Investigation of liver reconstruction mechanism by bone marrow transplantation

骨髓移植肝脏重建机制的探讨

• 批准号: 13307037
• 负责人: TAKAYAMA Tadatoshi
• 金额: $ 30.7万
• 依托单位: Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13307037/
• 关键词: Bone marrow cell; Transdifferentiation; Liver failure; Cytokine; Methylation; Partial hepatectomy; Hematopoietic progenitor cell; 分化誘導; Acetoaminofluorene; ケモカイン; Gene Chip; 脱メチル化; Cell fusion; 骨髄細胞; 肝細胞; 間葉系幹細胞; 共培養; オンコスタチンM; HGF; Aza

Background/Aim : The plasticity of bone marrow cells (BMCs) is shown by their ability to differentiate into mesenchymal as well as endodermal and ectodermal lineages. Transdifferentiation of BMCs into hepatocytes has also been demonstrated, both in vitro and in vivo. At first, we investigated the effects of liver nonparenchymal cells (NPCs) and sera from liver failure patients (HSLF) on the in vitro transdifferentiation of murine BMCs into hepatocytes. Next, we investigated the mechanisms underlying transdifferentiation of BMCs into hepatocytes processes. We have focused on the initial events occurring in bone marrow in response to liver injury.Methods : Liver NPCs from wild type mice, and 5-azacytidine-treated BMCs from green fluorescence protein transgenic mice, were cocultured in medium containing HSLF in combination with several cytokines. Hepatocyte-specific gene expression in BMCs was identified by immunocytochemistry and RT-PCR. Next, mice were given 2-acetyl aminofluorene (AAF) … More intraperitonealy for one, week, followed by two-thirds partial hepatectomy. Other mice underwent hepatectomy without AAF administration. Hepatic and endodermal differentiation of bone marrow cells was evaluated by measuring hepatocyte-related gene expression by real time RT-PCR before and after hepatectomy.Results : Bone marrow cell-derived hepatocyte-like colonies appeared after several days of coculture in medium containing HSLF, oncostatin M (OSM) and hepatocyte growth factor (HGF). These colonies expressed hepatocyte-specific genes. Transdifferentiation was enhanced by 5-azacytidine treatment, and by HSLF, OSM and HGF. It did not take place when the BMCs were separated, from the NPCs in a dual chamber dish, or cultured with other mesenchymal cells. Partial hepatectomy induced expression of several early hepatic genes such as alpha-fetoprotein (AFP) and hepatocyte nuclear factor 3. Expression of these genes was enhanced by the administration of AAF. We identify the sub-population of bone marrow cells responsible for transdifferentiation in the Lin, CD34^+, c-kit^+, Sca-1^+, CD49f^+ and CD45^+ fractions, corresponding to hematopoietic progenitors.Conclusions : Direct interaction of murine BMCs with liver NPCs, as well as soluble factors in the HSLF and a demethylating agent, strongly stimulate transdifferentiation into hepatocytes. Early endodermal and hepatic differentiation occurs in bone marrow in response to hepatectomy, especially when regeneration of the remnant liver is suppressed. Circulating signals generated by severe liver injury may stimulate this differentiation. Less

背景/目的：骨髓细胞 (BMC) 的可塑性通过其分化为间充质以及内胚层和外胚层谱系的能力来体现。 BMC 向肝细胞的转分化也已在体外和体内得到证实。首先，我们研究了肝非实质细胞（NPC）和肝衰竭患者（HSLF）血清对小鼠 BMC 体外转分化为肝细胞的影响。接下来，我们研究了 BMC 转分化为肝细胞过程的机制。我们重点关注骨髓中响应肝损伤而发生的初始事件。方法：将来自野生型小鼠的肝脏 NPC 和来自绿色荧光蛋白转基因小鼠的经 5-氮杂胞苷处理的 BMC 共培养在含有 HSLF 和多种细胞因子的培养基中。通过免疫细胞化学和 RT-PCR 鉴定 BMC 中肝细胞特异性基因的表达。接下来，小鼠腹膜内注射 2-乙酰氨基芴 (AAF) 一星期，然后进行三分之二的部分肝切除术。其他小鼠在未施用 AAF 的情况下接受了肝切除术。通过实时 RT-PCR 测量肝切除前后肝细胞相关基因的表达来评估骨髓细胞的肝和内胚层分化。结果：在含有 HSLF、制瘤素 M (OSM) 和肝细胞生长因子 (HGF) 的培养基中共培养数天后，出现骨髓细胞来源的肝细胞样集落。这些集落表达肝细胞特异性基因。 5-氮杂胞苷处理以及HSLF、OSM和HGF可增强转分化。当 BMC 与双室培养皿中的 NPC 分离或与其他间充质细胞一起培养时，这种情况不会发生。部分肝切除诱导了一些早期肝脏基因的表达，例如甲胎蛋白（AFP）和肝细胞核因子3。这些基因的表达通过施用AAF而增强。我们确定了 Lin、CD34^+、c-kit^+、Sca-1^+、CD49f^+ 和 CD45^+ 部分中负责转分化的骨髓细胞亚群，对应于造血祖细胞。结论：小鼠 BMC 与肝脏 NPC 以及 HSLF 中的可溶性因子和去甲基化剂的直接相互作用， 强烈刺激肝细胞转分化。响应肝切除术，骨髓中发生早期内胚层和肝分化，特别是当残余肝脏的再生受到抑制时。严重肝损伤产生的循环信号可能会刺激这种分化。较少的

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