發(fā)布時間：2018-01-07 04:18

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

  更多相關(guān)文章： EGCG Notch 人巨噬細胞 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)或促進因素,如何調(diào)控炎癥反應逐漸成為干預這些疾病的重要關(guān)注點。炎癥反應的機理已有廣泛的研究,其中,NF-κB是炎癥反應中關(guān)鍵的調(diào)節(jié)因子,MAPK信號通路在炎癥的發(fā)生、發(fā)展過程中發(fā)揮著重要作用。近年的研究表明,在機體對LPS刺激產(chǎn)生的炎癥反應過程中,本底水平的Notch活化信號對于炎癥反應的信號放大是必要的,抑制Notch的活化能夠有效降低炎癥反應的強度。茶葉中的表沒食子兒茶素沒食子酸酯(EGCG)有降低炎癥反應的效果,其調(diào)控炎癥反應的機理已有較多的研究。然而,這些研究集中在EGCG處理6h以上的細胞生理反應上,細胞在6h或更長時間的過程中經(jīng)過了多輪信號傳導,且在此過程中,細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與細胞轉(zhuǎn)導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理仍不清楚。為了探究EGCG調(diào)節(jié)炎癥反應的初始作用點和作用機制,我們以人急性單核細胞白血病細胞THP-1來源的人巨噬細胞為實驗模型,研究EGCG即時處理對LPS誘導的早期炎癥的影響。在THP-1來源的人巨噬細胞上,我們研究了EGCG即時處理對LPS誘導的早期炎癥因子分泌的影響,通過Elisa和人炎癥因子芯片檢測巨噬細胞上清中炎癥因子的分泌情況。結(jié)果表明,EGCG即時處理可以顯著抑制LPS誘導的早期炎癥因子分泌�，F(xiàn)有研究認為,EGCG通過下調(diào)NF-κB,MAPK(p38,Erk,JNK)磷酸化水平來抑制LPS誘導的炎癥反應。為了探究該機理是否適用于EGCG即時處理抑制LPS誘導的早期炎癥。我們使用EGCG或LPS處理THP-1來源的巨噬細胞,通過Western Blot檢測不同處理間NF-κB,MAPK磷酸化水平。我們發(fā)現(xiàn)EGCG即時處理不能下調(diào)NF-κB,MAPK磷酸化,說明EGCG即時處理抑制LPS誘導的早期炎癥,不是通過NF-κB,MAPK信號通路所介導的。Notch信號通路在調(diào)節(jié)炎癥反應過程中起到重要的作用。通過EGCG處理,我們發(fā)現(xiàn)EGCG可以顯著抑制Notch蛋白的表達且這種效果具有瞬時性和濃度依賴性。蛋白酶體抑制劑MG132處理可以抑制EGCG誘導的Notch蛋白的降解,說明EGCG通過蛋白酶體途徑降解Notch蛋白。67LR是EGCG在小鼠細胞膜上的受體,為了探究EGCG即時處理抑制炎癥反應與67LR的關(guān)系,我們使用67LR封閉性抗體(與文獻中使用的抗體、使用濃度、使用方法完全一致)阻斷EGCG與67LR的結(jié)合,通過Elisa和人炎癥因子芯片檢測LPS誘導的炎癥因子分泌情況。在THP-1來源的人巨噬細胞上,我們發(fā)現(xiàn)EGCG即時處理降低炎癥因子分泌的效應并沒有消失,甚至沒有降低,說明EGCG即時處理抑制LPS誘導的炎癥反應不依賴于67LR。同時這些結(jié)果提示我們EGCG在細胞膜上除67LR外,還有新的作用靶點。為了驗證EGCG即時處理調(diào)節(jié)炎癥反應是通過Notch信號通路所介導的必要性。我們通過RNA干擾將THP-1細胞中Notch1/2蛋白表達沉默,將其分化成巨噬細胞,通過人炎癥因子芯片進行研究,發(fā)現(xiàn)EGCG即時處理抑制LPS誘導的炎癥效應消失或顯著減弱。這些結(jié)果證實了,在炎癥發(fā)生的早期階段,EGCG通過Notch信號通路調(diào)節(jié)炎癥反應。THP-1細胞是人急性單核細胞白血病細胞,在調(diào)節(jié)炎癥反應中,THP-1細胞可能存在著與正常細胞不同的作用機理。我們從健康人外周血中分離出單核細胞(PBMC),將其分化成巨噬細胞并進行研究,發(fā)現(xiàn)EGCG抑制LPS誘導的早期炎癥和抑制Notch蛋白的表達在健康人來源的巨噬細胞上同樣適用。綜上所述,本論文闡明了表沒食子兒茶素沒食子酸酯(EGCG)通過Notch信號通路抑制早期炎癥反應,為更深入地了解EGCG抑制炎癥的作用機理提供依據(jù)。研究結(jié)果,對于人們飲茶和服用富含EGCG的茶產(chǎn)品提供科學指導,將為人們通過科學地飲茶進行炎癥相關(guān)疾病的防治提供理論依據(jù),同時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)代科學研究提供經(jīng)驗。
[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.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R364.5

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