【作者】 何建波；

【导师】 罗凌飞；

【作者基本信息】 西南大学 ， 发育生物学， 2014， 博士

【摘要】 肝脏是人体最大的内脏器官,具有分泌胆汁、消化和代谢脂肪、合成和分泌氨基酸、合成和储存糖原、储存和过滤血液、解毒及排除废物、分泌血清白蛋白以维持体内环境的稳定等多种重要功能。肝脏发挥正常生理功能是保障人口健康的基本要素之一。对于恶性肝脏疾病如肝癌,迄今为止最有效的治疗和防止病情恶化的方式就是手术切除癌变部分肝脏,但是由此会导致患者的肝脏功能大大减弱,因此促进肝脏再生以使得受损肝脏恢复功能至关重要。与脊椎动物的其它器官相比,肝脏的再生能力较强,在正常肝脏中的肝细胞虽然是静默不分裂的,但是肝细胞却拥有强大的分裂增殖能力并可在肝损伤后迅速进入细胞分裂周期。肝脏经手术切除三分之二的大鼠或小鼠可在7-10天内恢复100%的肝脏重量。当肝脏细胞的增殖能力被抑制或者肝脏受到严重损伤时,一部分肝脏前体细胞会参与肝脏再生的修复,但是对这些前体细胞鉴定和分离至今尚有突破性进展。因此,建立更大程度的甚至肝脏完全损伤后再生的动物模型,对于鉴定肝脏再生过程中发挥关键作用的内源性肝脏前体细胞并深入揭示肝脏再生的分子调控机制具有重要意义。在肝脏再生过程中,成熟的胆管细胞是否会转分化为肝脏细胞到目前为止还没有报道,在本研究中我们主要以斑马鱼为动物模型探究肝脏严重受损后胆管细胞是否会转分化为肝脏细胞来参与肝脏再生过程。在本研究中,我们利用斑马鱼作为模式生物来研究肝脏再生的细胞来源和分子机制,首先我们构建了药物特异性诱导损伤肝脏细胞的转基因斑马鱼肝脏再生模型,通过大量的品系筛选和药物优化发现Mtz处理后可以使近100%的肝细胞诱导凋亡并在撤去药物后肝脏能够恢复正常再生。BODIPY FL C5和糖原显色PAS反应实验检测到新生的肝脏细胞具有脂肪代谢、胆酸分泌和糖原储存能力。因此我们构建的斑马鱼肝脏再生模型是一种功能性的再生模型。当肝脏细胞被药物特异性诱导损伤后,胆管变粗,胆管表皮细胞收缩同时有一小部分胆管细胞开始表达肝细胞的标志物,再生中后期,所有的胆管细胞都失去了正常的形态并表达肝脏特异性的分子标记,这表明胆管对肝脏再生起了重要作用。EdU标记实验发现新生肝脏区域出现大量的增殖现象并且这些增殖细胞主要集中在胆管中。而再生的开始阶段,胆管细胞的增殖能力非常低,同时TUNEL实验未能检测到胆管细胞在再生过程中发生凋亡,因此胆管细胞在肝脏受损的过程中保持了完整性,这表明胆管细胞在肝脏受损后可以转分化为肝脏细胞。为了进一步确定胆管细胞的向肝脏细胞的转分化现象,我们利用Cre-Loxp系统来追踪胆管来源的肝脏细胞,再生后期70%的新生肝脏细胞都来源于胆管细胞。这些现象表明肝脏受损再生过程中,胆管细胞形态发生变化同时增殖转分化为肝脏细胞并对肝脏再生起了主要的贡献作用。为了进一步确定胆管细胞在肝脏再生过程中的功能作用,我们在胚胎发育的早期抑制Notch信号来特异性的抑制胆管系统的发育,胆管系统发育受损的肝脏再生明显受到抑制。同时我们利用胆管突变体证明了胆管细胞对肝脏再生的重要性。另外,在斑马鱼中用胆管毒性药物处理早期胚胎发现即使对胆管微弱的毒副作用也会抑制胆管细胞向肝脏的转分化和肝脏的正常再生。肝脏再生初期胆管细胞开始表达一些内胚层和肝脏早期的特异性标记因子,这也说明胆管细胞转分化会伴随一个去分化为多功能细胞的过程。荧光原位杂交偶联抗体显色发现除了早期内胚层相关的转录因子外sox9b也会在胆管细胞中上调表达,再生初期胆管细胞并未发现增殖现象但是sox9b的表达已经上调。在sox9b突变体中,胆管细胞向肝脏细胞的转分化受到抑制同时肝脏再生减慢,这说明sox9b对胆管细胞的转分化具有重要作用。已有研究表明Sox9b是Notch信号途径的一个靶基因,当抑制了Notch后肝脏再生减慢同时sox9b的表达下调。在肝脏受损的过程中,Notch的靶基因hes5在胆管中上调,这说明Notch通过介导sox9b来参与了胆管细胞的转分化过程。综上所述,本研究成功构建了斑马鱼肝脏严重受损模型,鉴定了与肝脏受损相关的信号机制,并首次表明斑马鱼肝脏在儿乎100%受损后,乃然可进行再生；并揭示在受损肝脏的再生修复过程中,成熟胆管细胞的转分化起了重要作用；同时也证明胆管细胞的转分化依赖于Notch信号途径。本研究关于新生肝脏再生的来源和机制的研究对肝病的治疗能够提供一定的临床指导意义和参考价值。更多还原

【Abstract】 The liver is the largest human visceral organs, with bile secretion, digestion and metabolism of fatty, synthesis and secretion of amino acid, the synthesis and storage of glycogen, the storage and filtering blood, detoxification and elimination of waste, the secretion of serum albumin to maintain a stable internal environment and other important functions. Liver function is one of the basic elements of security of population health. For malignant liver diseases such as liver cancer, so far the most effective treatment and prevention the exacerbation of operation is to remove the cancerous portion of the liver, but it will greatly reduce the patient’s liver function, thus to promote liver regeneration in the damaged liver is important. Compared with other organs of vertebrates, liver has greatly ability of regeneration, in normal liver cells is not split the silent, but liver cells have strong proliferation ability and can quickly enter the cell division cycle after liver injury. Liver by operation was performed in2/3rats or mice can recover100%of liver weight in7-10days. When the proliferation of hepatocyte is inhibited or liver damage, a portion of the liver precursor cells may participate in the repair of liver regeneration, but the identification and isolation of precursor cell still has difficult up to know. Therefore, to establish a greater degree of even fully liver injury animal model is of great significance. Identification of endogenous precursor cells and regulatory mechanism in liver regeneration process is very mportant. The liver has high regenerative capacity, but it is not clear whether most biliary cells (particularly larger cholangiocytes) transdifferentiate into hepatocytes in regenerating liver. We investigated how this process might contribute to liver regeneration in zebrafish.In this study, we use zebrafish as a model organism to explore the cell sources and mechanisms in liver regeneration, first we construct a transgenic zebrafish whose liver can be killed specificity induced by Mtz. We found that the liver can be induced to cell death close to100%and return to normal regeneration after withdraw the Mtz. By BOD1PY FL C5and glycogen color PAS reaction assays, we find the new regenerated liver cells have the ability to fat metabolization, bile acid secretion and glycogen storage. Therefore zebrafish liver injury model we established is a functional regeneration model.When the liver cells were injuried specificly, bile duct coarsens, contraction and a small part of the bile duct cells begain to express hepatic cells markers in regeneration, later, all of the bile duct cells lose their normal morphology and express the specific markers of hepatocyte, suggesting that the bile duct played an important role in liver regeneration. EdU labeling experiments found that a large number of proliferation cells are mainly concentrated in the bile duct cells. And at the beginning of regeneration, the proliferation of bile duct is very low, while the TUNEL experiments failed to detect the apoptosis of bile duct cells in the regeneration, so bile duct cells maintain integrity in the process of liver regeneration. In order to further determine whether the bile duct cell transdifferentiate into hepatocyte, we use Cre-Loxp systems to track the destiny of bile duct,70%new hepatocyte regeneration are derived from the bile duct cells. These phenomena indicate after liver damage during regeneration, the bile duct cells changed their morphology, transdifferentiate into liver cells and play a major contribution on liver regeneration.In order to further define the function of bile duct cells during liver regeneration, inhibiting Notch signal to specifically reduce the development of bile duct system in the early embryonic development, liver regeneration is impaired after the development of bile duct system was significantly inhibited. At the same time, we use a bile duct mutant to further prove the roles of biliary cells on liver regeneration. In addition, in zebrafish embryos with biliary toxicity by drug treatment found that even weak toxicity also inhibits bile duct cells to transdifferentiation. At the beginning of regeneration, bile duct cells began to express some early endoderm and liver specific markers, which also show that the bile duct cells will be accompanied by a process of dedifferentiation to an intermediate of precursor cells.At the same time, we used fluorescence in situ hybridization coupled antibody staining found early endodermal transcription factors are up regulated in bile duct cells, sox9b also upregulated in bile duct cells in liver regeneration. There is no proliferation in bile duct but the expression of sox9b has been raised after liver damaged. In sox9b mutants, we found that the bile duct cells to the hepatocyte transdifferentiation is restrained and liver regeneration slowed down, indicating that sox9b on bile duct cell transdifferentiation is important. Sox9b is one of the target gene of Notch signal pathway, when the Notch signaling suppressed, the transcription of sox9b is also suppressed. At the same time, we can detect the up regulation of another Notch target gene Hes5in the biliary system during the Mtz treatment, indicating that Notch mediated liver regeneration through the sox9b to participate in the transdifferentiation of bile duct cell.In summary, we have successfully constructed the liver injury model, identied the signal mechanisms associated with liver damage and firstly show that almost100%of zebrafish liver injured, it can be recovered to normal regeneration; we also revealed in the process of regeneration and repair of damaged liver, the transdifferentiation of mature biliary duct cells play a very important role; at the same time we validate that the transdifferentiation of bile duct cells is dependent on Notch signaling pathway. Study on the sources and mechanisms of liver regeneration can provide some clinical significance for liver disease.更多还原