实验性胆汁淤积性胆管增生中羊膜上皮细胞衍生的胆管细胞。

Amniotic epithelial cell-derived cholangiocytes in experimental cholestatic ductal hyperplasia.

机构信息

Division of Gastroenterology, Tohoku University Graduate School of Medicine, Miyagi, Japan; and Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, California, USA.

出版信息

Hepatol Res. 2007 Apr;37(4):286-94. doi: 10.1111/j.1872-034X.2007.00049.x.

DOI:10.1111/j.1872-034X.2007.00049.x

PMID:17397517

Abstract

AIM

Bile duct paucity, ductopenia, is a feature of end-stage chronic cholangiopathies such as primary biliary cirrhosis. The limited proliferative ability of cholangiocytes after specific injury is thought to be the principal cause of ductopenia, although the detailed mechanisms involved are unclear. It has been reported that human amniotic epithelial cells (AEC) express differentiation markers of hepatic parenchymal cells, suggesting a resemblance of AEC to hepatic progenitor cells. The aim of the present study was to develop a mouse model of experimental cholestasis to assess the capability of mouse AEC to trans-differentiate into cholangiocytes.

METHODS

Enhanced green fluorescent protein (EGFP)-transgenic C57BL/6 pregnant female mice were used as the source of AEC. At 11.5 gestational days, 1 x 10(5) AEC were isolated from EGFP-transgenic mouse embryos and transferred into C57BL/6 mice. Chronic cholestasis was induced by 0.1%alpha-naphthylisothiocyanate (ANIT) feeding immediately after the transfer of AEC. The proliferation of cholangiocytes in the livers was assessed morphologically and immunohistochemically (cytokeratin 7; CK7). The proliferative activity was also quantified immunohistochemically by proliferating cell nuclear antigen (PCNA) protein expression. EGFP of transferred AEC was confirmed by fluorescent laser microscopy and immunofluorescent staining for EGFP. Also, Notch2 and Hes1 expression was evaluated to examine the roles of the differentiation markers in this process.

RESULTS

Marked proliferation of cholangiocytes was observed in ANIT-fed mice confirmed by quantitative CK7 (3-4 fold vs control) and PCNA (11-20 fold vs control) staining. EGFP and CK7 double positive cells in interlobular bile ducts were confirmed in the livers of AEC-transferred recipients. Positivity of EGFP was further confirmed by the immunofluorescent staining for EGFP. Moreover, both Notch2 and Hes1 expression was confirmed in the proliferative bile duct in this model.

CONCLUSIONS

Significant ductular proliferation was observed in ANIT-fed mice. EGFP-positive cholangiocytes were confirmed in this chronic cholestasis model. AEC transfer was able to contribute to the repopulating of proliferating cholangiocytes under cholestasis, suggesting AEC might be a candidate cell source for stem cell administration in future clinical applications to re-model interlobular bile ducts.

摘要

目的

胆管稀少症（ductopenia）是原发性胆汁性肝硬化等终末期慢性胆管疾病的特征。虽然涉及的详细机制尚不清楚，但人们认为，胆管细胞在特定损伤后的有限增殖能力是胆管稀少症的主要原因。据报道，人羊膜上皮细胞（AEC）表达肝实质细胞的分化标志物，这表明 AEC 与肝祖细胞相似。本研究的目的是建立一种实验性胆汁淤积的小鼠模型，以评估小鼠 AEC 向胆管细胞转分化的能力。

方法

使用增强型绿色荧光蛋白（EGFP）转基因 C57BL/6 孕鼠作为 AEC 的来源。在 11.5 天的妊娠期，从 EGFP 转基因鼠胚胎中分离出 1x10(5)个 AEC，并转移到 C57BL/6 小鼠体内。在转移 AEC 后立即给予 0.1%α-萘基异硫氰酸酯（ANIT）喂养，诱导慢性胆汁淤积。通过细胞角蛋白 7（CK7）免疫组织化学染色（免疫组化）评估肝内胆管细胞的增殖情况。增殖细胞核抗原（PCNA）蛋白表达的免疫组化也对增殖活性进行了定量。通过荧光激光显微镜和 EGFP 的免疫荧光染色证实了转染 AEC 的 EGFP。还评估了 Notch2 和 Hes1 的表达，以检查这些分化标志物在该过程中的作用。

结果

在 ANIT 喂养的小鼠中观察到胆管细胞的显著增殖，这一点通过定量 CK7（是对照组的 3-4 倍）和 PCNA（是对照组的 11-20 倍）染色得到了证实。在 AEC 转移受者的肝脏中，在小叶间胆管中确认了 EGFP 和 CK7 双阳性细胞。通过 EGFP 的免疫荧光染色进一步证实了 EGFP 的阳性。此外，在该模型的增殖性胆管中还证实了 Notch2 和 Hes1 的表达。