MMP-1、TIMP-1在肝纤维化组织中的表达和外周血浓度与肝病理分级的相关性研究
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摘要
【目的】探讨慢性乙型肝炎、乙肝后肝硬化患者肝组织中基质金属蛋白酶-1(Matrix metalloproteinase,MMP-1)、基质金属蛋白酶抑制因子-1(Tissue inhibitors of metalloproteinase ,TIMP-1)的表达及患者外周静脉血中MMP-1、TIMP-1的浓度,分析两者与临床病理肝脏炎症活动度及纤维化程度的相关性,为寻找新的判断肝纤维化程度的血清学指标代替有创性的肝脏穿刺活组织检查法提供理论依据。
【方法】选择2007年10月至2008年5月广西医科大学第一附属医院门诊及住院收治的慢性乙型肝炎、乙肝后肝硬化患者共28例,均行肝穿刺活组织病理检查并进行肝脏炎症活动度分级(G)及纤维化程度分期(S),同步收集患者血清,并收集同期47例健康体检者血清作为对照。用免疫组织化学二步法测定肝纤维化组织中MMP-1、TIMP-1阳性率及染色程度的方法进行病理分析,用双抗体夹芯酶联免疫吸附实验检测血清MMP-1和TIMP-1。
【结果】
①慢性乙型肝炎、乙肝后肝硬化患者肝组织中TIMP-1蛋白的阳性表达,与肝纤维化分期呈正相关(r=0.386,P<0.05);慢性乙型肝炎、乙肝后肝硬化患者肝组织中MMP-1蛋白的阳性表达,随着肝纤维化程度的加重无显著相关(P>0.05);慢性乙型肝炎、乙肝后肝硬化患者肝组织中MMP-1/TIMP-1比值与纤维化分期无显著相关(r=-0.366,P=0.056)。
②随着肝脏纤维化程度加重,血清MMP-1逐渐降低,而血清TIMP-1逐渐升高, MMP-1、TIMP-1测定在正常组与肝纤维化各期的两两比较结果显示,相邻两组间的比较均无显著性差异,但不相邻的各组间比较仅有个别无显著性差异(α=0.05);MMP-1/TIMP-1比值在各组两两比较中无规律可循。
③随着肝脏炎症活动加重,血清MMP-1含量逐渐减少,TIMP-1含量逐渐增加。MMP-1、TIMP-1测定在正常组与各炎症分级的两两比较结果显示,相邻两组间的比较均无显著性差异,但不相邻的各组间比较均有显著性差异(α=0.05); MMP-1/TIMP-1比值在各组两两比较中无规律可循。。
④血清TIMP-1与肝纤维化分期程度呈正相关(r=0.646,P<0.01),与肝脏炎症活动度分级呈正相关(r=0.622,P<0.01);血清MMP-1与肝纤维化分期程度呈高度负相关(r=-0.83,P<0.01),与肝脏炎症活动分级呈高度负相关(r=-0.821,P<0.01);血清MMP-1/TIMP-1比值与肝纤维化分期程度呈高度负相关(r=-0.776,P<0.01),与肝脏炎症活动度分级呈高度负相关(r=-0.757,P<0.01)。
⑤血清MMP-1、TIMP-1水平分别与组织中的MMP-1、TIMP-1表达无显著性相关(P>0.05)。
【结论】
(1).TIMP-1在肝纤维化组织中的检测是一个有用的指标,对反映肝纤维化程度有重要意义。MMP-1在肝纤维化组织中的检测不能作为判断肝纤维化的指标。肝组织中MMP-1/TIMP-1比值不能作为诊断肝纤维化及其分期的指标。
(2).血清MMP-1、TIMP-1含量、MMP-1/TIMP-1比值均不能很好地区分肝纤维化各期和炎症活动各分级,不适合动态判断肝纤维化程度和肝脏炎症活动度。但三者与肝纤维化分期及肝脏炎症活动度均有很好的相关性,均能间接反映慢性活动性肝炎肝脏损伤程度,对于诊断及预后判断有重要参考作用。
(3).血清MMP-1含量与肝纤维化的相关性可能比TIMP-1更高。血清TIMP-1、MMP-1含量分别与肝纤维化分期的相关程度和与炎症活动分级的相关程度相近。
(4).血清MMP-1、TIMP-1含量分别与肝组织中的MMP-1、TIMP-1表达无相关性。
Objective:To approach the expression of Matrix metalloproteina- se-1(MMP-1) and Tissue inhibitors of metalloproteinase-1(TIMP-1) in the hepatic tissue from patients who suffered chronic hepatitis B or cirrhosis after Hepatitis B and the concentration of MMP-1 and TIMP-1 in peripheral venous blood from the patients above, to analyse the correlation about the two things above separately associated with the liver inflammatory activity grade and fibrosis staging in the aspect of clinic patholog, in order to provide theoretical basis for the thing that is to find new serum index which can judge the degree of hepatic fibrosis to take the place of the invasive liver puncture biopsy.
Methods: To Choose 28 cases of patients who suffered chronic hepatitis B or cirrhosis after hepatitis B and were treated in hospital or in out-patient clinic in The First Affiliated Hospital of Guangxi Medical University from October 2007 to May 2008 .All patients were punctured out a bit of liver tissue by liver puncture biopsy for pathological examination,the latter can judge liver inflammatory activity grade (G) and fibrosis staging (S), simultaneously collecting the serum of all the patients above, and collecting 47 cases of serum from healthy examination persons in the same period as control serum..To determine positive rate and degree of staining about the expression of MMP-1 and TIMP-1 in liver cells of liver fibrosis tissue by two-step method which is a immunohistochemical method,and determine the concentration of MMP-1 and TIMP-1 in serum by using double-antibody sandwich enzyme-linked immunosorbent assay.
Results:①The expression of TIMP-1 protein in the liver tissues from paients who suffered chronic hepatitis B or cirrhosis after Hepatitis B was positively correlated with liver fibrosis staging (r = 0.386, P = 0.042); with the heavier degree of liver fibrosis, The expression of MMP-1 protein in the liver tissues above showed no significant correlation with liver fibrosis staging (P> 0.05); the Statistics about the expression ratio of MMP-1 and TIMP-1 also showed no significant correlation with liver fibrosis staging (r =- 0.366, P = 0.056).
②With the heavier degree of liver fibrosis, the concentration of MMP-1 in serum gradually decreased, while the concentration of TIMP-1 in serum gradually increased.Comparing of the normal group and each group of liver fibrosis staging, the results about the determination of MMP-1 and TIMP-1 indicate that all of the Comparisons between the each pair of next-to-next groups had no significant difference, but the Comparisons between the each pair of nonadjacent groups showed that only a few reslults indicated no significant difference. The Statistics of the comparisons about the concentration ratio of MMP-1 and TIMP-1 between each two groups were irregular.
③With liver inflammation activity increasing, the concentration of MMP-1 in serum decreased, while the concentration of TIMP-1 in serum gradually increased. Comparing of the normal group and each group of inflammation grade, the results about the determination of MMP-1 and TIMP-1 indicated that all of the Comparisons between the each pair of next-to-next groups had no significant difference, but the Comparisons between the each pair of nonadjacent groups were all significantly different (α= 0.05); The results of the comparisons about the concentration ratio of MMP-1 and TIMP-1 between each two groups were irregular.
④the concentrations of TIMP-1 in serum were positively correlated with the degree of liver fibrosis staging (r = 0.646, P = 0.000), and were positively correlated with liver inflammation activity grade (r = 0.622, P = 0.000); the concentrations of MMP-1 in serum and were highly negatively correlated with the degree of liver fibrosis staging (r =- 0.83, P = 0.000), and were highly negatively correlated with liver inflammation activity grade (r =- 0.821, P = 0.000); the concentration ratio of MMP-1 and TIMP-1 in serum were highly negatively correlated with the degree of liver fibrosis staging (r =- 0.776, P = 0.000), and were highly negatively correlated with liver inflammation activity grade (r =- 0.757, P = 0.000)
⑤the concentrations of MMP-1 and TIMP-1 in serum had no significant correlation with the expression of the MMP-1 and TIMP-1 in liver tissues respectively.(P> 0.05).
Conclusions:(1) Determination of TIMP-1 in liver tissues of liver fibrosis is a useful index, and has important significance for reflecting the degree of liver fibrosis. Determination of MMP-1 in liver fibrosis can not be regarded as a index of hepatic fibrosis judgment. The index of the expression ratio of MMP-1 and TIMP-1 in liver tissues can not be used for diagnosing hepatic fibrosis and judging the liver fibrosis staging.
(2)The concentration of MMP-1 and TIMP-1 and the concentration ratio of MMP-1 and TIMP-1 in serum all can not well discriminate each staging of liver fibrosis and each grade of liver inflammation activity, and are not suitable to dynamicly judge the degree of liver fibrosis and liver inflammation activity grade.However, all of the three indexes are well correlated with liver fibrosis staging and liver inflammation activity grade,and can indirectly reflect liver damage degree of chronic active hepatitis,and can offer important reference for diagnosis and prognosis judgement.
(3) The concentration of MMP-1 in serum may have higher correlation with hepatic fibrosis than TIMP-1. The concentrations of MMP-1 and TIMP-1 in serum respectively have the the correlativity with liver fibrosis staging close to with liver inflammation activity grade.
(4) The concentrations of MMP-1 and TIMP-1 in serum respectively have no correlation with the expression of MMP-1 and TIMP-1 in liver tissues.
【方法】选择2007年10月至2008年5月广西医科大学第一附属医院门诊及住院收治的慢性乙型肝炎、乙肝后肝硬化患者共28例,均行肝穿刺活组织病理检查并进行肝脏炎症活动度分级(G)及纤维化程度分期(S),同步收集患者血清,并收集同期47例健康体检者血清作为对照。用免疫组织化学二步法测定肝纤维化组织中MMP-1、TIMP-1阳性率及染色程度的方法进行病理分析,用双抗体夹芯酶联免疫吸附实验检测血清MMP-1和TIMP-1。
【结果】
①慢性乙型肝炎、乙肝后肝硬化患者肝组织中TIMP-1蛋白的阳性表达,与肝纤维化分期呈正相关(r=0.386,P<0.05);慢性乙型肝炎、乙肝后肝硬化患者肝组织中MMP-1蛋白的阳性表达,随着肝纤维化程度的加重无显著相关(P>0.05);慢性乙型肝炎、乙肝后肝硬化患者肝组织中MMP-1/TIMP-1比值与纤维化分期无显著相关(r=-0.366,P=0.056)。
②随着肝脏纤维化程度加重,血清MMP-1逐渐降低,而血清TIMP-1逐渐升高, MMP-1、TIMP-1测定在正常组与肝纤维化各期的两两比较结果显示,相邻两组间的比较均无显著性差异,但不相邻的各组间比较仅有个别无显著性差异(α=0.05);MMP-1/TIMP-1比值在各组两两比较中无规律可循。
③随着肝脏炎症活动加重,血清MMP-1含量逐渐减少,TIMP-1含量逐渐增加。MMP-1、TIMP-1测定在正常组与各炎症分级的两两比较结果显示,相邻两组间的比较均无显著性差异,但不相邻的各组间比较均有显著性差异(α=0.05); MMP-1/TIMP-1比值在各组两两比较中无规律可循。。
④血清TIMP-1与肝纤维化分期程度呈正相关(r=0.646,P<0.01),与肝脏炎症活动度分级呈正相关(r=0.622,P<0.01);血清MMP-1与肝纤维化分期程度呈高度负相关(r=-0.83,P<0.01),与肝脏炎症活动分级呈高度负相关(r=-0.821,P<0.01);血清MMP-1/TIMP-1比值与肝纤维化分期程度呈高度负相关(r=-0.776,P<0.01),与肝脏炎症活动度分级呈高度负相关(r=-0.757,P<0.01)。
⑤血清MMP-1、TIMP-1水平分别与组织中的MMP-1、TIMP-1表达无显著性相关(P>0.05)。
【结论】
(1).TIMP-1在肝纤维化组织中的检测是一个有用的指标,对反映肝纤维化程度有重要意义。MMP-1在肝纤维化组织中的检测不能作为判断肝纤维化的指标。肝组织中MMP-1/TIMP-1比值不能作为诊断肝纤维化及其分期的指标。
(2).血清MMP-1、TIMP-1含量、MMP-1/TIMP-1比值均不能很好地区分肝纤维化各期和炎症活动各分级,不适合动态判断肝纤维化程度和肝脏炎症活动度。但三者与肝纤维化分期及肝脏炎症活动度均有很好的相关性,均能间接反映慢性活动性肝炎肝脏损伤程度,对于诊断及预后判断有重要参考作用。
(3).血清MMP-1含量与肝纤维化的相关性可能比TIMP-1更高。血清TIMP-1、MMP-1含量分别与肝纤维化分期的相关程度和与炎症活动分级的相关程度相近。
(4).血清MMP-1、TIMP-1含量分别与肝组织中的MMP-1、TIMP-1表达无相关性。
Objective:To approach the expression of Matrix metalloproteina- se-1(MMP-1) and Tissue inhibitors of metalloproteinase-1(TIMP-1) in the hepatic tissue from patients who suffered chronic hepatitis B or cirrhosis after Hepatitis B and the concentration of MMP-1 and TIMP-1 in peripheral venous blood from the patients above, to analyse the correlation about the two things above separately associated with the liver inflammatory activity grade and fibrosis staging in the aspect of clinic patholog, in order to provide theoretical basis for the thing that is to find new serum index which can judge the degree of hepatic fibrosis to take the place of the invasive liver puncture biopsy.
Methods: To Choose 28 cases of patients who suffered chronic hepatitis B or cirrhosis after hepatitis B and were treated in hospital or in out-patient clinic in The First Affiliated Hospital of Guangxi Medical University from October 2007 to May 2008 .All patients were punctured out a bit of liver tissue by liver puncture biopsy for pathological examination,the latter can judge liver inflammatory activity grade (G) and fibrosis staging (S), simultaneously collecting the serum of all the patients above, and collecting 47 cases of serum from healthy examination persons in the same period as control serum..To determine positive rate and degree of staining about the expression of MMP-1 and TIMP-1 in liver cells of liver fibrosis tissue by two-step method which is a immunohistochemical method,and determine the concentration of MMP-1 and TIMP-1 in serum by using double-antibody sandwich enzyme-linked immunosorbent assay.
Results:①The expression of TIMP-1 protein in the liver tissues from paients who suffered chronic hepatitis B or cirrhosis after Hepatitis B was positively correlated with liver fibrosis staging (r = 0.386, P = 0.042); with the heavier degree of liver fibrosis, The expression of MMP-1 protein in the liver tissues above showed no significant correlation with liver fibrosis staging (P> 0.05); the Statistics about the expression ratio of MMP-1 and TIMP-1 also showed no significant correlation with liver fibrosis staging (r =- 0.366, P = 0.056).
②With the heavier degree of liver fibrosis, the concentration of MMP-1 in serum gradually decreased, while the concentration of TIMP-1 in serum gradually increased.Comparing of the normal group and each group of liver fibrosis staging, the results about the determination of MMP-1 and TIMP-1 indicate that all of the Comparisons between the each pair of next-to-next groups had no significant difference, but the Comparisons between the each pair of nonadjacent groups showed that only a few reslults indicated no significant difference. The Statistics of the comparisons about the concentration ratio of MMP-1 and TIMP-1 between each two groups were irregular.
③With liver inflammation activity increasing, the concentration of MMP-1 in serum decreased, while the concentration of TIMP-1 in serum gradually increased. Comparing of the normal group and each group of inflammation grade, the results about the determination of MMP-1 and TIMP-1 indicated that all of the Comparisons between the each pair of next-to-next groups had no significant difference, but the Comparisons between the each pair of nonadjacent groups were all significantly different (α= 0.05); The results of the comparisons about the concentration ratio of MMP-1 and TIMP-1 between each two groups were irregular.
④the concentrations of TIMP-1 in serum were positively correlated with the degree of liver fibrosis staging (r = 0.646, P = 0.000), and were positively correlated with liver inflammation activity grade (r = 0.622, P = 0.000); the concentrations of MMP-1 in serum and were highly negatively correlated with the degree of liver fibrosis staging (r =- 0.83, P = 0.000), and were highly negatively correlated with liver inflammation activity grade (r =- 0.821, P = 0.000); the concentration ratio of MMP-1 and TIMP-1 in serum were highly negatively correlated with the degree of liver fibrosis staging (r =- 0.776, P = 0.000), and were highly negatively correlated with liver inflammation activity grade (r =- 0.757, P = 0.000)
⑤the concentrations of MMP-1 and TIMP-1 in serum had no significant correlation with the expression of the MMP-1 and TIMP-1 in liver tissues respectively.(P> 0.05).
Conclusions:(1) Determination of TIMP-1 in liver tissues of liver fibrosis is a useful index, and has important significance for reflecting the degree of liver fibrosis. Determination of MMP-1 in liver fibrosis can not be regarded as a index of hepatic fibrosis judgment. The index of the expression ratio of MMP-1 and TIMP-1 in liver tissues can not be used for diagnosing hepatic fibrosis and judging the liver fibrosis staging.
(2)The concentration of MMP-1 and TIMP-1 and the concentration ratio of MMP-1 and TIMP-1 in serum all can not well discriminate each staging of liver fibrosis and each grade of liver inflammation activity, and are not suitable to dynamicly judge the degree of liver fibrosis and liver inflammation activity grade.However, all of the three indexes are well correlated with liver fibrosis staging and liver inflammation activity grade,and can indirectly reflect liver damage degree of chronic active hepatitis,and can offer important reference for diagnosis and prognosis judgement.
(3) The concentration of MMP-1 in serum may have higher correlation with hepatic fibrosis than TIMP-1. The concentrations of MMP-1 and TIMP-1 in serum respectively have the the correlativity with liver fibrosis staging close to with liver inflammation activity grade.
(4) The concentrations of MMP-1 and TIMP-1 in serum respectively have no correlation with the expression of MMP-1 and TIMP-1 in liver tissues.
引文
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[8] Gruber BL, Marchee MJ, Suzuki K, et al.Synovial rocollagenase activation by humanmast cell tryptase dependence upon matrix metalloproteinase 3 activation[J].J Clin Invest, 1989, 84(5): 1657～1662
[9] Trim JE, Samra SK, ArthurMJ,et al.Upstream tissue inhibitor of metalloproteinases-1(TIMP-1) element-1, a novel and essential regulatory DNA motif in the human TIMP-1 gene promoter, directly interacts with a 30-kDa nuclear protein[J]. J Biol Chem, 2000,275(9):6657～6663.
[10] Zhong ZD, Hammani K, Bae WS,et al.NF-Y and Sp1 cooperate for the transcriptional activation and cAMP response of human tissue inhibitor of metalloproteinases-2[J]. J Biol Chem,2000,275(24):18602～18610.
[11] Li J,Rosman AS.Leo MA.et al.Tissue inhibitor of metalloproteinase is increased in the serum of precirrhotic and cirrhotic alcoholic patients and can serve as a marker of fibrosis[J].Hepatology 1994.19(6):1418～23
[12] Arthur MJP.FibrogenesisⅡ.Metalloproteinases and their inhibitors in liver fibrosis[J].Am J Physiol Gastrointest Liver Physiol, 2000,279(2): G245～G249.
[13] Herbst H, Heinrichs O, Schuppan D, et al.Temporal and spatial patterns of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis[J].Am J Pathol, 1997, 150(5):1647～1659.
[14]王宪波,刘平,唐志鹏,等.大鼠肝纤维化形成中基质金属蛋白酶2、9活性变化的病理意义[J].中华肝脏病杂志,2004,12(5):267～270.
[15] Sakaida I , Hironaka K, Terai S et al . Gadolinium chloride reverses dimethylnitrosamine(DMN-) induced rat liver fibrosis with increased matrix metalloproteinases(MMPs) of Kupffer cells[J]. Life Sci , 2003 ,72(8) :943～59.
[16] El-Gindy I,El Rahnan AT,EI-Alim MA,et a1.Diagnostic potential of serum matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-l as non-invasive markers of hepatic fibrosis inpatients with HCV related chronic liver disease[J].Egypt J Immunol,2003,10(1):127～35.
[17] Ninomiya T,Yoon S,Nagano H,et a1.Significance of serum matrix metalloproteinases and their inhibitors on the antifibrogenetic effect of interferon-alfa in chronic hepatitis C patients[J].Intervirology 2001.44(4):227～231.
[18] Yoshiji H, Kuriyama S, Miyamoto Y,et al. Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model[J].Hepatology 2000;32(6):1248～1254.
[19]马万里,李元桂,辛建保.基质金属蛋白酶及金属蛋白酶组织抑制剂在肺纤维化中的变化[J].华中科技大学学报.医学版,2002,31(5):527～529,533.
[20] SELMAN M ,RUIZ V ,CABRERA S,et a1.TIM P-1,-2,-3,and-4 in idiopathic pulmonary fibrosis.A prevailing nondeg- radative lung microenvironment[J].Am J Physiol Lung Cell Mo1 Physiol,2000,279(3):L562～L574.
[21] Swiderski RE, Dencoff JE, Floerchinger CS, et a1.Differential express of extracellular matrix remodeling genes in a murine model of bleomycininduced pulmonary fibrosis[J].Am J Pathol,1998.152(3):821～828
[22] Perez-Ramos-J,de-Lourdes-segura-Valdez-M.Vandez-B,et a1.Matrix metalloproteinases 2 , 9 , and 13 , and tissue inhibitors of metalloproteinases 1 and 2 in experimental lung silicosis [J].Am J Respir Crit Care Med.1999,160(4):1274～1282
[23] Keeling J,Herrera GA.Matrix metalloproteinases and mesangial remodeling in light chain-related glomerular damage[J].Kidney Int,2005,68(4):1590～1603.
[24] Portik-Dobos V ,Harris AK ,Song W ,et a1.Endothelin antagonism prevents early EGFR transactivation but not increased matrix metalloproteinase activity in diabetes[J].Am J Physiol Regul Integr Comp Physiol,2006,290(2):R435～441.
[25] Lutz J,Yao Y,Song E,et a1.Inhibition of matrix metalloproteinases during chronic allograft nephropathy in rats[J].Transplantation,2005,79(6):655～661.
[26] Duymelinck C,Deng JT,Dauwe SE,et a1.Inhibition of the matrix metalloproteinase system in a rat model of chronic cyclosporine nephropathy[J].Kidney Int,1998,54(3):804～818
[27] Nakamura T, Ushiyama C, Suzuki S, et al. Elevation of serum levels of metalloproteinase-1,tissue inhibitor of metalloproteinase-1 and typeⅣcollagen,and plasma levels of metalloproteinase- 9 in polycystic kidney disease[J]. Am J Nephrol,2000,20(1):32～36.
[28] Li YY,McTieman CF,Feldman AM,et al.Interplay of matrix metalloproteinases,tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling Cardiovasc Res,2000,46:214～224.
[29] Sivasubramanian N,Coker ML, Kurrelmeyer KM,et al.Left ventricular remodeling in transgenic mice with cardiac restricted overexpression of tumor necrosis factor [J].Circulation,2001,104(7):826.
[30] Li H,Simon H,Bocan TM,et al.MMP/TIMP expression in spontaneously hypertensive heart failure rats: the effect of ACE and MMP-inhibition[J].Cardiovasc Res,2000,46(2):298～30