發(fā)布時(shí)間：2018-01-07 04:18

  本文關(guān)鍵詞：EGCG通過(guò)Notch信號(hào)通路抑制早期炎癥的研究　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： EGCG Notch 人巨噬細(xì)胞 層粘連蛋白受體 炎癥

【摘要】：炎癥是糖尿病、腫瘤、心血管疾病等多種疾病的誘發(fā)或促進(jìn)因素,如何調(diào)控炎癥反應(yīng)逐漸成為干預(yù)這些疾病的重要關(guān)注點(diǎn)。炎癥反應(yīng)的機(jī)理已有廣泛的研究,其中,NF-κB是炎癥反應(yīng)中關(guān)鍵的調(diào)節(jié)因子,MAPK信號(hào)通路在炎癥的發(fā)生、發(fā)展過(guò)程中發(fā)揮著重要作用。近年的研究表明,在機(jī)體對(duì)LPS刺激產(chǎn)生的炎癥反應(yīng)過(guò)程中,本底水平的Notch活化信號(hào)對(duì)于炎癥反應(yīng)的信號(hào)放大是必要的,抑制Notch的活化能夠有效降低炎癥反應(yīng)的強(qiáng)度。茶葉中的表沒(méi)食子兒茶素沒(méi)食子酸酯(EGCG)有降低炎癥反應(yīng)的效果,其調(diào)控炎癥反應(yīng)的機(jī)理已有較多的研究。然而,這些研究集中在EGCG處理6h以上的細(xì)胞生理反應(yīng)上,細(xì)胞在6h或更長(zhǎng)時(shí)間的過(guò)程中經(jīng)過(guò)了多輪信號(hào)傳導(dǎo),且在此過(guò)程中,細(xì)胞因子的釋放以及反饋調(diào)節(jié)已經(jīng)發(fā)生。因此,現(xiàn)有的研究結(jié)果可能是EGCG與細(xì)胞轉(zhuǎn)導(dǎo)通路的中間蛋白的相互作用,而EGCG處理的直接或最原始的機(jī)理仍不清楚。為了探究EGCG調(diào)節(jié)炎癥反應(yīng)的初始作用點(diǎn)和作用機(jī)制,我們以人急性單核細(xì)胞白血病細(xì)胞THP-1來(lái)源的人巨噬細(xì)胞為實(shí)驗(yàn)?zāi)Ｐ?研究EGCG即時(shí)處理對(duì)LPS誘導(dǎo)的早期炎癥的影響。在THP-1來(lái)源的人巨噬細(xì)胞上,我們研究了EGCG即時(shí)處理對(duì)LPS誘導(dǎo)的早期炎癥因子分泌的影響,通過(guò)Elisa和人炎癥因子芯片檢測(cè)巨噬細(xì)胞上清中炎癥因子的分泌情況。結(jié)果表明,EGCG即時(shí)處理可以顯著抑制LPS誘導(dǎo)的早期炎癥因子分泌�，F(xiàn)有研究認(rèn)為,EGCG通過(guò)下調(diào)NF-κB,MAPK(p38,Erk,JNK)磷酸化水平來(lái)抑制LPS誘導(dǎo)的炎癥反應(yīng)。為了探究該機(jī)理是否適用于EGCG即時(shí)處理抑制LPS誘導(dǎo)的早期炎癥。我們使用EGCG或LPS處理THP-1來(lái)源的巨噬細(xì)胞,通過(guò)Western Blot檢測(cè)不同處理間NF-κB,MAPK磷酸化水平。我們發(fā)現(xiàn)EGCG即時(shí)處理不能下調(diào)NF-κB,MAPK磷酸化,說(shuō)明EGCG即時(shí)處理抑制LPS誘導(dǎo)的早期炎癥,不是通過(guò)NF-κB,MAPK信號(hào)通路所介導(dǎo)的。Notch信號(hào)通路在調(diào)節(jié)炎癥反應(yīng)過(guò)程中起到重要的作用。通過(guò)EGCG處理,我們發(fā)現(xiàn)EGCG可以顯著抑制Notch蛋白的表達(dá)且這種效果具有瞬時(shí)性和濃度依賴性。蛋白酶體抑制劑MG132處理可以抑制EGCG誘導(dǎo)的Notch蛋白的降解,說(shuō)明EGCG通過(guò)蛋白酶體途徑降解Notch蛋白。67LR是EGCG在小鼠細(xì)胞膜上的受體,為了探究EGCG即時(shí)處理抑制炎癥反應(yīng)與67LR的關(guān)系,我們使用67LR封閉性抗體(與文獻(xiàn)中使用的抗體、使用濃度、使用方法完全一致)阻斷EGCG與67LR的結(jié)合,通過(guò)Elisa和人炎癥因子芯片檢測(cè)LPS誘導(dǎo)的炎癥因子分泌情況。在THP-1來(lái)源的人巨噬細(xì)胞上,我們發(fā)現(xiàn)EGCG即時(shí)處理降低炎癥因子分泌的效應(yīng)并沒(méi)有消失,甚至沒(méi)有降低,說(shuō)明EGCG即時(shí)處理抑制LPS誘導(dǎo)的炎癥反應(yīng)不依賴于67LR。同時(shí)這些結(jié)果提示我們EGCG在細(xì)胞膜上除67LR外,還有新的作用靶點(diǎn)。為了驗(yàn)證EGCG即時(shí)處理調(diào)節(jié)炎癥反應(yīng)是通過(guò)Notch信號(hào)通路所介導(dǎo)的必要性。我們通過(guò)RNA干擾將THP-1細(xì)胞中Notch1/2蛋白表達(dá)沉默,將其分化成巨噬細(xì)胞,通過(guò)人炎癥因子芯片進(jìn)行研究,發(fā)現(xiàn)EGCG即時(shí)處理抑制LPS誘導(dǎo)的炎癥效應(yīng)消失或顯著減弱。這些結(jié)果證實(shí)了,在炎癥發(fā)生的早期階段,EGCG通過(guò)Notch信號(hào)通路調(diào)節(jié)炎癥反應(yīng)。THP-1細(xì)胞是人急性單核細(xì)胞白血病細(xì)胞,在調(diào)節(jié)炎癥反應(yīng)中,THP-1細(xì)胞可能存在著與正常細(xì)胞不同的作用機(jī)理。我們從健康人外周血中分離出單核細(xì)胞(PBMC),將其分化成巨噬細(xì)胞并進(jìn)行研究,發(fā)現(xiàn)EGCG抑制LPS誘導(dǎo)的早期炎癥和抑制Notch蛋白的表達(dá)在健康人來(lái)源的巨噬細(xì)胞上同樣適用。綜上所述,本論文闡明了表沒(méi)食子兒茶素沒(méi)食子酸酯(EGCG)通過(guò)Notch信號(hào)通路抑制早期炎癥反應(yīng),為更深入地了解EGCG抑制炎癥的作用機(jī)理提供依據(jù)。研究結(jié)果,對(duì)于人們飲茶和服用富含EGCG的茶產(chǎn)品提供科學(xué)指導(dǎo),將為人們通過(guò)科學(xué)地飲茶進(jìn)行炎癥相關(guān)疾病的防治提供理論依據(jù),同時(shí)也有助于人們以茶作為中藥材的類似物,為傳統(tǒng)中藥的現(xiàn)代科學(xué)研究提供經(jīng)驗(yàn)。
[Abstract]:Inflammation is diabetes, cancer, cardiovascular disease and other diseases or promoting factors, how to regulate inflammation has gradually become an important focus of intervention for these diseases. The study has extensive inflammatory reaction mechanism, NF- kappa B is a critical regulator of the inflammatory response in MAPK pathway in inflammation, play an important role in the process of development. Recent studies suggest that, in the process of inflammatory reaction in LPS induced, the level of activation of Notch signal for signal amplification of the inflammatory response is necessary, activation can effectively reduce the intensity of the inflammatory response. The inhibition of Notch in tea epigallocatechin gallate (EGCG) reduce inflammation effect, to study the regulation of inflammatory reaction mechanism more. However, these studies focused on the physiology of cells treated with EGCG above 6h reaction, cell In the process of 6h or longer after many rounds of signal transduction, and in the process, the release of cytokines and feedback regulation has occurred. Therefore, the existing research results may be the interaction among protein EGCG and cell transduction pathway, while EGCG directly or the original mechanism is not clear. In order to initial action point and to explore the mechanism of EGCG regulating inflammatory reaction, we used the human acute monocytic leukemia cell THP-1 from human macrophages as experimental model of EGCG instant effect on early inflammation induced by LPS in THP-1. The source of human macrophages, we investigated the EGCG effects on LPS induced immediate treatment the early inflammatory factor secretion, through the secretion of inflammatory cytokines Elisa and inflammatory cytokines in the supernatant of macrophage chip detection. The results showed that EGCG treatment can significantly inhibit the instant The early inflammatory factor secretion system induced by LPS. The existing studies suggest that EGCG downregulation by NF- kappa B, MAPK (p38, Erk, JNK) to inhibit the phosphorylation of LPS induced inflammation. In order to explore the mechanism of EGCG is suitable for real-time processing of early inflammation induced suppression of LPS. We use EGCG or LPS treated macrophages THP-1 the source, through the Western Blot detection among different treatments NF- kappa B, phosphorylation of MAPK. We found that EGCG treatment can not immediately down-regulation of NF- kappa B, MAPK phosphorylation, EGCG real-time early inflammation induced suppression of LPS, not by NF- kappa B,.Notch signaling pathway mediated by MAPK signaling pathway play an important the role in regulating the inflammatory reaction process. By EGCG, we found that EGCG can significantly inhibit the expression of Notch protein and this effect is transient and concentration dependent manner. The proteasome inhibitor MG132 can inhibit EG The degradation induced by CG Notch protein, EGCG Notch protein degradation via the proteasome pathway.67LR is EGCG in mouse receptors on the cell membrane, in order to explore the relationship between EGCG of immediate treatment inhibits the inflammatory reaction and 67LR, we use 67LR blocking antibody (anti body literature use concentration, using the identical method) blocking the combination of EGCG and 67LR, through the secretion of inflammatory cytokines Elisa and inflammatory factors induced by LPS chip in THP-1. The source of human macrophages, we found that EGCG treatment decreased the effect of immediate secretion of inflammatory cytokines did not disappear, even not reduced, EGCG real-time processing and inhibit the inflammatory reaction induced by LPS is not dependent on 67LR. at the same time, these results suggest that EGCG in the cell membrane in addition to 67LR, as well as new targets. In order to verify the EGCG real-time regulation of the inflammatory response through Notch signaling The necessity of road mediated by RNA interference. We will silence the expression of Notch1/2 protein in THP-1 cells to differentiate into macrophages, through the research of human inflammatory factor EGCG chip, found that immediate treatment inhibited LPS induced inflammatory effects disappeared or decreased. These results confirmed that in the early stage of inflammation, EGCG regulation of inflammation in.THP-1 cells through Notch signaling pathway is a human acute monocytic leukemia cell, in the regulation of the inflammatory response in THP-1 cells may have different mechanisms and normal cells. We isolated mononuclear cells from healthy human peripheral blood (PBMC) and its differentiation into macrophages and study, found that EGCG inhibited LPS induced early inflammation and inhibit the expression of Notch is also applicable in the sources of healthy macrophages. In summary, this paper illustrates the catechins epigallocatechin no food Sub acid (EGCG) inhibits the inflammatory reaction by Notch signal pathway, and provide the basis for a deeper understanding of the mechanism of EGCG inhibiting inflammation. Research results provide scientific guidance for people taking EGCG rich tea and tea products, will provide theoretical basis for the prevention and treatment for people through scientific drinking tea in inflammation related diseases, at the same time it also helps people with tea as analogues of Chinese medicinal materials, provide experience for modern scientific research of traditional Chinese medicine.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R364.5

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