﹒正常的 albumin 對腎臟細胞誘導 TNF-α之影響 ..... 41
圖四﹒透析管蛋白誘導 HEK293 產生 TNF-α .............. 42
圖五﹒透析管蛋白誘導 HEK293T 產生 TNF-α ............. 43
圖六﹒透析管蛋白之選取膠片圖..................................... 44
圖七﹒證實 BSA 有氧化的現象....................................... 45
圖八﹒氧化 BSA 誘導 HEK293 & HEK293T 產生 TNF-α ..... 46
圖九﹒氧化 BSA 誘導 HEK293T COX-2 的表現........... 47
圖十﹒透析管蛋白誘導 HEK293 COX-2 的表現........... 48
圖十一﹒氧化 BSA 對 HEK293 細胞增生的影響.......... 49
附表一﹒LC MS/MS 鑑定透析管蛋白覽表 .................... 51
附表二﹒LC MS/MS 鑑定氧化蛋白覽表 ........................ 63

捌、參考文獻（References）........................................ 74

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