Hsieh Chih-Sung, Chuang Jiin-Haur, Huang Chao-Cheng, Chou Ming-Huei, Wu Chia-Lin, Lee Shin-Yi, Chen Chao-Long
Department of Pediatric Surgery, Chang Gung Memorial Hospital, Kaohsiung Hsien 833, Taiwan.
J Pediatr Surg. 2005 Oct;40(10):1568-73. doi: 10.1016/j.jpedsurg.2005.06.028.
BACKGROUND/PURPOSE: Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are major proteases responsible for remodeling the liver tissue, but their roles in biliary atresia (BA)--associated liver fibrosis are not clear.
A DNA microarray containing complementary DNA clones of 10 MMPs and 4 TIMPs was used to compare the expression profiles of the liver cytokines among 3 patients with BA at the time of Kasai procedure (KP) with 3 at the time of liver transplantation (LT). Liver samples from 2 children without liver fibrosis were used as normal controls. Those genes that were differentially expressed by more than 2-fold between groups were further quantified with real time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and validated with gel electrophoresis.
In normal human liver, messenger RNAs (mRNAs) of TIMP-1, -2, and -3, but not of TIMP-4 and none of the 10 MMPs studied, were expressed in DNA microarray. With progression of liver fibrosis, only mRNA of MMP-7, but not other MMPs, was induced to express at a significantly higher level in the array. Despite its low level of expression, MMP-9 mRNA was significantly upregulated in KP but downregulated in LT, whereas MMP-2, which was not showed in the array, was significantly upregulated in LT than in KP and control in real time QRT-PCR. There was a more than 2-fold increase in TIMP-1 and TIMP-2 mRNA expression in LT over control in the array, which was confirmed in subsequent real time QRT-PCR. The expression of TIMP-3 mRNA was significantly downregulated in KP than in control.
This study verified differential expression of MMPs and TIMPs in different stages of BA, with emphasis on the role of TIMP-1, -2, and -3 as well as MMP-2, -7, and -9 transcripts in remodeling of liver tissue during the progress of BA-associated liver fibrosis.
背景/目的：基质金属蛋白酶（MMPs）及其内源性组织抑制剂（TIMPs）是负责肝脏组织重塑的主要蛋白酶，但其在胆道闭锁（BA）相关肝纤维化中的作用尚不清楚。
使用包含10种MMPs和4种TIMPs互补DNA克隆的DNA微阵列，比较3例接受葛西手术（KP）的BA患者与3例肝移植（LT）患者肝脏细胞因子的表达谱。将2例无肝纤维化儿童的肝脏样本用作正常对照。对组间差异表达超过2倍的基因，进一步采用实时定量逆转录-聚合酶链反应（QRT-PCR）进行定量，并通过凝胶电泳进行验证。
在正常人类肝脏中，DNA微阵列显示TIMP-1、-2和-3的信使核糖核酸（mRNAs）有表达，但TIMP-4以及所研究的10种MMPs均无表达。随着肝纤维化进展，在微阵列中，仅MMP-7的mRNA被诱导表达至显著更高水平，而其他MMPs则未出现这种情况。尽管MMP-9 mRNA表达水平较低，但在KP中显著上调，而在LT中下调，而微阵列中未显示的MMP-2，在实时QRT-PCR中，LT中的表达显著高于KP和对照。在微阵列中，LT中TIMP-1和TIMP-2 mRNA表达比对照增加超过2倍，随后实时QRT-PCR予以证实。KP中TIMP-3 mRNA的表达比对照显著下调。
本研究证实了BA不同阶段MMPs和TIMPs的差异表达，强调了TIMP-1、-2和-3以及MMP-2、-7和-9转录本在BA相关肝纤维化进展过程中肝脏组织重塑中的作用。
