本文選題：肝臟損傷 + 動(dòng)物模型�。� 參考：《西南大學(xué)》2014年博士論文

【摘要】：肝臟是人體最大的內(nèi)臟器官,具有分泌膽汁、消化和代謝脂肪、合成和分泌氨基酸、合成和儲(chǔ)存糖原、儲(chǔ)存和過濾血液、解毒及排除廢物、分泌血清白蛋白以維持體內(nèi)環(huán)境的穩(wěn)定等多種重要功能。肝臟發(fā)揮正常生理功能是保障人口健康的基本要素之一。對(duì)于惡性肝臟疾病如肝癌,迄今為止最有效的治療和防止病情惡化的方式就是手術(shù)切除癌變部分肝臟,但是由此會(huì)導(dǎo)致患者的肝臟功能大大減弱,因此促進(jìn)肝臟再生以使得受損肝臟恢復(fù)功能至關(guān)重要。與脊椎動(dòng)物的其它器官相比,肝臟的再生能力較強(qiáng),在正常肝臟中的肝細(xì)胞雖然是靜默不分裂的,但是肝細(xì)胞卻擁有強(qiáng)大的分裂增殖能力并可在肝損傷后迅速進(jìn)入細(xì)胞分裂周期。肝臟經(jīng)手術(shù)切除三分之二的大鼠或小鼠可在7-10天內(nèi)恢復(fù)100%的肝臟重量。當(dāng)肝臟細(xì)胞的增殖能力被抑制或者肝臟受到嚴(yán)重?fù)p傷時(shí),一部分肝臟前體細(xì)胞會(huì)參與肝臟再生的修復(fù),但是對(duì)這些前體細(xì)胞鑒定和分離至今尚有突破性進(jìn)展。因此,建立更大程度的甚至肝臟完全損傷后再生的動(dòng)物模型,對(duì)于鑒定肝臟再生過程中發(fā)揮關(guān)鍵作用的內(nèi)源性肝臟前體細(xì)胞并深入揭示肝臟再生的分子調(diào)控機(jī)制具有重要意義。在肝臟再生過程中,成熟的膽管細(xì)胞是否會(huì)轉(zhuǎn)分化為肝臟細(xì)胞到目前為止還沒有報(bào)道,在本研究中我們主要以斑馬魚為動(dòng)物模型探究肝臟嚴(yán)重受損后膽管細(xì)胞是否會(huì)轉(zhuǎn)分化為肝臟細(xì)胞來參與肝臟再生過程。 在本研究中,我們利用斑馬魚作為模式生物來研究肝臟再生的細(xì)胞來源和分子機(jī)制,首先我們構(gòu)建了藥物特異性誘導(dǎo)損傷肝臟細(xì)胞的轉(zhuǎn)基因斑馬魚肝臟再生模型,通過大量的品系篩選和藥物優(yōu)化發(fā)現(xiàn)Mtz處理后可以使近100%的肝細(xì)胞誘導(dǎo)凋亡并在撤去藥物后肝臟能夠恢復(fù)正常再生。BODIPY FL C5和糖原顯色PAS反應(yīng)實(shí)驗(yàn)檢測(cè)到新生的肝臟細(xì)胞具有脂肪代謝、膽酸分泌和糖原儲(chǔ)存能力。因此我們構(gòu)建的斑馬魚肝臟再生模型是一種功能性的再生模型。 當(dāng)肝臟細(xì)胞被藥物特異性誘導(dǎo)損傷后,膽管變粗,膽管表皮細(xì)胞收縮同時(shí)有一小部分膽管細(xì)胞開始表達(dá)肝細(xì)胞的標(biāo)志物,再生中后期,所有的膽管細(xì)胞都失去了正常的形態(tài)并表達(dá)肝臟特異性的分子標(biāo)記,這表明膽管對(duì)肝臟再生起了重要作用。EdU標(biāo)記實(shí)驗(yàn)發(fā)現(xiàn)新生肝臟區(qū)域出現(xiàn)大量的增殖現(xiàn)象并且這些增殖細(xì)胞主要集中在膽管中。而再生的開始階段,膽管細(xì)胞的增殖能力非常低,同時(shí)TUNEL實(shí)驗(yàn)未能檢測(cè)到膽管細(xì)胞在再生過程中發(fā)生凋亡,因此膽管細(xì)胞在肝臟受損的過程中保持了完整性,這表明膽管細(xì)胞在肝臟受損后可以轉(zhuǎn)分化為肝臟細(xì)胞。為了進(jìn)一步確定膽管細(xì)胞的向肝臟細(xì)胞的轉(zhuǎn)分化現(xiàn)象,我們利用Cre-Loxp系統(tǒng)來追蹤膽管來源的肝臟細(xì)胞,再生后期70%的新生肝臟細(xì)胞都來源于膽管細(xì)胞。這些現(xiàn)象表明肝臟受損再生過程中,膽管細(xì)胞形態(tài)發(fā)生變化同時(shí)增殖轉(zhuǎn)分化為肝臟細(xì)胞并對(duì)肝臟再生起了主要的貢獻(xiàn)作用。 為了進(jìn)一步確定膽管細(xì)胞在肝臟再生過程中的功能作用,我們?cè)谂咛グl(fā)育的早期抑制Notch信號(hào)來特異性的抑制膽管系統(tǒng)的發(fā)育,膽管系統(tǒng)發(fā)育受損的肝臟再生明顯受到抑制。同時(shí)我們利用膽管突變體證明了膽管細(xì)胞對(duì)肝臟再生的重要性。另外,在斑馬魚中用膽管毒性藥物處理早期胚胎發(fā)現(xiàn)即使對(duì)膽管微弱的毒副作用也會(huì)抑制膽管細(xì)胞向肝臟的轉(zhuǎn)分化和肝臟的正常再生。肝臟再生初期膽管細(xì)胞開始表達(dá)一些內(nèi)胚層和肝臟早期的特異性標(biāo)記因子,這也說明膽管細(xì)胞轉(zhuǎn)分化會(huì)伴隨一個(gè)去分化為多功能細(xì)胞的過程。 熒光原位雜交偶聯(lián)抗體顯色發(fā)現(xiàn)除了早期內(nèi)胚層相關(guān)的轉(zhuǎn)錄因子外sox9b也會(huì)在膽管細(xì)胞中上調(diào)表達(dá),再生初期膽管細(xì)胞并未發(fā)現(xiàn)增殖現(xiàn)象但是sox9b的表達(dá)已經(jīng)上調(diào)。在sox9b突變體中,膽管細(xì)胞向肝臟細(xì)胞的轉(zhuǎn)分化受到抑制同時(shí)肝臟再生減慢,這說明sox9b對(duì)膽管細(xì)胞的轉(zhuǎn)分化具有重要作用。已有研究表明Sox9b是Notch信號(hào)途徑的一個(gè)靶基因,當(dāng)抑制了Notch后肝臟再生減慢同時(shí)sox9b的表達(dá)下調(diào)。在肝臟受損的過程中,Notch的靶基因hes5在膽管中上調(diào),這說明Notch通過介導(dǎo)sox9b來參與了膽管細(xì)胞的轉(zhuǎn)分化過程。 綜上所述,本研究成功構(gòu)建了斑馬魚肝臟嚴(yán)重受損模型,鑒定了與肝臟受損相關(guān)的信號(hào)機(jī)制,并首次表明斑馬魚肝臟在兒乎100%受損后,乃然可進(jìn)行再生；并揭示在受損肝臟的再生修復(fù)過程中,成熟膽管細(xì)胞的轉(zhuǎn)分化起了重要作用；同時(shí)也證明膽管細(xì)胞的轉(zhuǎn)分化依賴于Notch信號(hào)途徑。本研究關(guān)于新生肝臟再生的來源和機(jī)制的研究對(duì)肝病的治療能夠提供一定的臨床指導(dǎo)意義和參考價(jià)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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575
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本文編號(hào)：1841345