臺灣博碩士論文加值系統

高血糖是糖尿病的主要特徵，高血糖可能與發炎反應及氧化壓力之產生有關，而發炎反應及氧化壓力是造成糖尿病併發症之主要危險因子。本研究目的擬探討在高糖或發炎環境下對肝細胞損傷及氧化壓力之影響。實驗將人類肝癌細胞株 (HepG2) 培養於葡萄糖 (5, 10, 15, 25, 35 mM) 和/或腫瘤壞死因子-α (TNF-α) (0, 0.1, 1, 10, 20 ng/ml) 的溶液中，於培養後第24、72及120 hr時檢測細胞存活率、亞硝酸鹽含量、脂質過氧化物含量；並探討NF-κB蛋白之表現量；另外加入L-抗壞血酸或α-生育醇 (0, 1, 10, 50, 100 μM) ，於培養後第24、72及120 hr時，檢測細胞存活率及亞硝酸鹽含量。結果顯示，經25及35 mM 葡萄糖處理的HepG2細胞存活率皆顯著低於相同培養時間之控制組 (5 mM葡萄糖) 。經0.1,1及20 ng/ml TNF-α處理之HepG2細胞存活率於培養後第24 hr顯著低於0 ng/ml TNF-α處理組。經25 mM 葡萄糖伴隨 20 ng/ml TNF-α共同處理之HepG2細胞存活率在培養後第120 hr顯著高於25 mM 葡萄糖處理組。經35 mM 葡萄糖和/或20 ng/ml TNF-α處理之HepG2細胞，於培養後第24 hr時，NF-κB蛋白之表現量皆顯著高於控制組 (5 mM葡萄糖) ，但是亞硝酸鹽和脂質過氧化物含量，除了第120 hr時10 ng/ml TNF-α處理組之亞硝酸鹽含量顯著低於0 ng/ml TNF-α控制組外，各組之間皆無顯著差異。經100 μM L-抗壞血酸處理之HepG2細胞存活率，於培養後第120 hr時，顯著低於控制組 (5 mM葡萄糖) ；經1和10 μM α-生育醇處理之HepG2細胞存活率，於培養後第24 hr時，顯著低於控制組 (5 mM葡萄糖) ；而經50及100 μM α-生育醇處理之HepG2細胞亞硝酸鹽之生成量，於培養後第24及72 hr時，皆會顯著高於控制組 (5 mM 葡萄糖) 。綜上所述，
高濃度葡萄糖導致肝細胞損傷，其機轉可能與NF-κB蛋白表現有關。

Hyperglycemia which major characterizes diabetes, may be related to inflammation and oxidative stress. Inflammation and oxidative stress were of the major risk factor for diabetic complications. The purpose of this study was to investigate the effect of high glucose or inflammation on the cell injury and oxidative stress of liver cell. Human hepatocellular carcinoma cell line (HepG2) was cultured with (5, 10, 15, 25, 35 mM) glucose and/or 0, 0.1, 1, 10, 20 ng/ml tumor necrosis factor-α (TNF-α) in the experiment. Cell viability, nitrite content, MDA content, for 24, 72, 120 hr after treatment and NF-κB protein expression were measured. Additional L-ascorbic acid or α-tocopherol (0, 1, 10,
50, 100 μM) was added to measure cell viability and nitrite content.
Results were HepG2 cell viability of groups treated with 25, 35 mM glucose and/or 20 ng/ml TNF-α exhibited significant lower than control group (5 mM glucose) in the same culture time. HepG2 cell viability of groups treated with 0.1, 1, 20 ng/ml TNF-α exhibited significant lower than 0 ng/ml TNF-α treated group for 24 hr. HepG2 cell viability of groups treated with 25 mM glucose and 20 ng/ml TNF-α exhibited significant higher than 25 mM glucose treated group for 120 hr. HepG2 cell NF-κB protein expression of groups treated with 35 mM glucose and/or 20ng/ml TNF-α exhibited significant higher than control group (5 mM glucose) for 24 hr, but nitrite content and MDA were not significantly different with each other, except that HepG2 cell nitrite content of groups treated with 10 ng/ml TNF-α exhibited significant lower than control group (0 ng/ml TNF-α) for 120 hr. HepG2 cell viability of groups treated with 100 μM L-ascorbic acid exhibited significant lower than control group (5 mM glucose) for 120 hr; HepG2 cell viability of groups treated with 1, 10 μM α-tocopherol exhibited significant lower than control group (5 mM glucose) for 24 hr; HepG2 cell nitrite product of groups treated with 50, 100 μM α-tocopherol exhibited significant higher than control group (5 mM glucose) for 72 hr. Generally, high glucose concentration induced cell injury, with mechanism was correlated to NF-κB protein
expression.

目錄 頁次
中文摘要 ........................................................................................ I
英文摘要 ...................................................................................... Ⅲ
謝誌 .............................................................................................. V
目錄 ............................................................................................. VI
圖目錄 ........................................................................................ X
表目錄 ........................................................................................ XI
第一章 緒言 ................................................................................ 1
第二章 文獻回顧
第一節 糖尿病簡介 .............................................................. 3
一、糖尿病定義 ................................................................ 3
二、糖尿病之流行病學 ..................................................... 3
三、高血糖與肝損傷之相關性 .......................................... 4
四、人類肝癌細胞株 (HepG2) 之簡介 ............................ 6
第二節 高血糖患者體內發炎反應及氧化壓力狀態 ............... 6
一、自由基之簡介 ............................................................ 6
二、氧化性傷害與高血糖及發炎反應之相關性 ................. 8
第三節 抗氧化防禦系統 ....................................................... 9
一、維生素C ..................................................................... 9
二、維生素E ................................................................... 10
第三章 材料與方法
第一節 材料 ....................................................................... 12
一、細胞來源 ................................................................. 12
二、實驗藥品 ................................................................. 13
三、實驗儀器設備及器材 ............................................... 16
第二節 實驗設計 ................................................................ 17
一、實驗架構 ................................................................. 18
二、實驗設計流程圖 ....................................................... 19
第三節 細胞培養 ................................................................ 24
一、培養液之製備 .......................................................... 24
二、活化細胞 ................................................................. 24
三、繼代細胞 ................................................................. 24
四、保存細胞 ................................................................. 24
第四節 實驗分析方法 ........................................................ 25
一、細胞存活率之測定 ................................................. 25
二、NO含量之測定........................................................ 26
三、MDA含量之測定..................................................... 26
四、NF-κB蛋白表現量測定.......................................... 28
(一)蛋白質萃取方法 ................................................... 28
(二)蛋白質含量分析 ................................................... 29
(三)SDS-PAGE ........................................................... 29
(四)卸膠 ..................................................................... 30
(五)轉漬步驟 .............................................................. 30
(六)蛋白質變化量之偵測 ............................................ 31
第五節 統計分析 ................................................................ 32
第四章 結果
第一節 高濃度葡萄糖對HepG2細胞之影響 ...................... 33
第二節 TNF-α對HepG2之影響 ....................................... 34
第三節 高濃度葡萄糖合併TNF-α對HepG2之影響 .......... 35
第四節 MDA的變化 ........................................................... 35
第五節 L-抗壞血酸對HepG2之影響 .................................. 36
第六節 α-生育醇對HepG2之影響 .................................... 37
第七節 NF-κB的變化 ....................................................... 37
第八節 L-抗壞血酸、α-生育醇、G35及T20之影響 ............ 38
第五章 討論
第一節 不同濃度之葡萄糖對HepG2細胞之影響 ............... 41
第二節 TNF-α對HepG2之影響 ....................................... 42
第三節 高濃度葡萄糖合併TNF-α對HepG2之影響 .......... 43
第四節 MDA的變化 ........................................................... 43
第五節 L-抗壞血酸及α-生育醇對HepG2之影響 .............. 44
第六節 NF-κB的變化 ....................................................... 46
第六章 結論與未來展望 ............................................................ 47
第七章 參考文獻 ....................................................................... 48

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