本文選題：芹菜素 + 白楊素　； 參考：《南京大學(xué)》2012年碩士論文

【摘要】：巨噬細胞在炎癥和機體抵抗外來微生物入侵時起一個中樞的作用,同時巨噬細胞具有很高的可塑性。在不同的環(huán)境中,巨噬細胞接受不同的刺激能夠表現(xiàn)出不同的激活方式。在炎癥反應(yīng)中,巨噬細胞有三項功能：抗原呈遞、吞噬作用以及通過分泌各種細胞因子和生長因子發(fā)揮免疫調(diào)節(jié)功能。巨噬細胞分泌的一些活性分子會參與有益的炎癥反應(yīng),也會參與有害的炎癥反應(yīng)。所以對巨噬細胞及其分泌的產(chǎn)物進行干預(yù)治療,可為控制炎癥性疾病開啟新的途徑。 大量文獻研究表明,飲食中的黃酮類具有抗炎及抗氧化的作用。而且,近幾年研究顯示這些黃酮類化合物對腫瘤細胞具有很強的殺傷作用,表明黃酮類化合物具有抑制腫瘤細胞的作用。在本研究中,我們選取兩種黃酮類化合物,芹菜素和白楊素,來研究它們對巨噬細胞激活的調(diào)節(jié)作用,從而對黃酮類化合物的藥用價值有進一步的了解。我們的結(jié)果發(fā)現(xiàn)： (1)兩種化合物對巨噬細胞的毒性有濃度依賴性和時間依賴性,并且在相同情況下芹菜素毒性高于白楊素。 (2)LPS激活的巨噬細胞可以產(chǎn)生NO和MHCII表達上升,并釋放促炎因子。芹菜素和白楊素可以抑制LPS的這些作用：使NO降低,MHCⅡ表達下降,分泌的促炎因子降低。提示芹菜素和白楊素可以通過調(diào)節(jié)天然免疫和獲得性免疫反應(yīng),對巨噬細胞介導(dǎo)的炎癥相關(guān)疾病有潛在的治療作用。 (3)芹菜素和白楊素可以促進IL-4誘導(dǎo)的巨噬細胞替代活化。它們可以使精氨酸酶活上升,并且使Arg1、CD206、Ym1和Fizz1基因表達水平有不同程度的升高。通過對IL-4信號通路檢測發(fā)現(xiàn),芹菜素和白楊素也可以使JAK1和STAT6的磷酸化水平升高。PPARγ的轉(zhuǎn)錄水平?jīng)]有變化,但是細胞核中PPARγ含量升高,同時PPARγ下游基因CD36的表達也升高,提示芹菜素和白楊素可能會對PPARγ的轉(zhuǎn)錄活性有調(diào)節(jié)作用。 綜上所述,我們的實驗探討了芹菜素和白楊素對巨噬細胞功能及活化的調(diào)節(jié)。芹菜素和白楊素不僅能抑制LPS誘導(dǎo)的巨噬細胞經(jīng)典激活,還能促IL-4誘導(dǎo)的巨噬細胞替代活化,同時還能抑制單核細胞的分化。這些表明芹菜素和白楊素不僅能抑制炎癥、有助于炎癥的消除,還能緩解炎癥對組織造成的損傷從而有助于組織修復(fù)。 動物水平上,我們的前期研究結(jié)果還不是很充分。但是在小鼠腹腔巨噬細胞中的結(jié)果顯示,芹菜素和白楊素能夠降低肥胖引起的腹腔巨噬細胞的炎癥,同時對腹腔巨噬細胞的形態(tài)也有一定的影響。通過這些前期的基礎(chǔ)研究,為后面芹菜素和白楊素對肥胖小鼠炎癥的研究奠定了基礎(chǔ)。 腫瘤微環(huán)境中的巨噬細胞被稱為腫瘤相關(guān)巨噬細胞(tumor-associated macrophages, TAM)。細胞表型學(xué)研究發(fā)現(xiàn)：這類細胞高表達甘露糖受體、清除劑受體、I型誘導(dǎo)型精氨酸酶、選擇素CCL12和CCL17等,低表達促炎因子IL-12、 IL-23、TNFα等。TAM在促進腫瘤血管新生、組織修復(fù)、輔助Th2細胞應(yīng)答等方面起到關(guān)鍵的正向調(diào)控作用,其活化方式以替代活化(alternative activation)為主。 肝臟是個性別二態(tài)性的器官,性別能夠直接影響肝臟的生理、病理狀態(tài)。肝細胞癌(Hepatocellular carcinoma, HCC)發(fā)生發(fā)展過程具有明顯的性別差異現(xiàn)象。流行病學(xué)調(diào)查發(fā)現(xiàn)：男性發(fā)病率約是女性的3-5倍；臨床研究數(shù)據(jù)表明,男性中HCC預(yù)后較女性差。但HCC性別差異的內(nèi)在機制不明,特別是腫瘤微環(huán)境在其中的作用亟待揭示。 實驗室前期工作已發(fā)現(xiàn)： (1)在小鼠肝細胞癌原位模型中,與雄性小鼠相比雌性小鼠TAM的替代活化程度較低,而補充生理濃度的雌激素可以顯著抑制雄性小鼠腫瘤部位巨噬細胞的替代活化。 (2)通過體外實驗,雌激素顯著抑制巨噬細胞表面抗原CD206的表達和精氨酸酶活。ELISA法分析發(fā)現(xiàn)10nM雌激素抑制替代活化的巨噬細胞釋放IL-10,下調(diào)IL-10/IL-12的比例。 (3)在10nM雌激素時,可以明顯抑制腫瘤的遷移。 (4)通過使用雌激素激動劑和抑制劑,發(fā)現(xiàn)雌激素發(fā)揮作用是通過ERβ而實現(xiàn)的。 基于實驗室以上的研究,本研究立足于從雌激素對巨噬細胞活化及功能調(diào)控的角度,繼續(xù)探討肝細胞癌發(fā)展性別差異的機制,主要工作及結(jié)果包括： (1)構(gòu)建小鼠原位HCC模型,進一步驗證雌激素在HCC發(fā)展中的作用。 (2)雌激素介導(dǎo)的巨噬細胞替代活化的信號轉(zhuǎn)導(dǎo)及分子機制。 在本實驗中發(fā)現(xiàn),17β雌二醇(E2)可以通過調(diào)節(jié)腫瘤巨噬細胞的極化方式抑制腫瘤的生長。在給去勢的小鼠補充雌激素后能夠降低腫瘤的生長。通過體外實驗我們發(fā)現(xiàn),IL-4刺激巨噬細胞后ERβ會與線粒體膜上的ATP酶結(jié)合。用E2預(yù)處理后,破壞了ERβ與ATP酶的結(jié)合,同時JAK1/STAT6信號通路的抑制因子SOCS1表達升高,從而抑制了巨噬細胞的替代活化。
[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.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

【參考文獻】

中國期刊全文數(shù)據(jù)庫 前2條

1 ;Effects of Activin A on the Activities of the Mouse Peritoneal Macrophages[J];Cellular & Molecular Immunology;2005年01期

2 Ichiro Shimizu;Nao Kohno;Katsuyoshi Tamaki;Masayuki Shono;;Female hepatology:Favorable role of estrogen in chronic liver disease with hepatitis B virus infection[J];World Journal of Gastroenterology;2007年32期

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本文編號：1798988