中文摘要-1

慢性肝病病人的matrix metalloproteinase-2活動性與cystatin C血清濃度的相關性
Relationship between matrix metalloproteinase-2 activity and cystatin C levels in patients with hepatic disease

目的：先前吾等研究已顯示cystatin c的濃度與肝病嚴重度有直接相關性。本研究則在探討matrix metalloproteinase-2（先前已被証明與肝纖維化有關）與cystatin在肝病嚴重度的關係。

設計與方法：連續收集154位肝病病人的血清，測量其cystatin C，matrix metalloproteinase-2（MMP-2）及metalloproteinase-9的濃度，並測量其他肝病參數。與40位正常對照組比較。

結果：病人的MMP-2及cystatin C濃度顯著比對照組要高，而MMP-9則較低。線性回歸分析顯示cystatin C與MMP-2有直接關係（Y=83.39+270.56X，R=0.38, P＜0.001）。MMP-2及肝病嚴重度亦有同樣關係。

結論：本研究首次展示cystatin C及MMP-2的關係。表示在肝病病人中兩者有某種相互關係。

中文摘要-2

慢性C型肝炎細胞激素（cytokines）的血清濃度及基因多樣性（polymorphism）的衝激（impact）

造成C型肝炎病毒（HCV）清除或者持續的因素尚無法完全清楚界定，其中宿主的免疫反應的重要性特別受到注意。
尤其幾個細胞激素（cytokines）的血清濃度及基因多樣性（polymorphism）在C型肝炎病毒感染的結果值得多加研究。

利用酵素連結免疫吸附分析法（Enzyme-linked immunosorbent assay, ELISA）來測量72個C型肝炎病人及180個健康對照者的腫瘤壞死因子-α（tumor necrosis factor alpha, TNF-α）介白素-4（interleukin-4, IL-4）及介白素-10 (IL-10)的濃度。同時更進一步，利用聚合酶鏈反應（polymerase chain reaction, PCR）限制酶斷片長度多樣性（restriction fragment length polymorphism, RFLP）來分析這些細胞激素的基因的單一核苷酸多樣性（single nucleotide polymorphism, SNP）及C型肝炎病毒感染的相關性。

這些分析的細胞激素的SNPs包括TNF-αG238A, TNF-αG308A, IL-4C589T, IL-10A1082G, IL-10T819C及IL-10A592C。

C型肝炎病人的TNF-α,IL-4及IL-10的血清濃度比對照組的值要高（P＜0.001）。再者，TNF-α G308A基因型（genotype）及對偶質（allele）在二組之間有意義的差別（P＜0.05），但其他的細胞激素就沒有差別。這些資料顯示在健康人及慢性C型肝炎病人之間，TNF-αG308A的多樣性的分佈可能是不一樣的。

Abstract-1

Objectives: A direct relationship between cystatin C levels and the severity of hepatic disease has been revealed in our previous study. This study was aimed to consider whether a correlation exists between matrix metalloproteinases (MMPs), which have been proven to be involved in liver cirrhosis, and cystatin C to reflect the severity of hepatic disease.
Design and Methods: A total of 154 consecutive patients with various liver diseases were recruited to determine their serum levels of cystatin C, MMP-2 and-9, together with other hepatic parameters. These were compared with 40 normal controls.
Results: Average levels of MMP-2 and cystatin C were significantly higher in patients while MMP-9 was significantly lower, as compared to controls. A linear regression analysis has revealed a direct relationship between cystatin C and MMP-2 (Y = 83.39 + 270.56X, R = 0.38, P < 0.001), as well as between MMP-2 and the severity of liver diseases.
Conclusion: This is the first study to demonstrate a correlation between cystatin C and MMP-2, suggesting that there may be certain interactions between cystatin C and MMP-2 in patients with hepatic diseases.
○C 2005 The Canadian Society of Clinical Chemists. All rights reserved.

Keywords: MMP-2; Cystatin C; Hepatic disease

Abstract-2

The factors leading to clearance or persistent infection of hepatitis C virus (HCV) were not well defined, and the importance of the host immune response has been highlighted. Therefore, the impact of the serum concentration and genetic polymorphism of several cytokines on the outcome of HCV infection warrant additional study.
Enzyme-linked immunosorbent assay was employed to measure serum tumor necrosis factor alpha (TNF), interleukin (IL)-4, and IL-10 concentrations on 72 hepatitis C patients and 180 controls. Furthermore, the association between single nucleotide polymorphism (SNP) of genes of these cytokines and hepatitis C infection was evaluated by polymerase chain reaction- restriction fragment length polymorphism and statistical analysis.
These analyzed SNPs of cytokines included TNF-αG238A, TNF-α G308A, IL-4 C589T, IL-10 A1082G, IL-10 T819C, and IL-10 A592C. Serum levels of TNF-μ α, IL-4, and IL-10 were significantly higher in hepatitis C patients than that of controls (P < 0.001). Furthermore, the distribution of TNF-αG308A genotypes and alleles, but not others, was statistically different between patients and controls (P < 0.05). These data suggested the distribution of polymorphism at TNF-αG308A may be different between normal subjects and patients with chronic infection of hepatitis C. (Translational Research 2007;150:116-121)

Abbreviations: ALT = alanine aminotransferase; AST = aspartate aminotransferase; ELISA =enzyme-linked immunosorbent assay; HBV = hepatitis B virus; HCV = hepatitis C virus; HIV =human immunodeficiency virus; IFN = interferon; IL =interleukin; NK =natural killer; OR =odds ratio; PCR = polymerase chain reaction; RFLP =restriction fragment length polymorphism;RT =reverse transcriptase; TNF-α= tumor necrosis factor alpha.

Contents

Introduction in Chinese ---------------------------------------------------------------------- 4
Introduction in English----------------------------------------------------------------------- 5
Abstract in Chinese （cystatin C and MMP-2）--------------------------------------- 6
（serum levels and gene polymorphism of cytokines）------ 7

Abstract in English （cystatin C and MMP-2）----------------------------------------8
（serum levels and gene polymorphism of cytokines）------9
Chapter 1 Introduction part Ⅰ（matrix metalloproteinase and cystatin C）---------11
Introduction part Ⅱ（TNF-α,IL-4,IL-10）-----------------------------------13
Chapter 2 Materials and Methods-----------------------------------------------------------15
2. Ⅰ-1 Materials ----------------------------------------------------------------------------- 15
2. Ⅰ-2 Quantitative determination of serum MMP-2 and MMP-9 --------------------17
2. Ⅰ-3 Determination of serum cystatin C ----------------------------------------------- 17
2. Ⅰ-4 Statistical analysis--------------------------------------------------------------------18
2. Ⅱ-1 General design and study subjects ------------------------------------------------ 19
2. Ⅱ-2 Preparation of RNA and RT-PCR -------------------------------------------------19
2. Ⅱ-3 Measurements of INF-α, IL-4, IL-10 by ELISA---------------------------------20
2. Ⅱ-4 PCR-RILP for genetic polymorphism within promoter regions of TNF-α, IL-4 and IL-10------------------------------------------------------------------------------20
2. Ⅱ-5 Statistical analysis---------------------------------------------------------------------20

Chapter 3 Results
3.Ⅰ Results Ⅰ（cystatin and MMP-2 , MMP-9）-------------------------------------21
3.Ⅰ-1 Basic characteristics of study Subjects----------------------------------------------21
3.Ⅰ-2 Significantly higher cystatin C levels in hepatic disease -------------------------21
3 Ⅰ-3 Quantitative determinations of MMP-2 and MMP-9 and their correlations to cystatin C values in hepatic diseases ----------------------------------------------24
3 Ⅰ-4 Relationship between the severity of liver disease and MMP-2 -----------------27
3 Ⅱ-1 Characteristics of study subjects ----------------------------------------------------29
3 Ⅱ-2 ELISA analysis for serum levels of TNF-α, IL-4 and IL-10 -------------------- 29
3 Ⅱ-3 Genotype and allele distribution of promoter of TNF-α , IL-4 and
IL-10 gene -----------------------------------------------------------------------------------31

Chapter 4 Discussion Ⅰ-----------------------------------------------------------------------36
Discussion Ⅱ-----------------------------------------------------------------------40
Tables Ⅰ
Table 1 Clinical parameters of controls and patients with various hepatic diseases---16
Table 2 Cystatin C concentrations , MMP-2 and MMP-9 of patients with hepatic diseases compared with normal controls------------------------------------------23

Tables Ⅱ
Table 3 Sequences of primers and restriction enzymes used in cytokine gene polymorphism and expected sizes of resultant PCR-RFLP products ------- 32
Table 4 Genotype frequencies of TNF-α, IL-4 and IL-10 polymorphic site in controls and HCV patients--------------------------------------------------------------------34
Table 5 Allele frequencies of IL-4, IL-10 and TNF-α polymorphic site in controls and HCV patients-------------------------------------------------------------------------35

Figures Ⅰ
Figure 1 Cystatin C concentrations in normal controls and patients with hepatic diseases of various severity--------------------------------------------------------25
Figure 2 Shows representative serum MMPs zymographs of the subjects ------------26
Figure 3 Linear regression analysis of the relationship between cystatin concentrations and MMP-2 activity ------------------------------------------------------------ 27
Figure 4 MMP-2 activity in normal controls and patients with various severity of liver disease-----------------------------------------------------------------------------28
Figures Ⅱ
Figure 5 Serum levels of TNF-α, IL-4 and IL-10 in hepatitis c patiens and controls determined by ELISA kits------------------------------------------------------30
Figure 6 PCR-RFLP analysis of genetic polymorphism for various cytokines ----- 33

References ------------------------------------------------------------------------------------ 43

Published papers-------------------------------------------------------------------------------51

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