本文選題：叉頭蛋白O3a + 自噬�。� 參考：《重慶醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的:肝臟內(nèi)過多的脂質(zhì)沉積可阻斷Kupffer細(xì)胞(Kupffer cells,KCs)的自噬流,導(dǎo)致炎癥小體的激活,從而加重肝臟內(nèi)脂質(zhì)沉積導(dǎo)致的炎癥反應(yīng)。叉頭蛋白O3a(forkhead box protein O3a,Foxo3a)是連接炎癥反應(yīng)和能量代謝的重要轉(zhuǎn)錄因子。但是,在高脂飲食(high fat diet,HFD)的條件下,Foxo3a調(diào)節(jié)KCs自噬并抑制炎癥小體激活的機(jī)制尚不清楚。因此,本實(shí)驗(yàn)將研究非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)模型小鼠中,KCs中Foxo3a的變化以及調(diào)節(jié)KCs自噬流并抑制炎癥小體活性的機(jī)制。方法:1.梯度濃度的棕櫚酸(palmitic acid,PA)(0,0.16,0.32,0.64 m M)以及脂多糖(lipopolysaccharide,LPS)(100 ng/ml)聯(lián)合刺激體外的KCs12 h:1)蛋白印記法(western blotting assay,WB)檢測(cè):(1)自噬相關(guān)蛋白如Beclin 1和LC3;(2)NLRP3炎癥小體(nucleotide oligomerization domain(NOD)-like receptor family pyrin domain containing 3,NLRP3)相關(guān)蛋白如:NLRP3、caspase 1和凋亡相關(guān)斑點(diǎn)蛋白(apoptosis associated speck-like protein,ASC);(3)Foxo3a及上游相關(guān)蛋白如:腺苷酸活化蛋白激酶(adenosine monophosphate ativated protein kinase,AMPK)、磷脂酰肌醇-3-羥激酶(phosphatidyl inositol3-kinase,PI3K)和絲氨酸/蘇氨酸蛋白激酶B(protein serine threonine kinase B,Akt)的表達(dá)變化。(2)酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immuno sorbent assay,ELISA)檢測(cè)細(xì)胞上清白介素1β(interleukin-1β,IL-1β)和IL-18的含量。(3)利用2′7′-二乙酰二氯熒光素(dichlorofluorescein diacetate,DCFH-DA)熒光探針并熒光顯微鏡觀察KCs中活性氧產(chǎn)生的情況。2.(1)分別用Foxo3a過表達(dá)質(zhì)粒(Foxo3a overexpression plasmid,Foxo3a-OE)和Foxo3a-短發(fā)夾RNA質(zhì)粒(Foxo3a-short hairpin RNA plasmid,Foxo3a-sh RNA)轉(zhuǎn)染KCs 48 h后,再利用PA和LPS聯(lián)合刺激KCs 12 h:(1)WB檢測(cè)自噬相關(guān)蛋白及NLRP3炎癥小體相關(guān)蛋白的表達(dá)變化;(2)ELISA檢測(cè)細(xì)胞上清IL-1β和IL-18的含量;(3)透射電鏡觀察KCs自噬小體的分布情況。(2)m RFP-EGFP-LC3缺陷腺病毒感染KCs 48 h后,先利用Foxo3a激動(dòng)劑Iturin A預(yù)處理KCs 1 h,再利用PA和LPS聯(lián)合刺激KCs 12 h,最后利用熒光顯微鏡觀察LC3標(biāo)記的自噬小體的分布情況。3.(1)Foxo3a-OE轉(zhuǎn)染KCs 48 h后,利用PA和LPS聯(lián)合刺激KCs 12 h,再利用熒光定量聚合酶鏈反應(yīng)(real-time quantitative polymerase chain reaction,RT-PCR)檢測(cè)Foxo3a轉(zhuǎn)錄下游靶分子的m RNA表達(dá)情況。(2)利用Bim-OE及Bim-sh RNA體外轉(zhuǎn)染KCs 48 h后,先分別利用Foxo3a抑制劑SC97和激動(dòng)劑Iturin A預(yù)處理KCs 1 h,再利用PA和LPS聯(lián)合刺激KCs 12 h,檢測(cè):(1)WB檢測(cè)自噬相關(guān)蛋白及NLRP3炎癥小體相關(guān)蛋白的表達(dá)變化;(2)ELISA檢測(cè)細(xì)胞上清IL-1β和IL-18的含量。4.24只清潔級(jí)小鼠隨機(jī)分為3組,即正常飼料組(coarse food diet group,CFD),高脂飼料組(high fat diet,HFD)和高脂飼料聯(lián)合Iturin A腹腔注射組(Iturin A),每組8只。3組小鼠均喂養(yǎng)16周后,分別取小鼠的肝臟及外周血:(1)HE染色檢測(cè)肝臟脂肪變情況;(2)全自動(dòng)生化分析儀檢測(cè)小鼠肝功能及血脂水平;(3)透射電鏡觀察肝臟組織KCs中的自噬小體分布情況;(4)分離肝組織中KCs,并提取其總RNA和蛋白后:(1)WB檢測(cè)自噬相關(guān)蛋白及NLRP3炎癥小體相關(guān)蛋白的表達(dá)情況;(2)RT-PCR檢測(cè)IL-1β和IL-18的m RNA水平。結(jié)果:1.PA聯(lián)合LPS刺激體外KCs組與單獨(dú)LPS處理KCs組比較:(1)自噬相關(guān)蛋白Beclin1表達(dá)下降,LC3Ⅱ/Ⅰ的表達(dá)比例升高;(2)NLRP3炎癥小體相關(guān)分子NLRP3、ASC、caspase 1 p10、IL-1β和IL-18的表達(dá)水平升高;(3)Foxo3a表達(dá)下降,p-Foxo3a、p-Akt和p-PI3K的表達(dá)升高,而p-AMPK表達(dá)無(wú)變化;(4)ROS產(chǎn)生明顯增多。2.(1)KCs過表達(dá)Foxo3a后,可明顯促進(jìn)KCs自噬流,并抑制由PA聯(lián)合LPS誘導(dǎo)的NLRP3炎癥小體的激活;(2)KCs沉默F(xiàn)oxo3a后,可進(jìn)一步抑制KCs自噬流,以及進(jìn)一步促進(jìn)由PA聯(lián)合LPS誘導(dǎo)的NLRP3炎癥小體的激活。3.(1)KCs過表達(dá)Foxo3a后,PA聯(lián)合LPS刺激組中只有Bim的m RNA表達(dá)明顯低于單獨(dú)過表達(dá)Foxo3a組;(2)(1)沉默KCs中的Bim,即使給予Foxo3a激動(dòng)劑,KCs的自噬流仍然受阻,并促進(jìn)由PA聯(lián)合LPS誘導(dǎo)的NLRP3炎癥小體的激活;(2)過表達(dá)KCs中的Bim,即使給予Foxo3a抑制劑,KCs的自噬仍然順向進(jìn)行,并抑制由PA聯(lián)合LPS誘導(dǎo)的NLRP3炎癥小體的激活。4.Iturin A組小鼠與HFD組小鼠比較:(1)肝組織脂肪變及炎癥反應(yīng)較輕;(2)肝功能及血脂水平較低;(3)肝組織KCs中的自噬小體分布較多;(4)KCs中Foxo3a與Bim的表達(dá)明顯升高,自噬被激活,高脂飲食誘導(dǎo)的NLRP3炎癥小體的活性較低。結(jié)論:1.游離脂肪酸通過影響Foxo3a的磷酸化,阻斷KCs的自噬流,并促進(jìn)NLRP3炎癥小體的激活。2.Foxo3a通過恢復(fù)KCs的自噬流,抑制由游離脂肪酸誘導(dǎo)的NLRP3炎癥小體的活性。3.Foxo3a通過促進(jìn)Bim的轉(zhuǎn)錄活性,誘導(dǎo)KCs的自噬,并抑制由游離脂肪酸誘導(dǎo)的NLRP3炎癥小體的活性。4.Foxo3a激動(dòng)劑Iturin A通過促進(jìn)Bim的轉(zhuǎn)錄活性,減輕小鼠肝臟由于脂質(zhì)沉積導(dǎo)致的脂肪變和炎癥反應(yīng)。
[Abstract]:Objective: excessive lipid deposition in the liver blocks the autophagic flow of Kupffer cells (Kupffer cells, KCs), which leads to the activation of the inflammatory corpuscles and thus aggravates the inflammatory response in the liver. The forked head protein O3a (forkhead box protein O3a, Foxo3a) is an important transcription factor that connects the inflammatory response and energy metabolism. However, it is in high fat drink. Under the condition of high fat diet (HFD), the mechanism of Foxo3a to regulate autophagy and inhibit the activation of inflammatory corpuscles is not clear. Therefore, this study will study the changes in the non alcoholic fatty liver disease (nonalcoholic fatty liver disease, NAFLD) model mice and the mechanism of regulating the autophagic flow and inhibiting the activity of the inflammatory corpuscle in the KCs. Methods: palmitic acid, PA (PA) (0,0.16,0.32,0.64 m M) and LPS (lipopolysaccharide, LPS) (lipopolysaccharide, LPS) (100 ng/ml) combined stimulation of KCs12 h:1) assay: (1) autophagy related egg white such as 1 and 1; (2) inflammatory corpuscles (2) - Like receptor family pyrin domain containing 3, NLRP3) related proteins such as NLRP3, caspase 1 and apoptosis related speckle protein (apoptosis associated speck-like); (3) and upstream related proteins such as adenylate activation protein kinase, phosphatidylinositol Enzyme (phosphatidyl inositol3-kinase, PI3K) and serine / threonine protein kinase B (protein serine threonine kinase B, Akt). (2) enzyme linked immunosorbent assay (enzyme-linked immuno) detection of cell supernatant interleukin 1 beta and content. (3) use 2 '7' - two acetyl Two chlorofluorescein (dichlorofluorescein diacetate, DCFH-DA) fluorescence probe and fluorescence microscopy were used to observe the production of reactive oxygen species in KCs..2. (1) was transfected with Foxo3a overexpressed plasmids (Foxo3a overexpression plasmid, Foxo3a-OE) and Foxo3a- short hairpin RNA plasmids, respectively. PA and LPS were combined to stimulate KCs 12 h: (1) WB to detect the expression of autophagy related proteins and NLRP3 inflammatory corpuscle related proteins; (2) ELISA was used to detect the content of IL-1 beta and IL-18 in cell supernatant; (3) transmission electron microscopy was used to observe the distribution of autophagic corpuscles in KCs. (2) m RFP-EGFP-LC3 deficiency adenovirus infection 48 KCs 1 h, and then the combination of PA and LPS to stimulate KCs 12 h. Finally, the distribution of autophagic bodies marked by LC3 was observed by fluorescence microscopy.3. (1) Foxo3a-OE transfection KCs 48 h, using PA and joint stimulation 12, and then detected by fluorescence quantitative polymerase chain reaction. M RNA expression of downstream target molecules was transcribed. (2) after transfection of KCs 48 h with Bim-OE and Bim-sh RNA, Foxo3a inhibitor SC97 and activator Iturin A were pre treated with KCs 1 respectively, and then the combination stimulation was used to detect the changes in the expression of autophagy related protein and inflammatory corpuscle related protein; (2) The content of IL-1 beta and IL-18 in cell supernatant.4.24 mice was randomly divided into 3 groups, namely, normal diet group (coarse food diet group, CFD), high fat diet group (high fat diet, HFD) and high fat diet combined abdominal injection group, and each group of 8 rats were fed for 16 weeks, respectively, to take the liver and peripheral blood of mice, respectively: (1) dyeing Color detection of liver fatty change; (2) automatic biochemical analyzer to detect liver function and blood lipid level in mice; (3) transmission electron microscope to observe the distribution of autophagic corpuscles in liver tissue KCs; (4) separate KCs in liver tissue and extract its total RNA and protein: (1) WB detection of autophagy related protein and NLRP3 inflammatory corpuscle related protein expression; (2) R T-PCR detected the m RNA level of IL-1 beta and IL-18. Results: 1.PA combined with LPS stimulation in KCs group and single LPS treatment KCs group: (1) Beclin1 expression of autophagy related proteins decreased, LC3 II / I increased the expression ratio; (2) the expression level of inflammatory corpuscles, 1 The expression of oxo3a, p-Akt and p-PI3K increased, but the expression of p-AMPK was not changed. (4) ROS produced a significant increase in.2. (1) KCs over expression of Foxo3a, which could obviously promote the KCs autophagic flow and inhibit the activation of the NLRP3 inflammatory corpuscle induced by PA combined LPS; (2) after the silence, the autophagic flow can be suppressed and further promoted by the joint induction of PA. After the activation of.3. (1) KCs over expression of Foxo3a in the RP3 inflammatory body, the RNA expression of m in the PA combined LPS stimulus group was significantly lower than that of the single overexpressed Foxo3a group; (2) (1) the Bim in the silent KCs, even if given the agonist, was still blocked and promoted the activation of the inflammatory corpuscle induced by the combination. (2) Even if Foxo3a inhibitors were given, the autophagy of KCs was still in progress, and the activation of the NLRP3 inflammatory body induced by PA combined with LPS in the.4.Iturin A group was compared with that of the HFD mice: (1) the liver tissues were fat and the inflammatory response was lighter; (2) the liver function and blood lipid levels were low; (3) the autophagic bodies in the liver tissues were more distributed; (4) Foxo3a in KCs The expression of Bim was significantly elevated, autophagy was activated and the activity of NLRP3 inflammatory bodies induced by high fat diet was lower. Conclusion: 1. free fatty acids can inhibit the autophagic flow of KCs by affecting the phosphorylation of Foxo3a, and promote the activation of NLRP3 inflammatory corpuscle by restoring the autophagic flow of KCs and inhibiting the NLRP3 inflammatory body induced by free fatty acids. The active.3.Foxo3a induces autophagy by promoting the transcriptional activity of Bim and inhibits the autophagy of KCs, and inhibits the active.4.Foxo3a agonist, Iturin A, induced by free fatty acids, Iturin A, by promoting the transcriptional activity of Bim, and alleviating the fatty changes and inflammatory reactions in the liver of mice due to lipid deposition.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575

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