Investigation of liver reconstruction mechanism by bone marrow transplantation

骨髓移植肝脏重建机制的探讨

基本信息

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

项目摘要

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质以及内胚层和外胚层谱系的能力来体现，BMC 转分化为肝细胞的能力也已在体外和体内得到证实。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的初始事件。方法：来自野生型小鼠的肝脏 NPC 和来自 5-氮杂胞苷处理的 BMC。将绿色荧光蛋白转基因小鼠在含有 HSLF 的培养基中与多种细胞因子共培养，通过免疫细胞化学鉴定 BMC 中的肝细胞特异性基因表达。 RT-PCR. 接下来，小鼠腹腔注射 2-乙酰氨基芴 (AAF) 一周，然后对其他小鼠进行三分之二的部分肝切除术，并评估了骨髓细胞的肝和内胚层分化。通过实时RT-PCR测量肝切除术前后肝细胞相关基因的表达。结果：共培养几天后出现骨髓细胞来源的肝细胞样集落在含有 HSLF、制瘤素 M (OSM) 和肝细胞生长因子 (HGF) 的培养基中，这些集落表达的肝细胞特异性基因通过 5-氮杂胞苷处理而增强，而在 HSLF、OSM 和 HGF 处理下则不发生。在双室培养皿中将 BMC 与 NPC 分离，或与其他间充质细胞一起培养，部分肝切除诱导多种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 得到增强。我们鉴定了负责 Lin、CD34^+、c-kit^+ 中转分化的骨髓细胞亚群。、Sca-1^+、CD49f^+ 和 CD45^+ 分数，对应于造血祖细胞。结论：小鼠 BMC 与肝脏 NPC 的直接相互作用，如HSLF 中的可溶性因子和去甲基化剂，强烈刺激骨髓中发生早期内胚层和肝分化，以响应肝切除术，特别是当严重肝损伤产生的残余肝脏再生受到抑制时。较少刺激这种分化。