本文選題：肝再生 + T細胞��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2017年碩士論文

【摘要】：肝臟是機體的“生化工廠”,每時每刻都發(fā)生著各種生化反應,如營養(yǎng)物質的合成與儲存、血清蛋白的分泌、毒素以及代謝物的降解等。由于肝臟特殊的解剖學結構,其經常受到各種化學物質以及病原體的損傷。肝臟具有強大的再生能力,可以通過代償性增生恢復原有的結構與功能。肝再生的調控精密而復雜,涉及多種組織損傷修復分子,如補體系統(tǒng)、血小板、炎性細胞因子(TNF-α、IL-1β、IL-6)、生長因子(HGF、EGF、VGF)和抗炎因子(IL-10、TGF-β);多種細胞,如肝實質細胞、肝竇內皮細胞、枯否細胞、星狀細胞;多個生命活動,如增殖、分化、凋亡、代謝等等;是一個魯棒性極強的生物學過程。對肝再生的研究一方面可以為臨床上肝切除、肝移植以及人工肝等治療提供理論支持,為肝病的轉歸提供判斷依據;另一方面也可以為研究細胞的生長調控、腫瘤的發(fā)生發(fā)展、以及器官的發(fā)育等重要科學問題提供重要模型。在所有哺乳動物中,組織損傷均伴隨著機體免疫系統(tǒng)的激活,臨床上,肝再生也往往與肝臟急、慢性炎癥損傷同時存在,這意味著免疫系統(tǒng)可能在組織損傷修復過程中發(fā)揮重要作用。實驗室前期檢測了肝切除后不同時間點肝臟內單個核細胞各亞群的數量,發(fā)現(xiàn)肝切除后4h和168h肝臟內CD3+、CD3+CD4+CD8-、CD3+CD4-CD8+細胞數量顯著上升,這提示T細胞可能參與肝再生過程。隨后我們觀察了T細胞缺陷裸小鼠(BALB/c NUDE-athymic mice)70%肝切除后7天內的肝再生能力變化,發(fā)現(xiàn)裸小鼠相較于對照小鼠肝再生過程中肝重的恢復發(fā)生了明顯延緩,同時我們通過檢測Brd U滲入率以及PCNA與p-HDAC3的表達發(fā)現(xiàn)裸小鼠肝實質細胞的增殖高峰晚于對照小鼠,這表明T細胞缺陷裸小鼠的肝再生進程發(fā)生了延遲。細胞因子在肝再生調控中發(fā)揮關鍵作用,盡管已經發(fā)現(xiàn)了很多參與肝再生的調控因子,但肝再生是一個魯棒性很強的生物學過程,通常缺失一個基因并不會使肝再生完全停止。我們利用多因子免疫檢測技術分析了小鼠肝切除后不同時間點血清中細胞因子的變化,并結合實驗室前期積累的小鼠肝切除后不同時間點的轉錄組學數據進行分析,一方面驗證了目前對肝再生的部分認識,如TNF-α及其下游通路參與肝再生啟動,另一方面發(fā)現(xiàn)了新的可能參與肝再生的新的細胞因子及其下游通路,如IL-5。多因子免疫檢測技術結果顯示肝切除后血清中IL-5水平變化的幅度顯著,轉錄組學數據分析顯示IL-5下游靶基因在肝切除后不同時間點的變化趨勢與IL-5一致。此外,肝再生也能誘導肝臟中IL-5蛋白及m RNA水平、IL-5受體IL-5Rα及CSF2RB的蛋白及m RNA水平的顯著增加,提示IL-5可能參與肝再生調控。我們發(fā)現(xiàn)小鼠在外源注射IL-5可以促進70%肝切除小鼠肝臟再生過程,表現(xiàn)為:肝臟體重比恢復增快,Brd U滲入水平增強,p-HDAC3、PCNA、Cyclin A、Cyclin B1、C yclin D1、Cyclin E1表達增強。另外,我們通過小分子抑制劑YM90709抑制IL-5與其受體的相互作用,從而阻斷其生物學效應,我們發(fā)現(xiàn)注射YM90709后,肝切除小鼠Brd U滲入水平降低,p-HDAC3和PCNA的表達減弱,但肝重體重恢復未受影響。另外我們通過注射Anti-IL5中和小鼠體內IL-5,同樣發(fā)現(xiàn)Anti-IL5可以抑制肝切除小鼠肝再生能力。為探討IL-5促進肝再生的機制,我們利用兩步灌流法分離并體外培養(yǎng)小鼠原代肝實質細胞,我們分別用不同濃度的IL-5進行刺激,利用Ed U滲入的方法我們檢測到IL-5刺激后Ed U陽性細胞比例隨IL-5濃度升高而增加,這說明IL-5在體外可以明顯地刺激肝實質細胞DNA的合成。同時我們檢測了部分增殖相關通路中重要分子的磷酸化水平,包括JAK/STAT通路中的JAK1和STAT3、Ras/ERK通路中的ERK、PI3K/AK T通路中的AKT、p38MAPK通路中的p38,發(fā)現(xiàn)這些信號分子發(fā)生了活化,這提示我們IL-5可能通過這些通路促進肝實質細胞的增殖。由于有研究報道嗜酸性粒細胞可以通過分泌IL-4調節(jié)肝再生,且IL-5參與嗜酸性粒細胞的活化,我們檢測了注射IL-5對肝切除后肝臟中嗜酸性粒細胞過氧化物酶(EPX)以及IL-4的含量,發(fā)現(xiàn)外源注射了IL-5的小鼠肝臟中EPX和IL-4的含量均有所升高,這提示我們IL-5促進肝再生可能也依賴于嗜酸性粒細胞的途徑。綜上所述,我們以小鼠70%肝切除為模型,利用所收集的切除后不同時間點肝臟單個核細胞各亞群數量、血清細胞因子水平、肝臟轉錄組等組學數據,發(fā)現(xiàn)了調節(jié)肝再生過程的新分子——IL-5,并利用重組蛋白、小分子抑制劑以及中和抗體深入驗證了IL-5促進肝再生的作用;分子和細胞水平的實驗結果揭示了IL-5促肝細胞增殖的新機制,為嚴重肝病的治療提供了新的思路。本研究雖然表明IL-5可以促進肝再生,但目前還缺乏整體動物模型的支持,下一步我們將制備IL-5基因敲除小鼠,并進行相關研究,這將更有助于分析IL-5及其下游通路在肝臟損傷修復中的作用。此外,為了進一步闡明IL-5促肝再生的機制,我們還將利用中和抗體清除嗜酸性粒細胞,以明確IL-5促肝細胞增殖的直接作用。
[Abstract]:The liver is the "biochemical factory" of the body. Various biochemical reactions occur every time, such as the synthesis and storage of nutrients, the secretion of serum proteins, the degradation of toxins and metabolites. Due to the special anatomical structure of the liver, it is often damaged by various chemical substances and pathogens. The liver has a powerful regenerative ability. It can restore the original structure and function through compensatory hyperplasia. The regulation of liver regeneration is sophisticated and complex, involving a variety of tissue damage repair molecules, such as complement system, platelets, inflammatory cytokines (TNF-, IL-1 beta, IL-6), growth factors (HGF, EGF, VGF) and anti-inflammatory factors (IL-10, TGF- beta); many cells, such as liver parenchyma cells, hepatic sinusoidal endothelial cells Kupffer cells, stellate cells; multiple life activities, such as proliferation, differentiation, apoptosis, metabolism, and so on; it is a robust biological process. The study of liver regeneration can provide theoretical support for the treatment of clinical hepatectomy, liver transplantation and artificial liver, and provide a basis for the prognosis of liver diseases; on the other hand, it can also be used as a basis for the study of liver disease. In all mammals, tissue damage is accompanied by the activation of the immune system in all mammals. In clinical, liver regeneration is often associated with acute liver and chronic inflammatory damage, which means that the immune system may be in the tissue. The number of mononuclear cells in the liver at different time points after hepatectomy was detected in the laboratory. The number of CD3+, CD3+CD4+CD8-, CD3+CD4-CD8+ cells in the liver of 4H and 168h after hepatectomy was significantly increased after hepatectomy, which suggested that T cells may participate in the process of liver regeneration. Then we observed the T cells. The changes in the liver regeneration ability of BALB/c NUDE-athymic mice 70% after 7 days of hepatectomy, we found that the nude mice had a significant delay in the recovery of liver weight during the liver regeneration of the control mice. At the same time, we found that the proliferation peak of the nude mice liver parenchyma cells was later than the Brd U infiltration rate and the expression of PCNA and p-HDAC3. In the control mice, this indicates a delay in the process of liver regeneration in nude mice with T cells. Cytokines play a key role in the regulation of liver regeneration. Although many regulators have been found to be involved in liver regeneration, liver regeneration is a robust biological process, and the absence of one gene often does not completely stop the liver regeneration. We analyzed the changes in serum cytokines in different time points after hepatectomy in mice, and analyzed the transcriptional data of different time points accumulated in mice after liver resection in the laboratory. On the one hand, we verified the current partial understanding of the liver rebirth, such as TNF- alpha and its downstream pathway. On the other hand, new cytokines and their downstream pathways that may participate in liver regeneration are found on the other hand, and the results of IL-5. multifactor immunoassay show that the changes in serum levels of IL-5 in the serum after hepatectomy are significant. The transcriptional data analysis shows that the changes of the target genes of the downstream target of IL-5 at different time points after hepatectomy. The potential is consistent with IL-5. In addition, liver regeneration can also induce the level of IL-5 protein and m RNA in the liver, the significant increase of IL-5 receptor IL-5R alpha and CSF2RB protein and m RNA level, suggesting that IL-5 may participate in the regulation of liver regeneration. We found that the exogenous injection IL-5 can promote the process of liver regeneration in 70% hepatectomy mice, showing that the liver weight is more than recovery. Brd U infiltration level enhancement, p-HDAC3, PCNA, Cyclin A, Cyclin B1, C yclin D1, Cyclin expression enhancement. Furthermore, we inhibit the interaction between the receptor and its receptor by a small molecule inhibitor, thus blocking its biological effect. In addition, we also found that Anti-IL5 could inhibit liver regeneration in hepatectomy mice by injecting Anti-IL5 and IL-5 in mice. In order to explore the mechanism of IL-5 promoting liver regeneration, we used two step perfusion method to separate and culture the primary liver parenchyma cells in vitro. We used the different methods to separate the liver parenchyma cells in vitro. The concentration of IL-5 was stimulated. Using the method of Ed U infiltration, we detected that the proportion of Ed U positive cells increased with the increase of IL-5 concentration after IL-5 stimulation. This indicates that IL-5 can stimulate the synthesis of DNA in liver parenchyma in vitro. At the same time, we detected the phosphorylation level of important molecules in the part of the proliferation related pathway, including JAK/STAT. The JAK1 and STAT3, the ERK in the Ras/ERK pathway, the AKT in the PI3K/AK T pathway, and p38 in the p38MAPK pathway, found that these signaling molecules were activated, suggesting that we IL-5 may promote the proliferation of liver parenchyma through these pathways. In the activation of eosinophils, we detected the content of eosinophil peroxidase (EPX) and IL-4 in liver after hepatectomy by injection of IL-5. It was found that the contents of EPX and IL-4 in the liver of mice injected with IL-5 were increased, which suggests that IL-5 may promote the rebirth of the liver and may also depend on the eosinophil. In this paper, we used a model of 70% hepatectomy in mice. We found a new molecule, IL-5, which regulates the process of liver regeneration by using the collected data of each subgroup of the liver mononuclear cells, serum cytokine level and liver transcriptional group at different time points after the excision. The recombinant protein, small molecule inhibitor and neutralizing antibody were used in depth. The effect of IL-5 on promoting liver regeneration was demonstrated. The results of molecular and cellular levels revealed a new mechanism for the proliferation of hepatocytes by IL-5, which provided a new idea for the treatment of severe liver diseases. Although this study indicates that IL-5 can promote liver regeneration, there is still a lack of support from the whole animal model, and the next step will be to prepare the IL-5 gene knockout. The mice, and the related research, will be more helpful in analyzing the role of IL-5 and its downstream pathway in the repair of liver damage. In addition, in order to further clarify the mechanism of IL-5 promoting liver regeneration, we will also use neutralizing antibodies to clear eosinophils to clarify the direct effect of IL-5 on hepatocyte growth.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R657.3

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