發(fā)布時間：2018-05-19 06:57

  本文選題：UCH-L1 + THP-1��； 參考：《廣州醫(yī)科大學》2017年碩士論文

【摘要】：背景:膿毒癥在臨床上的致死率極高,多由細菌感染引起。當內(nèi)毒素脂多糖釋放入血液,激活炎癥反應,再通過MAPK和NF-κB途徑逐級擴大,最后產(chǎn)生瀑布效應,從而引起組織器官功能的損害。關于膿毒癥目前在臨床上仍缺乏有效的治療方法。基于對膿毒癥發(fā)病機制的理解,我們試圖從炎癥反應方面尋找治療膿毒癥的新靶點。泛素蛋白酶體系統(tǒng)能調(diào)節(jié)蛋白降解,影響細胞內(nèi)各種活動。泛素羥基末端水解酶L1(ubiquitin C-terminal hydrolase-L1,UCH-L1)作為去泛素蛋白酶體家族中的成員存在于人類許多組織結(jié)構(gòu)中,包括神經(jīng)細胞,內(nèi)皮細胞,平滑肌細胞等。目前許多研究證實了UCH-L1的異常表達與某些疾病有著內(nèi)在聯(lián)系,諸如神經(jīng)疾病、局部缺血、顱腦創(chuàng)傷、癌癥及帕金森等。UCH-L1也被證實在腎炎、血管內(nèi)皮細胞炎癥方面發(fā)揮一定調(diào)節(jié)作用,但UCH-L1與膿毒癥炎癥反應之間的關系尚未明確,有待我們進一步研究。目的:本研究通過LPS誘導人急性單核細胞白血病細胞株(THP-1)構(gòu)建膿毒癥體外細胞炎癥模型,探討UCH-L1與LPS誘導THP-1細胞炎癥反應之間的關系及其可能的機制。內(nèi)容與方法:1.利用UCH-L1的阻滯劑LDN57444抑制THP-1細胞的活性,采用ELISA法檢測LDN57444(10、20、30μM)對LPS(100ng/m L)刺激THP-1細胞6、12、24小時后,其釋放炎癥因子TNF-α、IL-1β和IL-6水平的影響。同時,利用MTS法檢測LDN57444(10、20、30μM)對LPS(100ng/m L)刺激THP-1細胞6、12、24小時后細胞活力的影響。用Western blot法檢測不同濃度LPS(0-200ng/m L)刺激THP-1后UCH-L1的表達。2.用Western blot法研究LDN57444(20μM)對LPS(100ng/m L)刺激THP-1細胞后MAPK通路中蛋白p-ERK、p-P38、p-JNK、ERK、P38、JNK的表達影響。用Western blot法探索NF-κB通路中胞核p65、胞漿p65、IκBα的表達影響。用免疫熒光共聚焦顯微鏡分析LDN57444(20μM)對LPS(100ng/m L)刺激THP-1細胞12小時后胞核、胞漿P65的表達。3.轉(zhuǎn)染UCH-L1 si RNA(40n M)進入THP-1細胞48小時后,采用Western blot法檢測UCH-L1的表達。轉(zhuǎn)染si RNA后,再用LPS(100ng/m L)培養(yǎng)12小時后提取上清,利用ELISA法檢測炎癥因子TNF-α、IL-1β和IL-6的水平。用MTS法檢測UCH-L1 si RNA對THP-1細胞活力的影響。4.采用Western blot法測UCH-L1 si RNA(40n M)對LPS誘導的THP-1中的MAPK通路中p-ERK、p-P38、P38、p-JNK、ERK、JNK的表達及NF-κB通路中胞核p65、胞漿p65、IκBα的表達。結(jié)果:1.誘導THP-1細胞的LPS濃度增加時,UCH-L1的表達上調(diào)。與LPS組相比,LDN57444+LPS組中THP-1細胞釋放的炎癥因子TNF-α、IL-1β、IL-6減少(p0.05)。LDN57444對細胞活力無明顯影響(p0.05)。2.與LPS組相比,LDN57444+LPS組中THP-1細胞p-ERK、p-P38、p-JNK的表達稍減少,P38的表達上調(diào)(p0.05),ERK、JNK的表達無明顯變化(p0.05)。Western blot中:與LPS組相比,LDN57444+LPS組中的胞核p65減少(p0.05),胞漿p65無明顯變化(p0.05),IκBα在15、30分鐘增加(p0.05)。共聚焦顯微鏡下:與LPS組相比,LDN57444+LPS組中的胞核p65有減少。3.與negative si RNA+LPS組相比,UCH-L1 si RNA+LPS組中釋放炎癥因子TNF-α、IL-1β和IL-6減少(p0.05)。UCH-L1 si RNA對THP-1細胞活力無明顯影響(p0.05)。4.與negative si RNA組相比,UCH-L1 si RNA組中UCH-L1的表達明顯減少(p0.05)。UCH-L1 si RNA組MAPK通路中p-ERK、p-P38、p-JNK的表達稍減少(p0.05),NF-κB通路中胞核p65的表達明顯減少(p0.05),IκBα的表達增加(p0.05),ERK、P38、JNK、胞漿p65的表達無明顯變化(p0.05)。結(jié)論:UCH-L1促進LPS誘導的THP-1細胞炎癥反應,其機制與MAPK通路蛋白及P65核轉(zhuǎn)位有關。背景:膿毒癥是宿主對炎癥反應失控引起的多器官功能障礙。膿毒癥的全球發(fā)病人數(shù)、患病率、致死率都呈逐年上升的趨勢,是目前ICU中致死率最高的疾病之一。其發(fā)生機制仍未完全闡明,但始終認為炎癥反應的失控是其基本機制。炎癥反應是通過炎癥通路逐級擴大的過程,最后促使巨噬細胞釋放出大量的炎癥因子,包括TNF-α、IL-1β、IL-6、基質(zhì)金屬蛋白酶(matrix metalloproteinases,MMPs)等。這些炎癥因子增加血管內(nèi)皮細胞通透性,進一步加大炎癥因子的釋放,最終使內(nèi)皮細胞損傷、缺氧、組織壞死。當LPS刺激炎癥細胞后,激活了MAPK和NF-κB通路,促使許多炎癥介質(zhì)基因的合成及相應的炎癥介質(zhì)的釋放。高良姜素(galangin)作為黃酮醇類化合物的一種,在缺血性損傷,抗病毒,抗腫瘤,鎮(zhèn)痛,抗氧化,抗炎等方面都有大量的研究。我們的前期研究表明高良姜素對脂多糖(LPS)誘導的THP-1細胞炎癥反應具有一定的保護作用,但高良姜素對LPS刺激巨噬細胞的炎癥反應的研究很少。本研究擴大了高良姜素對炎癥細胞的范圍的探究,為高良姜素干預膿毒癥的炎癥反應提供更多依據(jù)。目的:本研究采用LPS刺激的巨噬細胞作為體外炎癥模型,探索高良姜素是否能抑制該模型的炎癥反應。內(nèi)容與方法:1.用PMA(乙酰豆肉蔻佛波醇,Phorbol 12-myristate 13-acetate)誘導單核細胞(THP-1)成巨噬細胞。2.酶聯(lián)免疫吸附(ELISA)法檢測高良姜素對LPS誘導的巨噬細胞12、24小時后釋放TNF-α、IL-1β、IL-6的水平。3.MTS法(實時熒光?MT細胞活性檢測)檢測LPS處理12、24小時后高良姜素對巨噬細胞活性的影響。同時采用Annexin V-FITC/PI雙染流式細胞術檢測LPS誘導巨噬細胞6小時后巨噬細胞凋亡率。4.酶聯(lián)免疫吸附(ELISA)法檢測高良姜素對LPS誘導的巨噬細胞12、24小時后TIMP-1的釋放水平。用免疫印跡(Western blot)法檢測LPS處理12小時后高良姜素對LPS誘導的巨噬細胞MMP-2、MMP-9蛋白水平的變化。結(jié)果:1.LPS的用藥濃度為500ng/m L時,與LPS組相比,高良姜素+LPS組(2.5-20μM+500ng/m L)能有效抑制炎癥因子TNF-α、IL-1β、IL-6的釋放(P0.05)。2.高良姜素在0-20μmol/L范圍內(nèi)對LPS誘導的巨噬細胞活力無明顯影響(P0.05)。高良姜素在10、20μM時對LPS誘導的巨噬細胞凋亡率無明顯影響(P0.05)。3.與LPS組比,10、20μM的高良姜素能夠抑制LPS誘導的巨噬細胞MMP-2的表達(P0.05),20μM的高良姜素能夠抑制LPS誘導的巨噬細胞MMP-9的表達(P0.05),但對TIMP-1的表達無調(diào)節(jié)作用(P0.05)。結(jié)論:高良姜素能夠抑制LPS誘導的巨噬細胞炎癥反應,使得TNF-α、IL-1β、IL-6、MMP-2、MMP-9的表達下調(diào)。
[Abstract]:Background: sepsis is highly lethal in clinic and is caused by bacterial infection. When endotoxin lipopolysaccharide is released into the blood, it activates the inflammatory reaction, and then expands by MAPK and NF- kappa B. Finally, it produces the waterfall effect, which causes the damage of tissue and organ function. The clinical treatment of sepsis is still lacking in clinical treatment. Based on the understanding of the pathogenesis of sepsis, we try to find a new target for the treatment of sepsis from the inflammatory response. The ubiquitin proteasome system can regulate protein degradation and affect various activities in cells. The ubiquitin hydroxyl terminal hydrolase L1 (ubiquitin C-terminal hydrolase-L1, UCH-L1) is a member of the family of the ubiquitin proteasome. There are many human tissue structures, including nerve cells, endothelial cells, smooth muscle cells and so on. Many studies have confirmed that the abnormal expression of UCH-L1 has an inherent relationship with some diseases, such as nerve disease, local ischemia, craniocerebral trauma, cancer, and Parkinson, and.UCH-L1 are also confirmed in nephritis and inflammation of vascular endothelial cells. The relationship between UCH-L1 and the inflammatory response of sepsis is not clear. We need further study. Objective: to construct a human acute monocytic leukemia cell line (THP-1) to construct an inflammatory model of sepsis in vitro by LPS, and to explore the relationship between UCH-L1 and LPS induced THP-1 cell inflammation. The mechanism. Content and methods: 1. using the inhibitor LDN57444 of UCH-L1 to inhibit the activity of THP-1 cells, the effect of LDN57444 (10,20,30 M) on LPS (100ng/m L) stimulated THP-1 cell 6,12,24 hours after LPS (100ng/m L) was detected by ELISA method. L stimulated the cell viability after 6,12,24 hours in THP-1 cells. Western blot method was used to detect the expression of LPS (0-200ng/m L) stimulated by LPS (0-200ng/m L). The expression of p65, cytoplasmic p65, I kappa B alpha in NF- kappa B pathway was explored. The expression of LDN57444 (20 mu M) to LPS (100ng/m L) stimulated by LPS (100ng/m L) for 12 hours was analyzed by immunofluorescence confocal microscopy. After RNA, LPS (100ng/m L) was used to extract the supernatant after 12 hours, and the ELISA method was used to detect the level of TNF- alpha, IL-1 beta and IL-6. The expression of p65, cytoplasmic p65, I kappa B alpha in the NF- kappa B pathway. Results: 1. the expression of UCH-L1 increased when the LPS concentration increased in THP-1 cells. Compared with the LPS group, the inflammatory factor released by THP-1 cells in LDN57444+LPS group had no obvious effect on cell viability. The expression of THP-1 cells p-ERK, p-P38 and p-JNK decreased slightly in group PS, and the expression of P38 was up to up (P0.05), ERK and JNK had no obvious changes (P0.05).Western blot. Compared with the group of negative Si RNA+LPS in the group LDN57444+LPS, the release of inflammatory factors TNF- a in the UCH-L1 Si RNA+LPS group was less than that in the UCH-L1 Si RNA+LPS group. The expression of p-ERK, p-P38, p-JNK in the MAPK pathway of group RNA decreased slightly (P0.05), and the expression of p65 in the NF- kappa B pathway decreased significantly (P0.05), and I kappa B alpha expression increased. Background: sepsis is the multiple organ dysfunction caused by the host's runaway inflammatory response. The global incidence, morbidity and mortality of sepsis are increasing year by year. It is one of the most fatal diseases in ICU. The mechanism of its occurrence is still not fully elucidated, but it is believed that inflammation is the basic mechanism of the disease. The disease response is a gradual expansion of the inflammatory pathway, which eventually causes macrophages to release a large number of inflammatory factors, including TNF- alpha, IL-1 beta, IL-6, matrix metalloproteinase (matrix metalloproteinases, MMPs). These inflammatory factors increase the permeability of vascular endothelial cells, further increase the release of inflammatory factors, and eventually make endothelial cells Injury, hypoxia, tissue necrosis. When LPS stimulates the inflammatory cells, it activates the MAPK and NF- kappa B pathway, which promotes the synthesis of many inflammatory mediators and the release of the corresponding inflammatory mediators. As one of the flavonols, alpinin (galangin) has a large number of ischemic damage, antivirus, anti-tumor, analgesic, antioxidant, and anti-inflammatory. Our previous study showed that alpinin has a protective effect on the inflammatory response of THP-1 cells induced by lipopolysaccharide (LPS), but alpinin has few studies on the inflammation of macrophages stimulated by LPS. This study expanded the study of Takara Jiangso's model of inflammatory cells and the intervention of alpinin in sepsis. Objective: the purpose of this study was to explore whether alpinin could inhibit the inflammatory response of the model by using LPS stimulated macrophages as an in vitro model of inflammation. Content and methods: 1. using PMA (acetyl nutmeg, phorbol, Phorbol 12-myristate 13-acetate) induced.2. enzyme linked immunosorbent (E) macrophages in mononuclear cells (THP-1) (E). LISA method was used to detect the effect of alpinin on the release of TNF- alpha, IL-1 beta, IL-6 in 12,24 hours after 12,24 hours of LPS induced macrophages. The effect of alpinin on the activity of macrophages was detected after 12,24 hours of 12,24 hours of LPS treatment. Meanwhile, 6 small macrophages were detected by Annexin V-FITC/. .4. enzyme linked immunosorbent assay (ELISA) was used to detect the release level of TIMP-1 after 12,24 hours of LPS induced macrophages. The level of MMP-2 and MMP-9 protein of macrophages induced by LPS after LPS treatment was detected by immunoblotting (Western blot) method for 12 hours after LPS treatment. 500ng/m L, compared with the LPS group, the group of alpinin +LPS (2.5-20 mu L) could effectively inhibit the inflammation factor TNF- alpha, IL-1 beta, IL-6 release (P0.05).2. algomin had no obvious effect on the viability of macrophage induced by 0-20 micron. P0.05.3. and LPS group, 10,20 mu M can inhibit the expression of LPS induced macrophage MMP-2 expression (P0.05). 20 micron alpinin can inhibit MMP-9 expression of macrophage induced by LPS (P0.05), but it has no regulatory effect on the expression of macrophage. The expression of TNF-, IL-1, IL-6, MMP-2 and MMP-9 was downregulated.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R459.7

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