本文選題：Notch + RBP-J�。� 參考：《第四軍醫(yī)大學(xué)》2010年博士論文

【摘要】： 肝臟作為人體最大的實(shí)質(zhì)性臟器,在維持體內(nèi)新陳代謝中處于核心地位。肝組織的破壞,如慢性炎癥、纖維變等,往往造成全身性的、甚至是致命的代謝紊亂。但是,肝臟具有很強(qiáng)的再生能力,這可在一定程度上代償肝實(shí)質(zhì)的破壞。然而致病因素的長(zhǎng)期存在(如慢性肝炎),往往導(dǎo)致肝再生的異常,最終引起肝實(shí)質(zhì)的結(jié)構(gòu)異常和功能喪失,危及患者生命。因此,掌握肝臟再生的調(diào)控機(jī)制,不但具有重要的理論意義,而且具有緊迫的臨床需求。 肝臟損傷或肝部分切除后,剩余肝臟會(huì)代償性再生,肝臟再生的實(shí)質(zhì)是肝細(xì)胞的不斷增殖、擴(kuò)增。肝細(xì)胞增殖主要依靠肝細(xì)胞生長(zhǎng)因子(Hepatocyte growth factor,HGF)和白細(xì)胞介素6(interleukin-6,IL-6)的作用,而這兩種細(xì)胞因子主要由一類肝臟非實(shí)質(zhì)細(xì)胞——肝血竇內(nèi)皮細(xì)胞(Liver sinusoidal endothelial cell,LSEC)分泌。肝再生的過(guò)程伴有大量LSEC的增殖和更新。因此,LSEC對(duì)于維系肝細(xì)胞的功能狀態(tài)進(jìn)而影響肝臟再生發(fā)揮著重要的作用。此外,LSEC所構(gòu)成的肝血竇又是肝臟微循環(huán)的基礎(chǔ)結(jié)構(gòu),它的正常結(jié)構(gòu)對(duì)于維系肝臟功能至關(guān)重要。本課題我們將著重通過(guò)研究LSEC以及肝血竇的結(jié)構(gòu)和功能變化,來(lái)闡明其對(duì)于肝再生的基本作用和其中包含的分子調(diào)控機(jī)制。 此外,既往研究已證實(shí)骨髓來(lái)源的干、祖細(xì)胞也參與到肝再生過(guò)程中。截至目前,尚無(wú)證據(jù)表明干、祖細(xì)胞可以直接分化為肝細(xì)胞進(jìn)而促進(jìn)肝再生。然而骨髓來(lái)源的內(nèi)皮祖細(xì)胞(Endothelial progenitor cell,EPC)可以分化為成熟內(nèi)皮細(xì)胞,這在多個(gè)組織、器官的損傷和修復(fù)過(guò)程中已被廣為證實(shí)。而肝再生過(guò)程中,骨髓來(lái)源的干、祖細(xì)胞也被證實(shí)參與LSEC的更新,它們可能直接分化為成熟的LSEC或通過(guò)旁分泌等途徑促進(jìn)LSEC乃至肝細(xì)胞的增殖。由此看來(lái),肝再生的進(jìn)程可能是一個(gè)由肝細(xì)胞、LSEC等內(nèi)源性因素和由骨髓來(lái)源EPC等外源性因素共同作用的結(jié)果。深入闡明肝再生的機(jī)制,我們需要從這兩個(gè)方面著手研究。 Notch信號(hào)途徑是人體最重要的信號(hào)轉(zhuǎn)導(dǎo)通路之一,其分子廣泛表達(dá)于胚胎和成年個(gè)體組織中,主要由表達(dá)于相鄰細(xì)胞上的Notch配體和Notch受體、以及表達(dá)于細(xì)胞內(nèi)的轉(zhuǎn)錄因子、下游分子和其他調(diào)節(jié)分子組成。RBP-J是激活Notch信號(hào)通路所必需的一個(gè)核心轉(zhuǎn)錄因子,它的缺失意味著Notch信號(hào)的完全阻斷。Notch信號(hào)途徑對(duì)細(xì)胞的分化、增殖、凋亡有重要的調(diào)控作用,它廣泛參與組織器官的發(fā)育、生長(zhǎng)以及再生進(jìn)程,其功能的異常與很多疾病及腫瘤發(fā)生有關(guān)。我們前期研究發(fā)現(xiàn),肝再生的進(jìn)程伴有Notch信號(hào)通路分子的廣泛變化。Notch信號(hào)途徑對(duì)于維持肝臟的穩(wěn)態(tài)具有重要作用。 本課題我們首先利用小鼠肝部分切除模型模擬肝臟再生進(jìn)程,并利用RBP-J條件性基因剔除模型,研究了RBP-J介導(dǎo)的Notch信號(hào)途徑在肝再生過(guò)程中的具體調(diào)控作用:主要是Notch信號(hào)通路如何通過(guò)調(diào)節(jié)LSEC和肝血竇的結(jié)構(gòu)和功能來(lái)調(diào)控肝細(xì)胞的功能狀態(tài),進(jìn)而影響到肝再生進(jìn)程;以及Notch信號(hào)通路如何通過(guò)調(diào)節(jié)EPC的功能,從而影響LSEC、肝細(xì)胞的功能狀態(tài),最終導(dǎo)致肝再生進(jìn)程的改變;此外,我們還深入探討了這其中所包含的具體分子機(jī)制,特別是Notch信號(hào)通過(guò)VEGF受體分子對(duì)LSEC的調(diào)控,以及Notch通過(guò)CXCR4對(duì)EPC的調(diào)控。 我們的主要研究成果如下: 1、成功構(gòu)建了RBP-J條件性基因剔除的小鼠,即Notch/RBP-J信號(hào)缺失的小鼠模型; 2、我們發(fā)現(xiàn)Notch信號(hào)缺失會(huì)導(dǎo)致肝臟穩(wěn)態(tài)的喪失,具體表現(xiàn)為肝臟淤血樣改變,肝細(xì)胞、LSEC的異常增殖,正常肝小葉結(jié)構(gòu)遭到破壞; 3、我們發(fā)現(xiàn)阻斷Notch信號(hào)會(huì)導(dǎo)致肝再生功能的障礙,具體表現(xiàn)為肝細(xì)胞、LSEC增殖能力減弱,肝臟失代償性增大,LSEC去分化,肝血竇阻塞,肝細(xì)胞合成、分泌白蛋白能力減弱; 4、通過(guò)體外實(shí)驗(yàn)我們發(fā)現(xiàn),Notch信號(hào)直接影響肝細(xì)胞、LSEC的細(xì)胞生物學(xué)活性,Notch信號(hào)缺失,會(huì)導(dǎo)致肝細(xì)胞、LSEC的異常增殖,還會(huì)造成LSEC分泌VEGF、HGF、IL-6的功能障礙; 5、Notch信號(hào)會(huì)影響到肝再生進(jìn)程中,骨髓來(lái)源EPC向外周的動(dòng)員和向肝臟的定向募集,阻斷Notch信號(hào)會(huì)增加EPC向外周血的動(dòng)員,但動(dòng)員出的EPC更難于募集到再生的肝臟; 6、Notch信號(hào)缺失的骨髓細(xì)胞無(wú)法正常參與到肝臟再生并促進(jìn)肝細(xì)胞、LSEC的增殖; 7、Notch信號(hào)對(duì)于維持體外培養(yǎng)的骨髓EPC的細(xì)胞活性至關(guān)重要,阻斷該通路會(huì)導(dǎo)致EPC增殖、粘附、集落形成、遷移、成管腔能力的減弱; 8、Notch信號(hào)對(duì)骨髓來(lái)源EPC的調(diào)控是通過(guò)CXCR4通路而實(shí)現(xiàn)的,外源性過(guò)表達(dá)CXCR4可以挽救Notch缺失后EPC喪失的成管腔能力。 綜上所述,肝再生進(jìn)程是一個(gè)多細(xì)胞共同參與并相互作用的過(guò)程。Notch信號(hào)通路在此過(guò)程中扮演著重要角色,它可以直接調(diào)控肝細(xì)胞、LSEC的生物學(xué)活性,也可以通過(guò)影響骨髓EPC的功能,而改變肝再生的進(jìn)程。同時(shí)Notch信號(hào)途徑對(duì)于維持肝臟穩(wěn)態(tài)也至關(guān)重要。這些都為我們研究有關(guān)肝臟疾病的預(yù)防和治療奠定了重要的理論依據(jù)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

【共引文獻(xiàn)】

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