血清学诊断肝纤维化新指标——金属蛋白酶组织抑制因子检测方法学建立及应用评价

Detection of new diagnostic serum markers TIMPs of hepatic fibrosis and its evaluation

目的 自行建立一种改良固相致敏红细胞吸附技术 (SPASE)用于快速检测肝硬化病人血清中金属蛋白酶组织抑制因子 1,2 (TIMP 1和TIMP 2 ) ,同时研究TIMP 1和TIMP 2在肝硬化病人肝组织中的定位及表达状态 ,以及肝组织TIMPs与血清TIMPs的相关性。探讨血清中TIMP 1和TIMP 2可否作为肝纤维化的血清学诊断新指标。方法 应用自行建立的SPASE检测 10 82例肝病病人 (其中肝硬化 3 10例 )血清标本中的TIMP 1和TIMP 2。用原位杂交及免疫组织化学技术分别检测 40例肝硬化病人活检肝组织中TIMP 1和TIMP 2mRNA及相关抗原的表达 ,并与病人自身血清检测结果对比。另检测 2 0例正常肝组织作为对照。结果 SPASE法特异性中和抑制试验示中和抑制率均在 70 %以上 ,检测 3 10例肝硬化病人血清中TIMP 1和TIMP 2的阳性率分别为 79.68%和 65 .16%。 40例肝硬化病人肝组织中TIMP 1和TIMP 2mRNA及相关抗原表达阳性率为 10 0 % ,TIMP 1表达强度高于TIMP 2 (P <0 .0 1) ;阳性信号主要位于肝细胞胞质内 ,未见细胞核表达。 40例肝硬化肝活检病人血清检测结果 ,TIMP 1阳性率为 10 0 % ,TIMP 2阳性率为 77.5 % ( 3 1/4 0 )。正常肝组织无一例有TIMPs相关抗原表达。结论 TIMPs定位于肝硬化病人的肝细胞胞质内 。

Objective To set up a new method to detect the tissue inhibitors of metalloproteinase 1 and -2(TIMP 1 and TIMP 2) in sera of cirrhotic patient, and to investigate the expression and location of TIMP 1 and TIMP 2 in liver tissue of these patients, the correlation between TIMPs in liver and sera so as to discuss whether TIMPs can be used as diagnostic serum markers index for hepatic fibrosis. Methods Sera TIMP 1 or TIMP 2 of 1082 patients with liver diseases(310 patients with liver cirrhosis) was detected by solid phase absorption to sensitized erythrocytes(SPASE) set up by us. Meanwhile, with the method of in situ hybridization and immunohistochemistry, we studied the mRNA expression and location of antigen TIMP 1 and TIMP 2 in 40 cirrhotic livers confirmed pathologicaly and 20 normal control livers. Results With SPASE, the positive rates were 79.68% and 65.16% respectively in sera of hepatic cirrhosis patients, which was remarkably higher than those in chronic hepatitis and acute hepatitis. In 40 specimes of cirrhotic tissues, all showed 100% positive expression of TIMP 1 and TIMP 2 mRNA detected by immunohistochemistry or in situ hybridization. Expression of TIMPs in different degrees could be found in cirrhotic liver tissue, these were located in the cytoplasm of liver cells. There was a significant correlation between the levels of TIMPs in serum and in liver tissue. None of the normal liver tissues were positive.Conclusion SPASE is a useful method for detecting the TIMP 1 and TIMP 2 in sera of patient with hepatic cirrhosis, and TIMP 1 and TIMP 2 can be considered as useful diagnostic for serum markers of hepatic fibrosis, especially TIMP 1.