酒精性脂肪肝與C型肝炎引起肝纖維化之血清蛋白生物標記之探討

Explore on Serum Biomarkers of Alcoholic Fatty Liver and HCV-induced Liver Fibrosis

劉淑玲(Shu-Lin Liu)

指導教授 ： 陳凌雲

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2012年)

https://doi.org/10.6834/CSMU.2012.00128

摘要

酒精成癮及濫用會造成肝臟的負擔，導致產生酒精性肝病變，包括酒精性脂肪肝、酒精性肝炎及酒精性肝硬化，而酒精性脂肪肝為其早期病變，尋找早期酒精性肝臟疾病蛋白生物標記將有助於預防肝臟產生後續之病變，並可早期發現及早治療。另外，當肝臟發炎時會刺激星狀細胞hepatic stellate cells (HSCs)活化並引發纖維化，雖然許多生物標誌已經被報告與肝纖維化有關，但受限於血中大量的白蛋白與免疫球蛋白的干擾，因而降低血清生物標誌分析實驗的正確性。本論文研究目的有二，一為利用二維微差電泳分析技術(two-dimensional differential gel electrophoresis)與質譜儀搜尋新的酒精性脂肪肝血清蛋白生物標記，另一目的為利用懸浮式微陣列分析法 (flexible Multi-Analyte Profiling, xMAP)來探討細胞激素是否可做為肝纖維化生物標誌。實驗首先分別對大鼠餵食酒精、高脂食物及thioacetamide (TAA)去誘導產生酒精性脂肪肝、非酒精性脂肪肝、及肝纖維化三種不同肝疾病老鼠動物模式，再從這些老鼠血清以二維微差電泳分析技術與質譜儀鑑定出八種具差異性之酒精性脂肪肝蛋白生物標記，其中包括表現量上升之 C-reactive protein (CRP) 及表現量下降之Haptoglobin (Hp)，由西方墨點法證實CRP於酒精性脂肪肝血清中呈現上升趨勢(p<0.05)，而在非酒清性脂肪肝或肝纖維化老鼠血清中則無改變，表示CRP或可用來預測或診斷早期酒清性脂肪肝。另外， CRP及Hp於肝纖維化老鼠血清中皆呈現下降趨勢，利用xMAP定量後也證實臨床HCV肝纖維化病人血清中CRP及Hp與正常對照組相比表現量減少，表示CRP及Hp或可當作肝纖維化蛋白生物標記，用以協助提高臨床診斷正確性。
此外，利用xMAP及商品化之cytokines 27- 與 23-panels 來搜尋肝纖維化疾病病人血清中的可能性生物標記。從HCV感染的病人血清中篩選了五十種細胞激素，結果鑑定出IFN-α2, GRO-α, SCF與 SDF-1α四種血清細胞激素的蛋白表現量從F1 到 F4 階段隨著肝纖維化的發展而有上升趨勢。此研究說明了細胞激素與肝纖維化之間的關係，且IFN-α2, GRO-α, SCF 與SDF-1α或可用於作為預測肝纖維化之生物標誌。

關鍵字

酒精性脂肪肝 ； C反應蛋白 ； 二維微差電泳分析法 ； 蛋白生物標記 ； 細胞激素 ； 懸浮式微陣列分析 ； C型肝炎病毒 ； 肝纖維化

並列摘要

Excessive consumption of alcohol contributes to alcoholic liver disease. Fatty liver is the early stage of alcohol-related liver disease. The aim of this study was to search for specific serological biomarkers of alcoholic fatty liver (AFL). Search for early protein biomarkers will contribute to the prevention of alcoholic liver disease have a follow-up of liver lesions for early detection and treatment. Otherwise, Hepatic inflammation is the stimulator to activate hepatic stellate cells (HSCs) and triggers fibrogenesis. , although many biomarkers have been reported associated with liver fibrosis. In the past, the discovery of serological biomarkers was limited due to the abundant amount of albumin and IgG in serum, which would decrease the accuracy in analytical experiments.
There are two purposes of this thesis research , One for use two-dimensional differential gel electrophoresis and mass spectrometer search for new serum biomarkers of alcoholic fatty liver, and also using Bio-Plex suspension array system based on flexible Multi-Analyte Profiling (xMAP ) technique to explore whether cytokines can be Biomarkers of liver fibrosis.
First, to generate animals with AFL, non-AFL (NAFL), and liver fibrosis. Wistar rats were orally fed alcohol, fructose and thioacetamide (TAA)-containing drinking water, respectively. Eight differential putative biomarkers were identified, and the two most differentiated proteins, including upregulated C-reactive protein (CRP) and downregulated haptoglobin (Hp). Western blotting validated that CRP was dramatically higher in the serum of AFL compared to healthy controls and other animals with liver disease of NAFL or liver fibrosis (p < 0.05). Moreover, we found that CRP and Hp were both lower in liver fibrosis of TAA-induced rats and clinical hepatitis C virus-infected patients compared to healthy controls, suggesting that CRP and Hp are reliable biomarkers of liver fibrosis to assist the accuracy of clinical diagnosis.
Otherwide, the xMAP and commercial cytokines 27- and 23-panels were used to search for putative serum biomarkers of liver fibrosis. Totally 50 cytokines were screened using flexible multi-analyte profiling to discover differential biomarkers. Finally, levels of protein expressions of individual stages of liver fibrosis were measured. Four serum cytokines, including IFN-α2 (p=0.023), GRO-α (p=0.013), SCF (p=0.047) and SDF-1α (p=0.024), were identified and they were increased from F1 to F4 stage in hepatitis C virus (HCV) infected patients. This study reveals the relationship between cytokines and liver fibrosis, and demonstrated that IFN-α2, GRO-α, SCF and SDF-1α may be used as biomarkers to predict liver fibrosis.

並列關鍵字

alcoholic fatty liver ； biomarker ； C - reactive protein ； two-dimensional differential gel electrophoresis ； cytokine ； flexible multi-analyte profiling ； hepatitis C virus ； liver fibrosis

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