TIMP-1和TIMP-2在原发性肝癌生长、浸润及转移中的作用

目的：了解基质金属蛋白酶组织抑制因子-1(tissue inhibitou of metalloproteinase-1，TIMP-1)和基质金属蛋白酶组织抑制因子-2(TIMP-2)mRNA及相关抗原在肝癌组织中的定位和表达状态，探讨TIMP-1和TIMP-2在肝癌组织生长、浸润及转移中所起的作用。方法：以TIMP-1和TIMP-2探针及单克隆抗体（McAb）为试剂，采用原位杂交技术及免疫组织化学法检测原发性肝癌、肝高分化腺癌的肝组织中TIMP-1和TIMP-2mRNA及相关抗原的表达，并与10例正常肝组织做对照。结果：20例原发性肝癌患者的肝组织中TIMP-1和TIMP-1mRNA及相关抗原表达的阳性率为90%;9例肝高分化腺癌的腺癌组织中无TIMP-1和TIMP-2mRNA及相关抗原的表达；10例正常肝组织中TIMP-1和TIMP-2mRNA及相关抗原表达均为阴性；TIMP-1和TIMP-2mRNA及相关抗原阳性信号呈现为棕黄色颗粒状，分布在肝细胞浆内，未见细胞核着色；无论是原发性肝癌癌组织还是癌周组织中,TIMP-1的表达强于TIMP-2的表达，癌周组织中TIMP-1和TIMP-2mRNA和相关抗原的表达与肝组织病理改变相关，即肝硬化者表达强，慢性肝炎者表达弱，正常肝组织无表达。结论：原发性肝癌的癌组织中存在TIMP-1和TIMP-2mRNA及相关抗原的表达，其表达强度可能与原发性肝癌的分型有关，它可能通过抑制MMP的活性使ECM降解减少从而阻止癌细胞通过基底膜移出而抑制肝癌细胞向周围浸润及转移。     

活血化淤中药治疗对脂肪性肝炎患者肝组织TIMP-1和TIMP-2表达的影响

目的探索活血化淤中药对脂肪肝肝纤维化发生的干预作用,尤其是对肝组织TIMP-1和TIMP-2蛋白表达的影响。方法用免疫组化法分别测定15例脂肪性肝炎患者治疗前后肝组织TIMP-1和TIMP-2蛋白的表达。结果活血化淤药物治疗后TIMP-1和TIMP-2表达水平明显低于治疗前。对照组肝组织TIMP-1、TIMP-2表达水平无明显变化。结论TIMP-1、TIMP-2与肝纤维化形成密切相关,活血化淤中药治疗对脂肪肝患者肝内TIMP-1和TIMP-2蛋白的表达有一定的抑制作用。     

基质金属蛋白酶组织抑制因子mRNA及蛋白在肝硬化组织中的定位

目的：了解基质金属蛋白酶组织抑制因子TIMP－1和TIMP－2蛋白及mRNA在肝硬化组织中的表达和分布状态。方法：以抗TIMP－1和TIMP－2单克隆抗体及TIMP－1和TIMP－2 cDNA探针为试剂,采用免疫组织化学法和原位杂交技术检测肝硬化组织中TIMP－1和TIMP－2蛋白和mRNA,结果：40例肝硬化患者肝组织中TIMP－1和TIMP－2蛋白以及mRNA表达的阳性率为100％,TIMP－1阳性的组织TIMP－2亦为阳性,且TIMP－1强度高于TIMP－2,而正常肝组织未见阳性表达,TIMP－1和TIMP－2蛋白及mRNA表达的阳性信号均位于肝细胞胞浆中,细胞核中无表达,结论：肝硬化患者的肝细胞中存在TIMP－1和TIMP－2蛋白及mRNA的表达,其表达强度与肝组织病理改变呈正相关,这可能是通过抑制基质金属蛋白酶（MMPs)的活性,使得细胞外基质（ECM）降解减少而引起肝硬化。     

慢性肝病患者血清MMP-1及TIMP-1的检测及临床意义

目的研究基质金属蛋白酶-1(MMP-1)和基质金属蛋白酶组织抑制因子-1(TIMP-1)在慢性肝病患者肝纤维化过程中的表达和动态变化。方法采用双抗体"夹芯"酶联免疫吸附(ELISA)法检测64例慢性乙型肝炎和22例乙型肝炎肝硬化患者血清MMP-1和TIMP-1水平,并与20例正常体检者进行对照。结果血清MMP-1水平在慢性肝病患者肝纤维化过程中逐渐降低,而血清TIMP-1水平则逐渐增高,与正常对照组比较有显著性差异(P<0.01);其中慢性肝炎轻度患者血清MMP-1水平与中度、重度和肝硬化之间,中度与重度和肝硬化之间两两比较有显著性差异(P<0.05或P<0.01),而慢性肝炎重度与肝硬化之间比较无显著性相差(P>0.05);慢性肝炎轻度患者血清TIMP1水平与中度、重度和肝硬化之间,中度与重度和肝硬化之间,重度与肝硬化之间两两比较均有显著性差异(P<0.05或P<0.01)。结论血清MMP-1和TIMP-1在慢性肝病患者肝纤维化过程中可能起重要作用,能够被用来做为判断肝纤维化程度的指标,尤其是血清TIMP-1意义更大。     

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

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1082例肝病病人（其中肝硬化310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－2 mRNA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20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310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－2 mRNA及相关抗原表达阳性率为100％，TIMP－1表达强度高于TIMP－2（P＜0．01）；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100％，TIMP－1阳性率为77．5％（31／40）。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，在肝硬化组织内呈程度不等的阳性表达；血清中TIMPs与肝组织中TIMPs表达有明显相关性，因而血清中TIMP－1和TIMP－2可作为肝纤维化较为有用的血清学诊断新指标，尤其是TIMP－1的诊断意义更大。SPASE法检测血清中TIMPs具有较好的特异性。     

转化生长因子-1和基质金属蛋白酶抑制因子-1在肝硬化组织中的表达及其临床意义

目的了解转化生长因子-1（TGFβ1）和基质金属蛋白酶组织抑制因子-1（TIMP1）在肝硬化组织中的表达和分布状态。方法采用免疫组化法（S—P）。检测10例对照肝组织,57例肝硬化患者肝组织中TGFβ1、TIMP1的表达。结果57例肝硬化患者肝组织中TGFβ1、TIMP1蛋白表达的阳性率分别为90．38％、100％,而正常肝组织未见阳性表达,差异有统计学意义（P〈0．01）。TGFβ1、TIMP1表达的阳性信号均位于肝细胞胞浆中,细胞核中无表达。结论（1）肝硬化患者肝细胞中存在TGFβ1、TIMP1的表达,其表达强度与肝组织病理改变相关。提示TGFβ1与TIMP1的表达与肝纤维化活动状态密切相关,在肝纤维化的形成和发展中具有重要意义。（2）检测TGFβ1、TIMP1的表达可对肝硬化病变从分子水平进行评估,也可作为早期诊断及临床治疗和判断预后的分子病理学指标。     

抗病毒治疗对HBeAg阴性CHB患者TGF-β1、TIMP-1、MMP-1水平的影响

目的 探讨TGF-β1、TIMP-1、MMP-1在HBeAg阴性CHB患者肝纤维化诊断中的作用以及抗乙肝病毒治疗对其影响.方法 以正常体检健康者为对照组,HBeAg阴性CHB分轻度、中度、重度组,依据抗病毒治疗48周后HBV DNA低于检测下限分为治疗有效组和治疗无效组,ELISA法测定各组血清中TGF-β1、TIMP-1、MMP-1含量,免疫组化检测肝组织TIMP-1、MMP-1表达.结果 HBeAg阴性慢性乙型肝炎外周血TGF-β1、TIMP-1水平明显高于正常对照组(P＜0.01),而血清MMP-1则明显低于正常对照组(P＜0.01).随慢性肝炎轻度-重度逐渐发展,血清TGF-β1、TIMP-1的水平逐渐增高,且差异有统计学意义(P＜0.05).TIMP-1蛋白在肝纤维化组织的阳性表达,随肝纤维化程度的加重而增强,呈正相关(rs=0.618,P=0.002);MMP-1蛋白在肝纤维化组织的阳性表达与肝纤维化程度无相关性(rs=0.077,P=0.732).抗病毒治疗48周后有效组血清TGF-β1、TIMP-1的含量明显下降,MMP-1水平上升(P＜0.01).无效组血清TGF-β1、TIMP-1、MMP-1水平变化不明显(P＞0.05).结论 联合检测TGF-β1、TIMP-1、MMP-1是判断慢性肝病患者有无纤维组织增生敏感而可靠的指标,动态观察上述指标的变化,对临床判断肝纤维化程度和活动度有着重要价值,同时也为临床疗效的判断提供有效的实验依据.     

慢性乙型肝炎患者肝组织和血清TIMP-2水平对肝纤维化程度的评估

目的了解慢性乙型肝炎患者肝组织和外周血清基质金属蛋白酶组织抑制物-2（TIMP-2）的表达情况,以进行对肝脏纤维化程度的评估。方法对105例慢性乙型肝炎患者肝活检组织进行肝纤维化分期。通过免疫组化技术检测患者肝组织TIMP-2的表达水平,采用ELISA法测定患者血清TIMP-2水平。结果随着慢性乙型肝炎患者肝纤维化程度的加重,肝组织TIM-2表达水平逐步上升,各期之间表达差异有统计学意义（P〈0．05）;与健康对照者比,S1-S4期患者血清TIMP-2水平均升高（P〈0．05）,除S1与S2期之间无显著性差异外,其余各期之间TIMP-2水平有统计学差异。结论肝组织和外周血TIMP-2表达水平可以较好地反映肝脏的纤维化程度。     

慢性肝病患者外周血细胞间质胶原酶(MMP-1)及其抑制剂(TIMP-1)基因表达的研究

采用RT-PCR法及Northem杂交，检测38例慢性肝炎患者及20例正常成人外周血单个核细胞中MMP-1及TIMP-1mRNA的表达。结果发现正常成人外周血单个核细胞中存在MMP-1及TIMP-1的表达，慢肝患者外周血单个核细胞中MMP-1及TIMP-1的表达升高，MMP-1/TIMP-1比值显著升高，并且与肝纤维化严重程度呈正相关。与肝纤维化血清学指标HA、LN-C-Ⅳ、PCⅢ变化一致，提示外周血单个核细胞中MMP-1及TIMP-1mRNA的表达水平与肝纤维化严重程度关系密切。MMP-1/TIMP-1比值可为临床诊断提供一个新的血清学指标。     

痰上清液MMP-9、TIMP-1与慢性阻塞性肺疾病气道炎症的关系

目的探讨痰上清液基质金属蛋白酶(MMP)-9及基质金属蛋白酶组织抑制剂(TIMP-1)与慢性阻塞性肺疾病(COPD)气道炎症的关系。方法 COPD急性期患者47例,COPD稳定期患者50例,同期健康体检者50例为健康对照组,均采集痰液计算痰细胞总数,离心处理后经细胞涂片瑞氏染色计算每类细胞数,包括中性粒细胞(Neu)、淋巴细胞(Lym)、巨噬细胞(Macro)及嗜酸性细胞(Eos),同时采用双抗体夹心法对痰上清液MMP-9与TIMP-1进行检测,分析COPD急性期MMP-9、TIMP-1与气道炎症的相关性。结果 COPD患者的细胞总数、Neu、Lym、Macro、Eos明显高于对照组(P<0.05),急性期组细胞总数、Neu、Neu/白细胞计数(WBC)明显高于稳定期组(P<0.05),而Lym/WBC、Macro/WBC、Eos/WBC明显低于稳定期组与对照组(P<0.05);急性期组的痰MMP-9、TIMP-1水平及MMP-9/TIMP-1比值均明显高于稳定期组与对照组(P<0.05),且稳定期组明显高于对照组(P<0.05);COPD急性期痰上清液中除TIMP-1外,MMP-9水平及MMP-9/TIMP-1均与Neu、Lym、Macro及Eos呈正相关(P<0.05)。而COPD稳定期仅痰上清液TIMP-1与Macro呈正相关(P<0.05)。结论 MMP-9与COPD气道炎症的关系紧密,TIMP-1与MMP-9水平失衡可能是COPD急性发作的重要原因。     

Methodologic research on TIMP—1,TIMP—2 detection as a new diagnostic index for hepatic fibrosis and its significance

AIM: To set up a new method to detect tissue inhibitors of metalloproteinase-1 and -2(TIMP-1 and TIMP-2) in sera of patients with hepatic cirrhosis, and to investigate the expression and location of TIMP-1 and TIMP-2 in liver tissue of patients with hepatic cirrhosis, and the correlation between TIMPs in liver and those in sera so as to discuss whether TIMPs can be used as a diagnosis index of hepatic fibrosis. METHODS: The monoclonal antibodies (McAbs) of TIMP-1 and TIMP-2 were used to sensitize erythrocytes, and solid-phase absorption to sensitized erythrocytes (SPASE) was used to detect TIMP-1 and TIMP-2 in the sera of patients with hepatic cirrhosis. Meanwhile, with the method of in situ hybridization and immunohistochemistry, we studied the mRNA expression and antigen location of TIMP-1 and TIMP-2 in the livers of 40 hepatic cirrhosis patients with pathologic diagnosis. RESULTS: With SPASE, they were 16.4% higher in the acute hepatitis group, 33.3% higher in the chronic hepatitis group, and the positive rates were 73.6% and 61.2% respectively in sera of hepatic cirrhosis patients, which were remarkably higher than those in chronic hepatitis and acute hepatitis group (P<0.001). In 40 samples of hepatic cirrhosis tissues, all of them showed positive expression of TIMP-1 and TIMP-2 mRNA detected with immunohistochemistry or in situ hybridization (positive rate was 100%). Expression of TIMPs in different degrees could be found in liver tissue with cirrhosis. TIMPs were located in cytoplasm of liver cells of patients with hepatic cirrhosis. There was a significant correlation between serum TIMPs level and liver TIMPs level. CONCLUSION: SPASE is a useful method to detect the TIMP-1 and TIMP-2 in sera of patients with hepatic cirrhosis, and TIMP-1 and TIMP-2 can be considered as a useful diagnostic index of hepatic fibrosis, especially TIMP-1.     

Expression of TIMP-1 and TIMP-2 in rats with hepatic fibrosis

AIM: To investigate the location and expression of TIMP-1 and TIMP-2 in the liver of normal and experimental hepatic fibrosis in rats. METHODS: The rat models of experimental immunity hepatic fibrosis (n=20) were prepared by the means of immunologic attacking with human serum albumin (HSA),and normal rats (n=10) served as control group. Both immunohistochemistry and in situ hybridization methods were respectively used to detect the TIMP-1 and TIMP-2 mRNA and related antigens in liver. The liver tissue was detected to find out the gene expression of TIMP-1 and TIMP-2 with RT-PCR. RESULTS: The TIMP-1 and TIMP-2 related antigens in livers of experimental group were expressed in myofibroblasts and fibroblasts (TIMP-1: 482&#177;65 vs 60&#177;20; TIMP-2:336&#177;48 vs 50&#177;19, P&lt;0.001). This was the most obvious in portal area and fibrous septum. The positive signals were located in cytoplasm, not in nucleus. Such distribution and location were confirmed bysitu hybridization (TIMP-1/β-actin: 1.86&#177;0.47 vs 0.36&#177;0.08; TIMP-2/β-actin: 1.06&#177;0.22 vs 0.36&#177;0.08,P&lt;0.001). The expression of TIMP-1 and TIMP-2 was seen in the liver of normal rats, but the expression level was very low. However, the expression of TIMP-1 and TIMP-2 in the liver of experimental group was obviously high. CONCLUSION: In the process of hepatic fibrosis, fibroblasts and myofibroblasts are the major cells that express TIMPs.The more serious the hepatic fibrosis is in the injured liver,the higher the level of TIMP-1 and TIMP-2 gene expression.     

Clinicopathological correlations of TIMP-1 and TIMP-2 in Hodgkin's lymphoma

OBJECTIVES: The influence of matrix-tumour interactions in Hodgkin's lymphoma is poorly characterised, although a large part of the tumour often consists of reactive tissue. The aim of the present study was to assess the clinicopathological role of two main inhibitors of matrix metalloproteinases, TIMP-1 and TIMP-2, in Hodgkin's lymphoma. MATERIALS AND METHODS: The TIMP-1 and TIMP-2 protein expressions were studied from paraffin-embedded tumour sections of 68 patients with Hodgkin's lymphoma by using immunostaining with TIMP-1 and TIMP-2-specific antibodies. The results of the stainings were compared with the clinicopathological disease characteristics. RESULTS: A total of 33.3% of the tumour tissue sections expressed TIMP-1 and 46.8% expressed TIMP-2. The expression of the TIMP-1 protein was found to be strongly associated with the nodular sclerosis subtype (P = 0.015) and the existence of a bulky tumour (P = 0.004) in Hodgkin's lymphoma. The expression of the TIMP-2 protein, on the other hand, correlated with the occurrence of B symptoms (P = 0.032). CONCLUSIONS: These results provide the first clinical evidence suggesting that TIMP-1 could promote growth of Hodgkin's lymphoma, and may be linked to connective tissue turnover in the nodular sclerosis subtype. However, TIMP-2 is shown to correlate with systemic symptoms.     

HGV/GBV-C与HCV混合感染者肝组织中相关病毒表达的免疫组化研究

目的 了解HGV/GBV-C与HCV混合感染者肝组织HGV/GBV-C相关抗原的分布状况,探讨HGV/GBV-C对肝脏的损害机制。方法 以抗HGV/GBV-C NS5单克隆抗体或抗HCV NS3单克隆抗体为试剂,采用免疫组织化学方法检测肝炎病人肝组织中HGV/GBV-C、HCV相关抗原表达。结果 56例肝炎病肝组织中HGV/GBV-C相关抗原表达阳性率为26.79％(15/56);HCV NS3抗原表达阳性率为39.29％(22/56)。HGV/GBV-C NS5抗原表达阳性信号主要位于肝细胞胞浆中,染色阳性细胞周围可见淋巴细胞浸润。结论 肝细胞中存在HGV/GBV-C相关抗原表达,其编码产物可能作为一种靶抗原,诱发免疫病理反应,免疫损伤可能是其发病机制之一。     

固相致敏红细胞吸附技术快速检测肝硬化患者血清中TIMP-1

目的 建立一种新的固相致敏红细胞吸附技术(SPASE)用于快速检测肝硬化患者血清中TIMP-1。方法 用TIMP-1单克隆抗体包被微量血凝板,加热冲洗后加入待检血清标本,最后加入单克隆抗体致敏红细胞。应用致敏红细胞作为指示系统,观察红细胞吸附状况来判定结果。结果 SPASE法全过程仅需要2.5～3小时,408例肝病患者血清检测TIMP-1结果为急性肝炎组检出阳性率16.41％;慢性肝炎组阳性率为33.31％;肝硬化组阳性率为73.58％。结论 血清中TIMP-1的变化可作为肝纤维化较为有用的诊断指标。SPASE法具有较高的灵敏性、特异性和快速性,试剂用量少,且不需复杂的仪器设备,加之实验程序简单,更适于医院及基层医疗单位应用。     

Tissue inhibitor of metalloproteinase-1 in the liver of patients with chronic liver disease

Background/Aims: Tissue inhibitor of metalloproteinase (TIMP)-1 is an important regulator of matrix metalloproteinase activity. To clarify the changes in TIMP-1 in diseased livers, we measured TIMP-1 concentrations in liver tissue samples from patients with chronic liver disease. The relationship between serum and liver levels of TIMP-1 was also examined in some patients.Methods: The subjects were 68 patients who underwent liver biopsy. The liver TIMP-1 concentration was measured using an enzyme immunoassay after the extraction of TIMP-1 with 2 M guanidine.Results: As compared with the controls (n=10), the liver TIMP-1 level was increased 2.2-fold in the 24 chronic active hepatitis 2A patients, 2.9-fold in the 10 chronic active hepatitis 2B patients and 4.1-fold in the six liver cirrhosis patients, but no significant increase was observed among the 18 chronic persistent hepatitis patients. The liver TIMP-1 levels were closely correlated with the histological degrees of periportal necrosis, portal inflammation, and liver fibrosis. When the localization of TIMP-1 was examined immunohistochemically, TIMP-1 was stained mainly in hepatocytes, and the intensity was stronger in the livers of chronic active hepatitis and liver cirrhosis patients than in those of the chronic persistent hepatitis patients. The serum TIMP-1 and liver TIMP-1 levels were significantly correlated, indicating that serum TIMP-1 could reflect the change of liver TIMP-1 in patients with chronic liver disease.Conclusion: Liver TIMP-1 concentration increases with progression of the liver disease, when the degradation of extracellular matrix proteins is decreased, resulting in the development of liver fibrosis.     

ELISA法快速检测慢性肝病患者血清中TIMP-1

目的 应用酶联免疫吸附方法（ELISA）快速检测慢性肝病患者血清中基质金属蛋白酶组织抑制剂-1（TIMP-1），了解TIMP-1血清学检测与肝纤维化病程进展程度。方法 以抗人TIMP-1单克隆抗体为包被抗体，15％小牛血清为封闭液，辣根过氧化物酶（HRP）标记的羊抗人IgG作为酶结合物建立检测人血清TIMP-1 ELISA法，用于临床检测399例肝病患者血清和健康人血清105例。结果 临床检测表明肝硬化患者TIMP-1检出阳性率为76．9％，明显高于慢性肝炎（40．7％）和急性肝炎（19．4％）（P〈0．005），并随着纤维化程度加重而升高（P〈0．01）。结论 血清中TIMP-1的变化可作为肝纤维化较为有用的诊断指标。ELISA法具有较高的敏感性、特异性和快速性，试剂用量少，且不需复杂的仪器设备，加之实验程序简单，甚适于医院及基层医疗单位应用。