Mangasser-Stephan K, Gartung C, Lahme B, Gressner A M
Institute of Clinical Chemistry and Pathobiochemistry, Central Laboratory, RWTH-University Hospital, Aachen, Germany.
Liver. 2001 Apr;21(2):105-13. doi: 10.1034/j.1600-0676.2001.021002105.x.
BACKGROUND/AIMS: The activation of hepatic stellate cells (HSC) to extracellular matrix (ECM) producing myofibroblasts (MFB) is the key pathogenetic event in human liver fibrogenesis. Latent transforming growth factor beta binding protein (LTBP), a component of the profibrogenic large latent transforming growth factor (TGF)-beta complex, is suggested to be important for secretion, latency, storage and activation of TGF-beta in the ECM. This study was performed to identify the expression profile of all hitherto known LTBP isoforms and LTBP splice variants in conjunction with that of TGF-beta isoforms in cultured human liver MFB.
Cultured human MFB were analyzed for TGF-beta and LTBP using reverse-transcription polymerase chain reaction (RT-PCR), sequence analysis, immunofluorescence staining, metabolic labeling, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
Transcripts of all three TGF-beta isoforms, of all four LTBP isoforms and of nearly all splice variants of LTBP-1 and LTBP-4 so far known were detected. Metabolic labeling followed by immunoprecipitation with anti-LTBP-1 antibody revealed the synthesis of LTBP proteins. Secretion of free LTBP and LTBP integrated into the large latent TGF-beta complex was demonstrated by size-exclusion chromatography. Co-localization of LTBP-1 and -2 with fibronectin and collagen type I was observed by double immunofluorescence staining.
The expression of a complete profile of hitherto known LTBP proteins by cultured human MFB suggests a role in modulating the bioactivity of TGF-beta in the diseased liver.
背景/目的：肝星状细胞（HSC）激活成为产生细胞外基质（ECM）的肌成纤维细胞（MFB）是人类肝纤维化发生的关键致病事件。潜在转化生长因子β结合蛋白（LTBP）是促纤维化大潜在转化生长因子（TGF）-β复合物的一个组成部分，被认为对TGF-β在ECM中的分泌、潜伏、储存及激活很重要。本研究旨在确定在培养的人肝MFB中，所有已知LTBP亚型和LTBP剪接变体与TGF-β亚型的表达谱。
采用逆转录聚合酶链反应（RT-PCR）、序列分析、免疫荧光染色、代谢标记、免疫沉淀和酶联免疫吸附测定（ELISA）分析培养的人MFB中的TGF-β和LTBP。
检测到所有三种TGF-β亚型、所有四种LTBP亚型以及目前已知的LTBP-1和LTBP-4几乎所有剪接变体的转录本。代谢标记后用抗LTBP-1抗体进行免疫沉淀，显示LTBP蛋白的合成。通过尺寸排阻色谱法证明了游离LTBP和整合到大型潜在TGF-β复合物中的LTBP的分泌。通过双重免疫荧光染色观察到LTBP-1和-2与纤连蛋白和I型胶原的共定位。
培养的人MFB表达完整的已知LTBP蛋白谱，提示其在调节患病肝脏中TGF-β生物活性方面发挥作用。
