血小板来源的生长因子受体识别肽在肝纤维化无创诊断中的应用

It is critical for the diagnosis and management of chronic liver disease to diagnosis liver fibrosis as early as possible, but there is no effective noninvasive method to identify liver fibrosis at different stage at present. The key factors in hepatic fibrogenesis are the activation and proliferation of hepatic stellate cells (HSC). As a result of sustained or repeated liver injury, HSC undergo a process of activation and transform into myofibroblast-like cells, which are characterized by α-smooth muscle actin (α-SMA) expression. In our work supported by Grants from the National Nature Science Funds for Young Scholar, we found that most of the area positive for platelet derived growth factor receptor-β(PDGFR-β) was overlapped with the area positive for α-SMA. In addition, we prepared the pPB cyclic peptides which could specially recognize PDGFR-β. After the liposomes were modified with pPB, they could specially bind to activated HSC. Thus, in this study, we suppose to use radiolabeled pPB cyclic peptides to image hepatic PDGFR-β-expression in fibrotic livers in order to develop a noninvasive approach to diagnosis and stage liver fibrosis.

肝纤维化的早期诊断对于慢性肝病的诊治具有重要的意义，目前尚缺乏能区分各期肝纤维化的无创诊断方法。活化的肝星状细胞（HSC）是肝纤维化过程中纤维胶原的主要来源细胞，α-SMA被认为是活化HSC的标记物。在青年科学基金项目研究工作中，我们发现在纤维化的肝组织中，血小板来源生长因子受体-β（platelet derived growth factor receptor-β, PDGFR-β）分布的区域与α-SMA分布的区域在很大程度上相互重叠；更为重要的是，我们成功合成能特异识别PDGFR-β的pPB环肽，利用该环肽修饰脂质体后，能特异结合在活化的HSC上。因此，在本课题中，我们设想利用同位素标记的pPB环肽来显影纤维化肝组织中的PDGFR-β的表达情况，通过分子影像学的显影方法来诊断肝纤维化并尝试根据同位素结合量的差异区分不同程度的纤维化，从而为肝纤维化的无创诊断提供一种新方法。

慢性肝病的预后和治疗很大程度上取决于肝纤维化的程度。本项目通过胆总管结扎（BDL）、四氯化碳皮下注射（CCl4）和高脂饮食饲养（HFD）三种方法诱导了不同发病机制的大鼠肝纤维化模型，通过Real-time PCR和Western blot以及免疫荧光检测，发现血小板来源生长因子受体-β（PDGFR-β）在肝组织中的表达水平随着肝纤维化的加重而升高。PDGFR-β和肝非实质细胞标记物的双免疫荧光染色发现纤维化的肝组织中，表达PDGFR-β的细胞主要是表达α-SMA的活化肝星状细胞和表达CD31的肝窦内皮细胞。体外细胞培养证实人活化的肝星状细胞和肝窦内皮细胞表达PDGFR-β，这两种细胞可以特异结合PDGFR-β的受体识别肽（pPB），并且这种结合是时间依赖和浓度依赖的。用钆（Gd）标记的pPB环肽作为示踪剂，通过核磁共振显像不同纤维化程度的肝组织中表达PDGFR-β水平的差异，成功地区分了肝纤维化的程度。本项目的实施，为慢性肝病肝纤维化的诊断提供了一种新的、无创伤的分子影像学的方法。通过今后该方法的进一步完善，将可能运用到临床，从而造福于广大的慢性肝病患者。