巨噬细胞替代活化及功能的小分子调控

发布时间：2018-04-25 00:34

  本文选题：芹菜素 + 白杨素　； 参考：《南京大学》2012年硕士论文

【摘要】：巨噬细胞在炎症和机体抵抗外来微生物入侵时起一个中枢的作用,同时巨噬细胞具有很高的可塑性。在不同的环境中,巨噬细胞接受不同的刺激能够表现出不同的激活方式。在炎症反应中,巨噬细胞有三项功能：抗原呈递、吞噬作用以及通过分泌各种细胞因子和生长因子发挥免疫调节功能。巨噬细胞分泌的一些活性分子会参与有益的炎症反应,也会参与有害的炎症反应。所以对巨噬细胞及其分泌的产物进行干预治疗,可为控制炎症性疾病开启新的途径。 大量文献研究表明,饮食中的黄酮类具有抗炎及抗氧化的作用。而且,近几年研究显示这些黄酮类化合物对肿瘤细胞具有很强的杀伤作用,表明黄酮类化合物具有抑制肿瘤细胞的作用。在本研究中,我们选取两种黄酮类化合物,芹菜素和白杨素,来研究它们对巨噬细胞激活的调节作用,从而对黄酮类化合物的药用价值有进一步的了解。我们的结果发现： (1)两种化合物对巨噬细胞的毒性有浓度依赖性和时间依赖性,并且在相同情况下芹菜素毒性高于白杨素。 (2)LPS激活的巨噬细胞可以产生NO和MHCII表达上升,并释放促炎因子。芹菜素和白杨素可以抑制LPS的这些作用：使NO降低,MHCⅡ表达下降,分泌的促炎因子降低。提示芹菜素和白杨素可以通过调节天然免疫和获得性免疫反应,对巨噬细胞介导的炎症相关疾病有潜在的治疗作用。 (3)芹菜素和白杨素可以促进IL-4诱导的巨噬细胞替代活化。它们可以使精氨酸酶活上升,并且使Arg1、CD206、Ym1和Fizz1基因表达水平有不同程度的升高。通过对IL-4信号通路检测发现,芹菜素和白杨素也可以使JAK1和STAT6的磷酸化水平升高。PPARγ的转录水平没有变化,但是细胞核中PPARγ含量升高,同时PPARγ下游基因CD36的表达也升高,提示芹菜素和白杨素可能会对PPARγ的转录活性有调节作用。 综上所述,我们的实验探讨了芹菜素和白杨素对巨噬细胞功能及活化的调节。芹菜素和白杨素不仅能抑制LPS诱导的巨噬细胞经典激活,还能促IL-4诱导的巨噬细胞替代活化,同时还能抑制单核细胞的分化。这些表明芹菜素和白杨素不仅能抑制炎症、有助于炎症的消除,还能缓解炎症对组织造成的损伤从而有助于组织修复。 动物水平上,我们的前期研究结果还不是很充分。但是在小鼠腹腔巨噬细胞中的结果显示,芹菜素和白杨素能够降低肥胖引起的腹腔巨噬细胞的炎症,同时对腹腔巨噬细胞的形态也有一定的影响。通过这些前期的基础研究,为后面芹菜素和白杨素对肥胖小鼠炎症的研究奠定了基础。 肿瘤微环境中的巨噬细胞被称为肿瘤相关巨噬细胞(tumor-associated macrophages, TAM)。细胞表型学研究发现：这类细胞高表达甘露糖受体、清除剂受体、I型诱导型精氨酸酶、选择素CCL12和CCL17等,低表达促炎因子IL-12、 IL-23、TNFα等。TAM在促进肿瘤血管新生、组织修复、辅助Th2细胞应答等方面起到关键的正向调控作用,其活化方式以替代活化(alternative activation)为主。 肝脏是个性别二态性的器官,性别能够直接影响肝脏的生理、病理状态。肝细胞癌(Hepatocellular carcinoma, HCC)发生发展过程具有明显的性别差异现象。流行病学调查发现：男性发病率约是女性的3-5倍；临床研究数据表明,男性中HCC预后较女性差。但HCC性别差异的内在机制不明,特别是肿瘤微环境在其中的作用亟待揭示。 实验室前期工作已发现： (1)在小鼠肝细胞癌原位模型中,与雄性小鼠相比雌性小鼠TAM的替代活化程度较低,而补充生理浓度的雌激素可以显著抑制雄性小鼠肿瘤部位巨噬细胞的替代活化。 (2)通过体外实验,雌激素显著抑制巨噬细胞表面抗原CD206的表达和精氨酸酶活。ELISA法分析发现10nM雌激素抑制替代活化的巨噬细胞释放IL-10,下调IL-10/IL-12的比例。 (3)在10nM雌激素时,可以明显抑制肿瘤的迁移。 (4)通过使用雌激素激动剂和抑制剂,发现雌激素发挥作用是通过ERβ而实现的。 基于实验室以上的研究,本研究立足于从雌激素对巨噬细胞活化及功能调控的角度,继续探讨肝细胞癌发展性别差异的机制,主要工作及结果包括： (1)构建小鼠原位HCC模型,进一步验证雌激素在HCC发展中的作用。 (2)雌激素介导的巨噬细胞替代活化的信号转导及分子机制。 在本实验中发现,17β雌二醇(E2)可以通过调节肿瘤巨噬细胞的极化方式抑制肿瘤的生长。在给去势的小鼠补充雌激素后能够降低肿瘤的生长。通过体外实验我们发现,IL-4刺激巨噬细胞后ERβ会与线粒体膜上的ATP酶结合。用E2预处理后,破坏了ERβ与ATP酶的结合,同时JAK1/STAT6信号通路的抑制因子SOCS1表达升高,从而抑制了巨噬细胞的替代活化。
[Abstract]:Macrophages play a central role in inflammation and the body's resistance to foreign microbe invasion, while macrophages have high plasticity. In different environments, macrophages receive different stimuli in different stimuli. In the inflammatory response, macrophages have three functions: antigen presentation, phagocytosis Some of the active molecules secreted by macrophages are involved in beneficial inflammatory reactions and are involved in harmful inflammatory reactions. Therefore, a new way to control inflammatory diseases is initiated by intervening in the treatment of macrophages and their secreted products.
A large number of literature studies have shown that flavonoids in the diet have anti-inflammatory and antioxidant effects. Moreover, recent studies have shown that these flavonoids have a strong killing effect on tumor cells, indicating that flavonoids have the effect of inhibiting tumor cells. In this study, we selected two flavonoids, apigenin and Aspen is used to study their regulatory effects on macrophage activation, so as to further understand the medicinal value of flavonoids.
(1) the toxicity of two compounds to macrophages was concentration dependent and time-dependent, and in the same condition, apigenin was more toxic than Aspen.
(2) LPS activated macrophages can produce an increase in the expression of NO and MHCII and release pro-inflammatory factors. Apigenin and poplar can inhibit these effects of LPS, decrease NO, decrease the expression of MHC II, and decrease the secretory proinflammatory factors. Inflammation related diseases lead to potential therapeutic effects.
(3) apigenin and poplar can promote IL-4 induced macrophage substitution activation. They can increase arginase activity and increase the level of Arg1, CD206, Ym1 and Fizz1 gene expression to varying degrees. The phosphorylation of Apigenin and poplar can also increase the phosphorylation level of JAK1 and STAT6 by IL-4 signal pathway detection. The transcriptional level did not change, but the content of PPAR gamma in the nucleus increased and the expression of the downstream gene CD36 of the PPAR gamma increased, suggesting that apigenin and poplar could regulate the transcriptional activity of PPAR gamma.
In summary, our experiment explored the regulation of Apigenin and poplar on function and activation of macrophages. Apigenin and poplar can not only inhibit the classical activation of macrophages induced by LPS, but also promote IL-4 induced macrophages to replace activation, but also inhibit the differentiation of monocytes. These indicate that apigenin and poplar are not only effective. It can inhibit inflammation, help to eliminate inflammation, relieve inflammation damage to tissues, and help tissue repair.
At animal levels, our previous studies were not very well, but the results in mouse peritoneal macrophages showed that apigenin and poplar could reduce the inflammation of peritoneal macrophages caused by obesity, and also have a certain influence on the morphology of peritoneal macrophages. The study on the inflammation of obese mice has laid a foundation.
Macrophages in tumor microenvironment are called tumor-associated macrophages (TAM). Cell phenotype studies have found that these cells express mannose receptor, scavenger receptor, I inducible arginase, selectin CCL12 and CCL17, low expression proinflammatory factors IL-12, IL-23, TNF alpha and other.TAM in promoting tumor Angiogenesis, tissue repair, Th2 cell response and so on play a key positive regulatory role. The activation method is the main alternative to activation (alternative activation).
The liver is a sex two state organ, and sex can directly affect the physiological and pathological state of the liver. The development process of Hepatocellular carcinoma (HCC) has obvious gender differences. The epidemiological survey found that the incidence of male is about 3-5 times of the female; the clinical data show that the prognosis of HCC in male is more than that of the male. Women are poor. However, the inherent mechanism of sex differences in HCC is unknown, especially the role of tumor microenvironment.
The early work of the laboratory has been found:
(1) in the mouse hepatocarcinoma in situ model, the replacement activation of TAM in female mice was lower than that of male mice, while estrogen supplemented with physiological concentration could significantly inhibit the replacement activation of macrophages in the tumor site of male mice.
(2) through in vitro experiments, estrogen significantly inhibited the expression of macrophage surface antigen CD206 and the analysis of arginase active.ELISA method, and found that 10nM estrogen inhibited the release of IL-10 from activated macrophages and reduced the proportion of IL-10/IL-12.
(3) when 10nM estrogen is used, it can significantly inhibit tumor migration.
(4) through the use of estrogen agonists and inhibitors, the role of estrogens is achieved through ER beta.
Based on the study above, this study continues to explore the mechanism of gender differences in the development of hepatocellular carcinoma from the point of view of estrogen's activation and function of macrophages. The main work and results include:
(1) in situ HCC model was constructed to further verify the role of estrogen in the development of HCC.
(2) signal transduction and molecular mechanism of estrogen mediated activation of macrophages.
In this experiment, 17 beta estradiol (E2) can inhibit the growth of tumor by regulating the polarization of tumor macrophages. It can reduce the growth of tumor after adding estrogen to the ovariectomized mice. In vitro, we found that after IL-4 stimulation of macrophages, ER beta will combine with the ATP enzyme on the mitochondrial membrane. After preconditioning with E2, it is broken. The binding of ER beta to ATP enzyme is impaired, and the expression of SOCS1, an inhibitor of JAK1/STAT6 signaling pathway, is increased, thus inhibiting the activation of macrophages.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】

中国期刊全文数据库 前2条

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本文编号：1798988