胆管转分化对斑马鱼肝脏严重受损后的修复作用

发布时间：2018-05-04 03:17

  本文选题：肝脏损伤 + 动物模型　； 参考：《西南大学》2014年博士论文

【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。
[Abstract]:The liver is the largest internal organ of the human body. It has many important functions, such as the secretion of bile, the digestion and metabolism of fat, the synthesis and secretion of amino acids, the synthesis and storage of glycogen, the storage and filtration of blood, the detoxification and elimination of waste, the secretion of serum albumin to maintain the stability of the body, and the normal physiological function of the liver as the basis for ensuring the health of the population. One of these factors. For malignant liver diseases such as liver cancer, the most effective treatment so far to prevent the deterioration of the disease is surgical removal of the cancerous part of the liver, but it will lead to a significant decrease in the liver function of the patient, thus promoting the regeneration of the liver to make the damaged liver function critical. The liver has a stronger ability to regenerate, while the liver cells in the normal liver are silent, but the liver cells have a strong ability to split and proliferate and can quickly enter the cell division cycle after liver injury. The liver's 2/3 rats or rats can recover 100% of the liver weight within 7-10 days after the liver resection. A part of the liver precursor cells will participate in the repair of liver regeneration when the proliferation ability of the visceral cells is suppressed or the liver is seriously damaged, but there is a breakthrough in the identification and separation of these precursors. Therefore, the animal model of a greater degree of regeneration after complete liver injury has been established for the identification of liver regeneration. In the course of liver regeneration, it is not reported that mature cholangiocytes turn into liver cells in the course of liver regeneration. In this study, we mainly use zebrafish as animal models to explore the liver. After severe injury, can bile duct cells differentiate into liver cells to participate in the process of liver regeneration.
In this study, we used zebrafish as a model organism to study the cell origin and molecular mechanism of liver regeneration. First, we constructed a transgenic zebrafish liver regeneration model with drug specific induced damage to liver cells. Through a large number of strain screening and drug optimization, we found that nearly 100% of the liver cells could be induced by Mtz treatment. The liver regeneration model of zebrafish is a functional regeneration model. The liver regeneration model of zebrafish is a functional regeneration model. The liver cells with.BODIPY FL C5 and glycogen chromogenic PAS reaction test showed that the newborn liver cells have fat metabolism, cholic acid secretion and glycogen storage ability.
When the liver cells are induced by the drug specific injury, the bile duct becomes thicker, the bile duct epidermal cells shrink and a small portion of the bile duct cells begin to express the markers of the liver cells. In the middle and late stages of the regeneration, all the bile duct cells lose the normal form and express the specific markers of the liver, which indicates that the bile duct plays an important role in the regeneration of the liver. A large number of proliferation phenomena in the newborn liver region were found and the proliferation cells were mainly concentrated in the bile duct. At the beginning of the regeneration, the proliferation ability of the bile duct cells was very low, and the TUNEL test failed to detect the apoptosis of the bile duct cells during the regeneration process, so the bile duct cells were damaged in the liver. In order to further determine the differentiation of bile duct cells to liver cells, we use the Cre-Loxp system to track the liver cells derived from the bile duct, and 70% of the newly born liver cells from the later stage of regeneration are derived from the bile duct cells. The image shows that during the process of liver regeneration, the morphology of bile duct cells is changed, and the proliferation and differentiation of liver cells become the main role of liver cells.
In order to further determine the function of bile duct cells in the process of liver regeneration, we inhibit the development of the bile duct system specifically at the early stage of embryonic development and inhibit the development of the bile duct system, and the liver regeneration, which is damaged by the biliary system development, is obviously suppressed. At the same time, we use the bile duct mutants to prove the importance of bile duct cells to the regeneration of the liver. In addition, the treatment of early embryos in zebrafish with bile duct toxic drugs found that even the weak toxic and side effects of the bile duct could inhibit the differentiation of the bile duct cells to the liver and the normal regeneration of the liver. In the early stage of the liver regeneration, bile duct cells began to express some specific markers of early endoderm and liver, which also indicated that bile duct cells were also indicated. Transdifferentiation is accompanied by a process of dedifferentiation into multifunctional cells.
The fluorescent in situ hybridization showed that the expression of sox9b in the bile duct cells was also up-regulated in addition to the early endoderm related transcription factors. The proliferation of bile duct cells was not found at the early stage of regeneration, but the expression of sox9b was up to up. In the sox9b mutant, the transdifferentiation of the bile duct cells to the liver cells was inhibited and the liver was reformed. Slow down, which indicates that sox9b plays an important role in the differentiation of bile duct cells. Sox9b is a target gene for the Notch signal pathway, and the expression of sox9b is down regulated when the Notch is slowed down after the inhibition of Notch. In the course of liver damage, the target gene Hes5 is up-regulated in the bile duct, indicating that Notch is mediated by sox9b. It participates in the process of differentiation of bile duct cells.
To sum up, this study successfully constructed a serious damage model of zebrafish liver, identified the signal mechanism associated with liver damage, and first showed that the liver of zebrafish could be regenerated after 100% damage, and revealed that the differentiation of mature bile duct cells played an important role in the process of regeneration and repair of damaged liver. It is also proved that the differentiation of bile duct cells is dependent on the Notch signal pathway. The research on the origin and mechanism of the regeneration of the newborn liver can provide some clinical guidance and reference value for the treatment of liver disease.

【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575
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本文编号：1841345