Notch信号通路调控肝再生的细胞生物学基础及其分子机制的研究

发布时间：2018-04-15 01:28

本文选题：Notch + RBP-J　；参考：《第四军医大学》2010年博士论文

【摘要】： 肝脏作为人体最大的实质性脏器,在维持体内新陈代谢中处于核心地位。肝组织的破坏,如慢性炎症、纤维变等,往往造成全身性的、甚至是致命的代谢紊乱。但是,肝脏具有很强的再生能力,这可在一定程度上代偿肝实质的破坏。然而致病因素的长期存在(如慢性肝炎),往往导致肝再生的异常,最终引起肝实质的结构异常和功能丧失,危及患者生命。因此,掌握肝脏再生的调控机制,不但具有重要的理论意义,而且具有紧迫的临床需求。肝脏损伤或肝部分切除后,剩余肝脏会代偿性再生,肝脏再生的实质是肝细胞的不断增殖、扩增。肝细胞增殖主要依靠肝细胞生长因子(Hepatocyte growth factor,HGF)和白细胞介素6(interleukin-6,IL-6)的作用,而这两种细胞因子主要由一类肝脏非实质细胞——肝血窦内皮细胞(Liver sinusoidal endothelial cell,LSEC)分泌。肝再生的过程伴有大量LSEC的增殖和更新。因此,LSEC对于维系肝细胞的功能状态进而影响肝脏再生发挥着重要的作用。此外,LSEC所构成的肝血窦又是肝脏微循环的基础结构,它的正常结构对于维系肝脏功能至关重要。本课题我们将着重通过研究LSEC以及肝血窦的结构和功能变化,来阐明其对于肝再生的基本作用和其中包含的分子调控机制。此外,既往研究已证实骨髓来源的干、祖细胞也参与到肝再生过程中。截至目前,尚无证据表明干、祖细胞可以直接分化为肝细胞进而促进肝再生。然而骨髓来源的内皮祖细胞(Endothelial progenitor cell,EPC)可以分化为成熟内皮细胞,这在多个组织、器官的损伤和修复过程中已被广为证实。而肝再生过程中,骨髓来源的干、祖细胞也被证实参与LSEC的更新,它们可能直接分化为成熟的LSEC或通过旁分泌等途径促进LSEC乃至肝细胞的增殖。由此看来,肝再生的进程可能是一个由肝细胞、LSEC等内源性因素和由骨髓来源EPC等外源性因素共同作用的结果。深入阐明肝再生的机制,我们需要从这两个方面着手研究。 Notch信号途径是人体最重要的信号转导通路之一,其分子广泛表达于胚胎和成年个体组织中,主要由表达于相邻细胞上的Notch配体和Notch受体、以及表达于细胞内的转录因子、下游分子和其他调节分子组成。RBP-J是激活Notch信号通路所必需的一个核心转录因子,它的缺失意味着Notch信号的完全阻断。Notch信号途径对细胞的分化、增殖、凋亡有重要的调控作用,它广泛参与组织器官的发育、生长以及再生进程,其功能的异常与很多疾病及肿瘤发生有关。我们前期研究发现,肝再生的进程伴有Notch信号通路分子的广泛变化。Notch信号途径对于维持肝脏的稳态具有重要作用。本课题我们首先利用小鼠肝部分切除模型模拟肝脏再生进程,并利用RBP-J条件性基因剔除模型,研究了RBP-J介导的Notch信号途径在肝再生过程中的具体调控作用:主要是Notch信号通路如何通过调节LSEC和肝血窦的结构和功能来调控肝细胞的功能状态,进而影响到肝再生进程;以及Notch信号通路如何通过调节EPC的功能,从而影响LSEC、肝细胞的功能状态,最终导致肝再生进程的改变;此外,我们还深入探讨了这其中所包含的具体分子机制,特别是Notch信号通过VEGF受体分子对LSEC的调控,以及Notch通过CXCR4对EPC的调控。我们的主要研究成果如下: 1、成功构建了RBP-J条件性基因剔除的小鼠,即Notch/RBP-J信号缺失的小鼠模型; 2、我们发现Notch信号缺失会导致肝脏稳态的丧失,具体表现为肝脏淤血样改变,肝细胞、LSEC的异常增殖,正常肝小叶结构遭到破坏; 3、我们发现阻断Notch信号会导致肝再生功能的障碍,具体表现为肝细胞、LSEC增殖能力减弱,肝脏失代偿性增大,LSEC去分化,肝血窦阻塞,肝细胞合成、分泌白蛋白能力减弱; 4、通过体外实验我们发现,Notch信号直接影响肝细胞、LSEC的细胞生物学活性,Notch信号缺失,会导致肝细胞、LSEC的异常增殖,还会造成LSEC分泌VEGF、HGF、IL-6的功能障碍; 5、Notch信号会影响到肝再生进程中,骨髓来源EPC向外周的动员和向肝脏的定向募集,阻断Notch信号会增加EPC向外周血的动员,但动员出的EPC更难于募集到再生的肝脏; 6、Notch信号缺失的骨髓细胞无法正常参与到肝脏再生并促进肝细胞、LSEC的增殖; 7、Notch信号对于维持体外培养的骨髓EPC的细胞活性至关重要,阻断该通路会导致EPC增殖、粘附、集落形成、迁移、成管腔能力的减弱; 8、Notch信号对骨髓来源EPC的调控是通过CXCR4通路而实现的,外源性过表达CXCR4可以挽救Notch缺失后EPC丧失的成管腔能力。综上所述,肝再生进程是一个多细胞共同参与并相互作用的过程。Notch信号通路在此过程中扮演着重要角色,它可以直接调控肝细胞、LSEC的生物学活性,也可以通过影响骨髓EPC的功能,而改变肝再生的进程。同时Notch信号途径对于维持肝脏稳态也至关重要。这些都为我们研究有关肝脏疾病的预防和治疗奠定了重要的理论依据。
[Abstract]:Liver is the largest organ, is at the core position in maintaining body. The new supersedes the old. liver damage, such as chronic inflammation, fibers, often resulting in systemic metabolic disorders, and even fatal. However, the liver has a very strong ability of regeneration, which can in some degree the previous compensation of liver parenchyma there is a long-term damage. However, pathogenic factors (such as chronic hepatitis), often leads to abnormal liver regeneration, resulting in abnormal structure of liver parenchyma and loss of function, endangering the lives of patients. Therefore, the control mechanism of liver regeneration, not only has important theoretical significance, but also has urgent clinical needs.
Liver injury or liver after partial hepatectomy, the remaining liver may be compensatory regeneration, the essence of liver regeneration is proliferating, liver cell amplification. Liver cell proliferation depends mainly on hepatocyte growth factor (Hepatocyte growth, factor, HGF) and interleukin 6 (interleukin-6, IL-6) and the role of the two kinds of cytokines is mainly composed of a class of liver nonparenchymal cells, liver sinusoidal endothelial cells (Liver sinusoidal endothelial cell, LSEC). The proliferation and secretion of liver regeneration process with a large number of updates of LSEC. Therefore, LSEC function for state maintaining liver cells affect liver regeneration plays an important role. In addition, a LSEC of the liver the blood sinus is the basis of the structure of the liver microcirculation, it is essential to maintain the normal structure of liver function. In this paper we will focus on the study of LSEC and the structure and function of hepatic sinusoid changes, to clarify the The basic function of liver regeneration and the molecular mechanism involved in it.
In addition, previous studies have shown that bone marrow derived stem and progenitor cells are also involved in the process of liver regeneration. Up to now, there is no evidence that stem progenitor cells can differentiate into hepatocyte and promote liver regeneration. However, bone marrow derived endothelial progenitor cells (Endothelial progenitor cell, EPC) can differentiate into mature endothelial cells, the in many organizations, the process of injury and repair of organs has been widely confirmed. And the process of liver regeneration, bone marrow derived stem and progenitor cells have been shown to participate in the LSEC update, they may directly differentiate into mature LSEC or through paracrine way to promote LSEC and liver cell proliferation. It seems that the process of liver regeneration may be a liver cell, LSEC and other endogenous factors and exogenous bone marrow derived by EPC factors results. To elucidate the mechanism of liver regeneration, we need from these two aspects Hand study.
Notch signal pathway is one of the most important signal transduction pathways in the human body, the molecules are widely expressed in embryonic and adult tissues, mainly expressed in adjacent cells by Notch ligands and Notch receptors, and the expression of intracellular transcription factors, downstream molecules and other regulatory molecules.RBP-J is a core transcription factor activation must the Notch signaling pathway, its absence means completely blocked the differentiation of.Notch signaling pathway on cell proliferation of Notch signal, apoptosis plays an important role, it is widely involved in tissue organ development, growth and regeneration process, and many abnormal diseases and tumors. Its function in our previous study we found that the changes of.Notch signal pathway is widely associated with the process of liver regeneration, Notch signaling pathway plays an important role in the homeostasis of the liver.
In this paper we first use the mouse partial hepatectomy model to simulate the process of liver regeneration, and the use of RBP-J conditional gene knockout model specific regulation of Notch mediated RBP-J signaling during liver regeneration: the main function of Notch signaling pathway to regulate liver cells through regulating the structure and function of LSEC and hepatic sinusoid the influence of liver regeneration process; and how Notch signaling by regulating the function of EPC, thus affecting the LSEC function of liver cells, resulting in changes in the process of liver regeneration; in addition, we also discussed the molecular mechanism which contains, in particular Notch signal through VEGF receptor molecules on the regulation of LSEC, Notch and CXCR4 through the regulation of EPC.
Our main research results are as follows:
1, the RBP-J conditional gene culling mice were successfully constructed, that is, the Notch/RBP-J signal deficient mouse model.
2, we found that the loss of Notch signal can lead to the loss of liver homeostasis, which is characterized by hepatic congestion, abnormal proliferation of LSEC and hepatocytes, and destruction of normal hepatic lobule.
3, we found that blocking Notch signaling can lead to dysfunction of liver regeneration, which is manifested as hepatocyte, LSEC proliferation, liver decompensation, LSEC dedifferentiation, hepatic sinusoid obstruction, hepatocyte synthesis and albumin secretion.
4, through in vitro experiments, we found that Notch signal directly affects the biological activity of hepatocytes, LSEC, and Notch signal loss, which can lead to abnormal proliferation of hepatocytes and LSEC, and also cause dysfunction of VEGF, HGF and IL-6 secreted by LSEC.
5, Notch signal will affect the mobilization of bone marrow EPC and directional recruitment to the liver during the process of liver regeneration. Blocking Notch signal will increase the mobilization of EPC to peripheral blood, but the mobilized EPC is harder to collect the regenerated liver.
6, the bone marrow cells with missing Notch signal can not participate in the normal liver regeneration and promote the proliferation of hepatocytes and LSEC.
7, Notch signal is essential for maintaining the activity of EPC in vitro. Blocking the pathway will lead to EPC proliferation, adhesion, colony forming, migration and lumen weakening.
8, the regulation of Notch signal on bone marrow derived EPC is achieved through CXCR4 pathway. Exogenous over expression of CXCR4 can save the lumen capacity of EPC lost after Notch deletion.
In summary, the process of liver regeneration is a multicellular participate and play an important role in the process of.Notch signal pathway in the process of interaction, it can directly regulate the liver cells, the biological activity of LSEC, can affect the bone marrow function of EPC, and the change of liver regeneration process. At the same time, the Notch signaling pathway to maintain liver homeostasis also these are crucial. For us to study the prevention and treatment of liver disease has laid an important theoretical basis.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

【共引文献】