Department of Gastroenterology, Zhongshan Hospital affiliated to Fudan University, 180 Feng Lin Road, Shanghai 200032, China.
Biochem Pharmacol. 2010 Jul 1;80(1):136-43. doi: 10.1016/j.bcp.2010.03.015. Epub 2010 Mar 18.
Activated hepatic stellate cells (HSCs) play a crucial role in the development of liver fibrosis. Noninvasive monitoring of the activation of HSCs has been challenging due to the lack of specific receptors or motifs on the cells. The present study provides the evidence that integrin alpha v beta 3 expressed on HSCs is a biomarker reflecting the activation of HSCs. Solid-phase synthesis of cRGDyK (Arg-Gly-Asp-(D)Tyr-Lys) peptide and FAM-conjugated peptide were employed for binding to integrin alpha v beta 3. The increased expression of integrin alpha v and beta 3 at mRNA and protein levels was detected during HSC activation. The affinity of cRGDyK to integrin alpha v beta 3 was examined by both radioligand binding assay and FAM-conjugated peptide binding measurements. Quantitative RT-PCR and Western blotting showed a less dramatic, but significant increase in alpha v and beta 3 integrin mRNA and protein expression following activation of rat HSCs. Radioiodinated cRGDyK binds to both purified and membrane-bound integrin alpha v beta 3 with high affinity in a dissociable manner. FAM-conjugated cRGDyK was coupled to activated HSCs in a time- and dose-dependent, receptor-mediated manner. Activated HSCs express sufficient number of integrin alpha v beta 3 receptor. cRGDyK peptide binds to both purified and membrane-bound integrin alpha v beta 3 with high affinity in a reversible fashion. Thus, the cRGDyK peptide represented a new agent potentially useful for the diagnosis of liver fibrosis.
活化的肝星状细胞（HSCs）在肝纤维化的发展中起着至关重要的作用。由于 HSCs 上缺乏特异性受体或基序，因此对其活化的非侵入性监测一直具有挑战性。本研究提供的证据表明，HSCs 上表达的整合素α v β 3 是反映 HSCs 活化的生物标志物。采用固相合成法合成 cRGDyK（精氨酸-甘氨酸-天冬氨酸-（D）酪氨酸-赖氨酸）肽和 FAM 缀合肽，用于与整合素α v β 3 结合。在 HSC 活化过程中，检测到整合素α v 和β 3 的 mRNA 和蛋白水平表达增加。通过放射性配体结合测定和 FAM 缀合肽结合测量检查 cRGDyK 对整合素α v β 3 的亲和力。定量 RT-PCR 和 Western blot 显示，大鼠 HSCs 活化后，α v 和β 3 整合素 mRNA 和蛋白表达均有明显但不显著的增加。放射性碘标记的 cRGDyK 以可解离的方式与纯化的和膜结合的整合素α v β 3 以高亲和力结合。FAM 缀合的 cRGDyK 以时间和剂量依赖性、受体介导的方式与活化的 HSCs 偶联。活化的 HSCs 表达足够数量的整合素α v β 3 受体。cRGDyK 肽以可逆的方式与纯化的和膜结合的整合素α v β 3 以高亲和力结合。因此，cRGDyK 肽代表了一种新的潜在有用的肝纤维化诊断试剂。
