Investigation of liver reconstruction mechanism by bone marrow transplantation

背景/目的：骨髓细胞的可塑性表现为其向间充质、内胚层和外胚层分化的能力。在体外和体内，BMCs向肝细胞的转分化也得到了证实。首先，我们研究了肝非实质细胞(NPC)和肝衰竭患者血清(HSLF)对小鼠骨髓细胞体外转分化为肝细胞的影响。接下来，我们研究了骨髓细胞转分化为肝细胞过程的机制。方法：将野生型小鼠肝神经干细胞与绿色荧光蛋白转基因小鼠经5-氮胞苷处理的小鼠骨髓细胞在含有多种细胞因子的HSLF培养液中进行共培养。免疫细胞化学和RT-PCR检测BMCs肝细胞特异性基因的表达。接下来，给小鼠注射2-乙酰氨基荧烯(AAF)…更多的腹膜腔内切除，一周，然后三分之二的肝部分切除。其他小鼠在没有注射AAF的情况下接受了肝切除。结果：在含有HSLF、OSM和肝细胞生长因子(HGF)的培养液中共培养数天后，出现了骨髓源性肝细胞样集落。这些克隆表达肝细胞特异性基因。5-氮胞苷、HSLF、OSM和HGF可促进转分化。当骨髓间充质细胞在双室培养皿中从NC中分离出来，或与其他间充质细胞培养时，不会发生这种情况。肝部分切除可诱导甲胎蛋白(AFP)和肝细胞核因子3等早期肝脏基因的表达，应用AAF可增强这些基因的表达。我们在LIN、CD34^、c-kit^、SCA-1^、CD49f^和CD45^组分中鉴定了负责转分化的骨髓细胞亚群，与造血祖细胞相对应。结论：小鼠骨髓细胞与肝脏NPC的直接相互作用，以及HSLF中的可溶性因子和去甲基化试剂，强烈刺激向肝细胞的转分化。早期的内胚层分化和肝脏分化发生在骨髓中，作为对肝切除的反应，特别是当残肝的再生受到抑制时。严重肝损伤产生的循环信号可能刺激这种分化。较少

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