IL5调节肝再生的作用及其机制研究

发布时间：2018-04-20 07:51

本文选题：肝再生 + T细胞　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：肝脏是机体的“生化工厂”,每时每刻都发生着各种生化反应,如营养物质的合成与储存、血清蛋白的分泌、毒素以及代谢物的降解等。由于肝脏特殊的解剖学结构,其经常受到各种化学物质以及病原体的损伤。肝脏具有强大的再生能力,可以通过代偿性增生恢复原有的结构与功能。肝再生的调控精密而复杂,涉及多种组织损伤修复分子,如补体系统、血小板、炎性细胞因子(TNF-α、IL-1β、IL-6)、生长因子(HGF、EGF、VGF)和抗炎因子(IL-10、TGF-β);多种细胞,如肝实质细胞、肝窦内皮细胞、枯否细胞、星状细胞;多个生命活动,如增殖、分化、凋亡、代谢等等;是一个鲁棒性极强的生物学过程。对肝再生的研究一方面可以为临床上肝切除、肝移植以及人工肝等治疗提供理论支持,为肝病的转归提供判断依据;另一方面也可以为研究细胞的生长调控、肿瘤的发生发展、以及器官的发育等重要科学问题提供重要模型。在所有哺乳动物中,组织损伤均伴随着机体免疫系统的激活,临床上,肝再生也往往与肝脏急、慢性炎症损伤同时存在,这意味着免疫系统可能在组织损伤修复过程中发挥重要作用。实验室前期检测了肝切除后不同时间点肝脏内单个核细胞各亚群的数量,发现肝切除后4h和168h肝脏内CD3+、CD3+CD4+CD8-、CD3+CD4-CD8+细胞数量显著上升,这提示T细胞可能参与肝再生过程。随后我们观察了T细胞缺陷裸小鼠(BALB/c NUDE-athymic mice)70%肝切除后7天内的肝再生能力变化,发现裸小鼠相较于对照小鼠肝再生过程中肝重的恢复发生了明显延缓,同时我们通过检测Brd U渗入率以及PCNA与p-HDAC3的表达发现裸小鼠肝实质细胞的增殖高峰晚于对照小鼠,这表明T细胞缺陷裸小鼠的肝再生进程发生了延迟。细胞因子在肝再生调控中发挥关键作用,尽管已经发现了很多参与肝再生的调控因子,但肝再生是一个鲁棒性很强的生物学过程,通常缺失一个基因并不会使肝再生完全停止。我们利用多因子免疫检测技术分析了小鼠肝切除后不同时间点血清中细胞因子的变化,并结合实验室前期积累的小鼠肝切除后不同时间点的转录组学数据进行分析,一方面验证了目前对肝再生的部分认识,如TNF-α及其下游通路参与肝再生启动,另一方面发现了新的可能参与肝再生的新的细胞因子及其下游通路,如IL-5。多因子免疫检测技术结果显示肝切除后血清中IL-5水平变化的幅度显著,转录组学数据分析显示IL-5下游靶基因在肝切除后不同时间点的变化趋势与IL-5一致。此外,肝再生也能诱导肝脏中IL-5蛋白及m RNA水平、IL-5受体IL-5Rα及CSF2RB的蛋白及m RNA水平的显著增加,提示IL-5可能参与肝再生调控。我们发现小鼠在外源注射IL-5可以促进70%肝切除小鼠肝脏再生过程,表现为:肝脏体重比恢复增快,Brd U渗入水平增强,p-HDAC3、PCNA、Cyclin A、Cyclin B1、C yclin D1、Cyclin E1表达增强。另外,我们通过小分子抑制剂YM90709抑制IL-5与其受体的相互作用,从而阻断其生物学效应,我们发现注射YM90709后,肝切除小鼠Brd U渗入水平降低,p-HDAC3和PCNA的表达减弱,但肝重体重恢复未受影响。另外我们通过注射Anti-IL5中和小鼠体内IL-5,同样发现Anti-IL5可以抑制肝切除小鼠肝再生能力。为探讨IL-5促进肝再生的机制,我们利用两步灌流法分离并体外培养小鼠原代肝实质细胞,我们分别用不同浓度的IL-5进行刺激,利用Ed U渗入的方法我们检测到IL-5刺激后Ed U阳性细胞比例随IL-5浓度升高而增加,这说明IL-5在体外可以明显地刺激肝实质细胞DNA的合成。同时我们检测了部分增殖相关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,发现这些信号分子发生了活化,这提示我们IL-5可能通过这些通路促进肝实质细胞的增殖。由于有研究报道嗜酸性粒细胞可以通过分泌IL-4调节肝再生,且IL-5参与嗜酸性粒细胞的活化,我们检测了注射IL-5对肝切除后肝脏中嗜酸性粒细胞过氧化物酶(EPX)以及IL-4的含量,发现外源注射了IL-5的小鼠肝脏中EPX和IL-4的含量均有所升高,这提示我们IL-5促进肝再生可能也依赖于嗜酸性粒细胞的途径。综上所述,我们以小鼠70%肝切除为模型,利用所收集的切除后不同时间点肝脏单个核细胞各亚群数量、血清细胞因子水平、肝脏转录组等组学数据,发现了调节肝再生过程的新分子——IL-5,并利用重组蛋白、小分子抑制剂以及中和抗体深入验证了IL-5促进肝再生的作用;分子和细胞水平的实验结果揭示了IL-5促肝细胞增殖的新机制,为严重肝病的治疗提供了新的思路。本研究虽然表明IL-5可以促进肝再生,但目前还缺乏整体动物模型的支持,下一步我们将制备IL-5基因敲除小鼠,并进行相关研究,这将更有助于分析IL-5及其下游通路在肝脏损伤修复中的作用。此外,为了进一步阐明IL-5促肝再生的机制,我们还将利用中和抗体清除嗜酸性粒细胞,以明确IL-5促肝细胞增殖的直接作用。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R657.3

【参考文献】