肝内胆管系の粘膜免疫機構とその破綻:自己免疫性胆管系疾患と肝内胆管癌を中心に

肝内胆管系统的粘膜免疫机制及其分解：聚焦自身免疫性胆管疾病和肝内胆管癌

• 批准号: 03J08618
• 负责人: 一瀬 久美子
• 金额: $ 1.15万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03J08618/
• 关键词: 原発性胆汁性肝硬変; ケモカイン; サイトカイン; 自己免疫性疾患; 肝内胆管; 自己免疫性胆管疾患; 粘膜免疫; リンパ球

【背景】Fractalkine(FRK)は細胞の遊走と接着の双方に関与するケモカインで,小腸でのFRK/CX36R1発現は腸管上皮層内リンパ球の維持とCrohn病の病態形成に関与する.原発性胆汁性肝硬変(PBC)は肝内小型胆管の選択的な破壊と消失を特徴とする自己免疫性肝疾患で,胆管周囲及び上皮層内へのリンパ球浸潤が病態形成に重要である.今回我々は,肝内小型胆管におけるFRK/CX3CR1発現とFRKの発現調節機構について検討した.【材料と方法】対象はPBC(1〜2期)17例,原発性硬化性胆管炎9例,閉塞性黄疸10例,C型慢性ウイルス性肝炎20例,組織学的正常肝18例,肝内小型胆管におけるFRK,CX3CR1発現を免疫組織化学染色(IHC)にて検討し,胆管周囲炎症細胞をCX3CR1とCD3,CD4,CD8との蛍光二重染色にて検討した.胆管上皮をmicrodissectしRT-PCRを行った.肝内胆管癌細胞株(HuCC-T1),ヒト肝内胆管細胞培養株(HIBEC)でのFRK,CX3CR1発現をIHC,RT-PCRにて検討し,細胞株に対する種々のサイトカイン刺激によるFRK発現の動態をreal time PCR, Western blot, ELISA法にて検討した.HuCC-T1を用いCX3CR1^+細胞株(THP-1)のchemotactic assayを行った.【結果】IHCでは対照群に比しPBCでの肝内小型胆管の有意なFRK発現亢進と門脈域内CX3CR1^+細胞数の増加を認め,CX3CR1^+T細胞の胆管上皮層内浸潤像を認めた.microdissectした胆管上皮からFRK/CX3CR1 mRNAを検出した.胆管培養細胞に対するLPS,IL-1β,IFN-γ,TNF-a刺激にてFRK mRNA発現が亢進し,培養細胞及び培養上清中のFRK蛋白の増加も認めた.IL-4,IL-6刺激によるFRK mRNA発現誘導は明らかではなかった.HuCCT1によるTHP-1遊走活性はLPS刺激で増加し,recombinant FRK,CX3CR1抗体投与で阻害された.【考察】PBCの障害胆管周囲の著明な炎症細胞浸潤はサイトカインネットワークの存在を示唆する.今回,in vivoにてPBCの障害胆管でFRK発現が亢進し,胆管周囲及び上皮層内にCX3CR1^+細胞の浸潤を多く認めた.in vitroにて培養胆管細胞でのLPS,IL-1β,IFN-γ,TNF-a刺激によるFRK mRNA,蛋白の発現亢進が認められ,CX3CR1^+細胞の遊走がFRK/CX3CR1系によることが示された.以上より,PBCの胆管周囲にみられる特異的な炎症が肝内小型胆管上皮のFRK過剰発現によることが明らかとされた.

FRACTalkine(FRK) is involved in cell migration and subsequent development. FRK/CX36R1 in the small intestine is involved in the development of Crohn disease. Primary biliary cirrhosis (PBC) is characterized by the disappearance of small intrahepatic bile ducts and plays an important role in the pathogenesis of autoimmune liver disease. In this paper, FRK/CX3CR1 development and FRK development regulatory mechanism in small intrahepatic bile ducts are discussed. Materials and Methods: Seventeen cases of PBC(stage 1 ~ 2), 9 cases of primary sclerosing cholangitis, 10 cases of red jaundice, 20 cases of chronic hepatitis C, 18 cases of histologically normal liver, small intrahepatic bile ducts with FRK, CX3CR1 were detected by immunohistochemical staining (IHC), and inflammatory cells around bile ducts with CX3CR1, CD3, CD4, CD8 were detected by double staining. Bile duct epithelium microdissection RT-PCR FRK, CX3CR1 expression in intrahepatic cholangiocarcinoma cell line (HuCC-T1), intrahepatic cholangiocarcinoma cell culture line (HIBEC) was investigated by IHC,RT-PCR, and the dynamics of FRK expression in cell line CX3CR1^+ cell line (THP-1) was investigated by real time PCR, Western blot, and ELISA. [Results] IHC microdissect FRK/CX3CR1 mRNA expression in intrahepatic small bile ducts and increase of CX3CR1 ^+ cell count in portal vein compared with PBC. FRK mRNA expression increased in bile duct culture cells stimulated by LPS,IL-1β,IFN-γ,TNF-a, FRK protein increased in culture cells and culture supernatant. FRK mRNA expression increased in response to IL-4,IL-6 stimulation. THP-1 migration activity increased in HuCCT1 cells stimulated by LPS, and recombinant FRK, CX3CR1 antibody inhibited FRK protein expression. [Investigation] The presence of inflammatory cell infiltration in the peripheral bile duct of PBC is indicated. In vivo, FRK expression in PBC-impaired bile ducts was increased, and infiltration of CX3CR1^+ cells in the peribiliary and supracortical areas was more frequent. In vitro, FRK mRNA and protein expression in cultured bile duct cells stimulated by LPS,IL-1β,IFN-γ and TNF-a were increased, and migration of CX3CR1 ^+ cells in FRK/CX3CR1 system was also observed. As mentioned above, the inflammation of the peripheral bile duct of PBC is specific to the FRK of small intrahepatic bile duct epithelium.

项目成果

Harada K, Ohira S, Isse K, Ozaki S, Zen Y, Sato Y, Nakanuma Y.: "Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells."Lab Invest. 83(11). 1657-1667 (2003)

Harada K、Ohira S、Isse K、Ozaki S、Zen Y、Sato Y、Nakanuma Y.：“脂多糖通过培养的胆管上皮细胞中的 Toll 样受体和相关分子激活核因子 kappaB。”Lab Invest。

一瀬久美子, 原田憲一, 中沼安二: "肝内胆管における上皮内リンパ球の構成 -原発性胆汁性肝硬変(PBC)を中心に-"消化器と免疫. 39. 106-108 (2002)

Kumiko Ichinose、Kenichi Harada、Yasuji Nakanuma：“肝内胆管中上皮内淋巴细胞的组成 - 关注原发性胆汁性肝硬化 (PBC) -”胃肠病学和免疫学。 39. 106-108 (2002)。