本文關(guān)鍵詞：敲除SIRT1基因?qū)π∈驛ML-12細(xì)胞中SFRS10、Lipin 1表達(dá)的影響　出處：《河北醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 酒精性肝病 沉默信息調(diào)節(jié)因子l 絲氨酸精氨酸二肽富含性剪切因子10 脂素1 酒精性脂肪性肝病 發(fā)病機(jī)理

【摘要】：目的:酒精性肝病(alcoholic liver disease,ALD)是因長(zhǎng)期過量飲酒引起的中毒性肝臟疾病,其中包括輕癥ALD、酒精性脂肪肝(alcohol fatty liver disease,AFLD)、酒精性肝炎、酒精性肝纖維化和酒精性肝硬化等類型。嚴(yán)重酗酒時(shí)可誘發(fā)廣泛肝細(xì)胞壞死,甚至肝功能衰竭。ALD病因明確,但其發(fā)病機(jī)制目前尚不完全清楚,可能與酒精及其代謝產(chǎn)物對(duì)肝臟的毒性作用、氧化應(yīng)激、脂質(zhì)過氧化、免疫反應(yīng)與內(nèi)毒素、細(xì)胞因子等密切相關(guān)。我們應(yīng)用酒精灌胃建立的大鼠ALD模型發(fā)現(xiàn),肝組織SIRT1 m RNA和蛋白表達(dá)逐漸減少的同時(shí)SFRS10 m RNA和蛋白的表達(dá)逐漸減弱,總Lipin 1及細(xì)胞質(zhì)中Lipin1-β的表達(dá)逐漸增多而細(xì)胞核中的Lipin1-α表達(dá)逐漸減少。Pihlajam?ki等研究發(fā)現(xiàn)在肥胖患者和高脂飲食小鼠肝組織中SFRS10表達(dá)減弱,且SFRS10的表達(dá)量與選擇性剪接表達(dá)Lipin 1的功能亞型有關(guān)。Yin等報(bào)道酒精以濃度依賴的方式抑制小鼠肝細(xì)胞株SIRT1表達(dá)的同時(shí)SFRS10表達(dá)減弱。因此,抑制SIRT1的表達(dá)對(duì)SFRS10以及Lipin 1表達(dá)的影響值得進(jìn)一步研究。本研究選用能代謝酒精的小鼠AML-12細(xì)胞,給予梯度酒精培養(yǎng)及si RNA干擾敲除SIRT1基因,應(yīng)用RT-q PCR和Western blot法檢測(cè)SFRS10,Lipin 1,Lipin1-β和Lipin1-α的m RNA和蛋白表達(dá);應(yīng)用油紅O染色觀察細(xì)胞內(nèi)脂質(zhì)沉積;應(yīng)用免疫熒光染色觀察Lipin 1的細(xì)胞定位。在細(xì)胞水平闡明抑制SIRT1的表達(dá)對(duì)SFRS10、Lipin 1表達(dá)的影響。方法:復(fù)蘇AML-12細(xì)胞用含10%胎牛血清、1%丙酮酸鈉、1‰的HEPES、1%青鏈霉素的高糖DMEM培養(yǎng)基在5%CO2,37℃條件下培養(yǎng)至對(duì)數(shù)生長(zhǎng)期,梯度酒精培育AML-12細(xì)胞,根據(jù)不同酒精濃度,分為0m M組(正常對(duì)照組)、40m M組、80m M組、120m M組;敲除SIRT1基因,分為空白對(duì)照組(Blank)、SIRT1si RNA陰性對(duì)照組(SIRT1si RNA NC)、SIRT1si RNA組、SIRT1si RNA+Ethanol組。1首先實(shí)驗(yàn)組分別用40m M、80m M、120m M濃度的酒精培育AML-12細(xì)胞,對(duì)照組常規(guī)培養(yǎng)并加入等體積無菌生理鹽水,培養(yǎng)24h后,吸取舊培養(yǎng)液,收集細(xì)胞,分別應(yīng)用實(shí)時(shí)熒光定量聚合酶連反應(yīng)(Real Time-quantitative Polymerase Chain Reaction,RT-q PCR)和Western blot法檢測(cè)SIRT1、剪切因子SFRS10和Lipin 1(核、漿)的m RNA和蛋白表達(dá)水平;用4%多聚甲醛固定,行油紅O染色觀察細(xì)胞內(nèi)脂質(zhì)沉積;行免疫熒光染色,觀察Lipin 1的胞漿或胞核分布。2實(shí)驗(yàn)組分別將SIRT1si RNA陰性對(duì)照序列、SIRT1si RNA轉(zhuǎn)染至AML-12細(xì)胞,以僅加相同體積的脂質(zhì)體作為空白對(duì)照組,培養(yǎng)6h后,更換為新鮮完全培養(yǎng)液繼續(xù)培養(yǎng)至24小時(shí),SIRT1si RNA+Ethanol組給予120m M濃度的酒精,余組加等體積的生理鹽水,繼續(xù)培養(yǎng)24h,收集細(xì)胞,分別應(yīng)用RT-q PCR和Western blot法檢測(cè)SIRT1、剪切因子SFRS10和Lipin 1(核、漿)的m RNA和蛋白表達(dá)水平;用4%多聚甲醛固定,行油紅O染色觀察細(xì)胞內(nèi)脂質(zhì)沉積;行免疫熒光染色,觀察Lipin 1的胞漿或胞核分布。結(jié)果:1梯度酒精(0m M、40m M、80m M、120m M)刺激AML-12細(xì)胞,各個(gè)指標(biāo)m RNA和蛋白表達(dá)變化及細(xì)胞病理學(xué)改變1.1 RT-q PCR法檢測(cè)SIRT1、SFRS10、總Lipin 1、Lipin1-β及Lipin1-αm RNA的表達(dá)變化:如Fig.1及Table 2所示,正常AML-12細(xì)胞中表達(dá)SIRT1,SFRS10較多,總的Lipin 1,細(xì)胞質(zhì)Lipin1-β及細(xì)胞核Lipin1-α表達(dá)均較少,隨著酒精濃度的增加,SIRT1表達(dá)逐漸減少,SFRS10的表達(dá)逐漸減弱;總的Lipin 1和細(xì)胞質(zhì)中Lipin1-β表達(dá)量均逐漸增多,而細(xì)胞核中Lipin1-α表達(dá)逐漸減少(F值分別為35.64,16.59,100.81,78.92,28.41,P均0.01)。如Fig.2及Table 3所示,隨著酒精濃度增加,Lipin1-β/α比值逐漸升高(F值為119.98,P均0.01)。1.2 Western blot法檢測(cè)SIRT1、SFRS10、總Lipin 1、Lipin1-β及Lipin1-α的蛋白水平表達(dá)變化:如Fig.4及Table 4所示,正常AML-12細(xì)胞中表達(dá)SIRT1,SFRS10較多,總的Lipin 1,細(xì)胞質(zhì)Lipin1-β及細(xì)胞核Lipin1-α表達(dá)均較少,隨著酒精濃度的增加,SIRT1表達(dá)逐漸減少,SFRS10的表達(dá)逐漸減弱;總的Lipin 1和細(xì)胞質(zhì)中Lipin1-β表達(dá)量均逐漸增多,而細(xì)胞核中Lipin1-α表達(dá)逐漸減少(F值分別為64.79,74.46,97.11,71.74,57.62,P均0.01)。1.3油紅O染色法觀察小鼠AML-12細(xì)胞內(nèi)的脂變:如Fig.5所示,正常對(duì)照組(0m M組)AML-12細(xì)胞內(nèi)未見脂肪變性,40m M濃度酒精刺激時(shí),AML-12細(xì)胞內(nèi)可見少量橘紅色脂滴,120m M濃度酒精刺激時(shí),AML-12細(xì)胞內(nèi)可見較多的橘紅色脂滴。1.4免疫熒光染色法觀察小鼠AML-12細(xì)胞中Lipin 1蛋白的細(xì)胞定位:如Fig.6所示,正常對(duì)照組示Lipin 1蛋白主要位于細(xì)胞質(zhì),細(xì)胞核也表達(dá)少量的Lipin 1蛋白。隨著酒精濃度的增加,細(xì)胞質(zhì)中Lipin 1蛋白表達(dá)逐漸增多,細(xì)胞核中的Lipin 1蛋白表達(dá)逐漸減少。2敲除小鼠AML-12細(xì)胞中SIRT1基因,各個(gè)指標(biāo)m RNA和蛋白表達(dá)變化及細(xì)胞病理學(xué)改變2.1敲除SIRT1基因通過RT-q PCR法檢測(cè)SIRT1、SFRS10、總Lipin 1、Lipin1-β及Lipin1-αm RNA的表達(dá):如Fig.7及Table 5所示,正常AML-12細(xì)胞中表達(dá)SIRT1、SFRS10較多,總的Lipin 1、細(xì)胞質(zhì)Lipin1-β及細(xì)胞核Lipin1-α表達(dá)均較少,敲除SIRT1基因,SIRT1si RNA組與空白對(duì)照組比較,SIRT1的表達(dá)降低約42%的同時(shí)SFRS10的表達(dá)明顯減少,總的Lipin 1和細(xì)胞質(zhì)中Lipin1-β表達(dá)量明顯增多,而細(xì)胞核中Lipin1-α表達(dá)明顯減少(P均0.01);在敲除SIRT1基因并加入酒精刺激后,SIRT1、SFRS10、Lipin1-α表達(dá)進(jìn)一步減少,總Lipin 1、細(xì)胞質(zhì)Lipin1-β的表達(dá)進(jìn)一步增加,差異均有統(tǒng)計(jì)學(xué)意義(P0.05或P0.01);SIRT1si RNA陰性對(duì)照組和空白對(duì)照組比較,各個(gè)指標(biāo)表達(dá)的變化,差異均無統(tǒng)計(jì)學(xué)意義(P均0.05)。如Fig.8及Table 6所示,Lipin1-β/α的比值,敲除基因SIRT1,SIRT1si RNA組與空白對(duì)照組比較,Lipin1-β/α的比值明顯增高,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。在敲除SIRT1基因并加入酒精刺激后,Lipin1-β/α的比值進(jìn)一步升高,SIRT1si RNA+Ethanol組與SIRT1si RNA組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。2.2敲除SIRT1基因通過Western blot法檢測(cè)SIRT1、SFRS10、總Lipin 1、Lipin1-β及Lipin1-α的蛋白水平表達(dá)變化:如Fig.10及Table 7所示,正常AML-12細(xì)胞中表達(dá)SIRT1、SFRS10較多,總Lipin 1、細(xì)胞質(zhì)Lipin1-β及細(xì)胞核Lipin1-α表達(dá)均較少,敲除SIRT1基因,SIRT1si RNA組與空白對(duì)照組比較,SIRT1的表達(dá)降低45%的同時(shí)SFRS10的表達(dá)明顯減少,總的Lipin 1和細(xì)胞質(zhì)中Lipin1-β表達(dá)明顯增多,而細(xì)胞核中Lipin1-α表達(dá)明顯減少(P均0.01);在敲除SIRT1基因并加入酒精刺激后,SIRT1、SFRS10、Lipin1-α表達(dá)進(jìn)一步減少,總Lipin 1、細(xì)胞質(zhì)Lipin1-β的表達(dá)進(jìn)一步增加,SIRT1si RNA+Ethanol組與SIRT1si RNA組比較,差異均有統(tǒng)計(jì)學(xué)意義(P均0.01)。而各個(gè)指標(biāo)中SIRT1si RNA陰性對(duì)照組和空白對(duì)照組比較,差異均無統(tǒng)計(jì)學(xué)意義(P均0.05)。2.3油紅O染色法觀察小鼠AML-12細(xì)胞內(nèi)的脂變:如Fig.11所示,油紅O染色示正常對(duì)照組及陰性對(duì)照組中AML-12細(xì)胞內(nèi)未見脂肪變性,敲除SIRT1基因,AML-12細(xì)胞內(nèi)可見較多的橘紅色脂滴,敲除SIRT1基因并加入120m M濃度酒精刺激,AML-12細(xì)胞內(nèi)可見大量的橘紅色脂滴。2.4免疫熒光染色法觀察小鼠AML-12細(xì)胞中Lipin 1蛋白的細(xì)胞定位:如Fig.12所示,正常對(duì)照組及陰性對(duì)照組示Lipin 1蛋白主要位于細(xì)胞質(zhì),細(xì)胞核也表達(dá)少量Lipin 1蛋白。敲除SIRT1基因,細(xì)胞質(zhì)中的Lipin 1蛋白表達(dá)明顯增多,細(xì)胞核中的Lipin 1蛋白表達(dá)明顯減少。敲除SIRT1基因并加入120m M濃度酒精刺激,細(xì)胞質(zhì)中的Lipin 1蛋白表達(dá)進(jìn)一步增多,細(xì)胞核中的Lipin 1蛋白表達(dá)進(jìn)一步減少。結(jié)論:SIRT1上游調(diào)節(jié)著SFRS10、Lipin 1的表達(dá),這可能為酒精性脂肪性肝病發(fā)生的重要機(jī)制之一。
[Abstract]:Objective: alcoholic liver disease (alcoholic liver, disease, ALD) is due to the long-term excessive drinking caused by poisoning liver disease, including mild ALD, alcoholic fatty liver (alcohol fatty liver disease, AFLD), alcoholic hepatitis, alcoholic hepatic fibrosis and liver cirrhosis and other types of heavy drinking can be induced. Extensive necrosis of liver cells, even.ALD liver failure etiology, but its pathogenesis is still unclear, may be related to alcohol and its metabolites on liver toxicity, oxidative stress, lipid peroxidation, immune response and endotoxin, cytokines and other closely related. We applied the ALD rat model of gastric alcohol was established at the same time, gradually reduce the expression of SFRS10 m RNA and SIRT1 M protein decreased RNA and protein expression in liver tissue, the expression of total Lipin and 1 in the cytoplasm of Lipin1- beta increased gradually and the nucleus of Lipin1 Alpha expression gradually decreased.Pihlajam? Ki study found in liver tissue in patients with obesity and high fat diet in mice in the reduced expression of SFRS10, expression of alternative splicing and the expression of SFRS10 Lipin 1 subtype.Yin reported on the function of alcohol in a concentration dependent manner inhibited the mouse liver cell line SIRT1 expression and SFRS10 expression decreased therefore, inhibiting the expression of SIRT1 is worthy of further study on the impact of SFRS10 and Lipin 1 expression. This study used to alcohol metabolism of mouse AML-12 cells, with gradient alcohol culture and Si RNA interference SIRT1 gene knockout, detection of SFRS10, PCR and Western blot method using RT-q Lipin 1, m RNA and protein expression of Lipin1- and beta Lipin1- alpha; observation of intracellular lipid deposition by oil red O staining; observe the cellular localization of Lipin 1 by immunofluorescence staining. The cell level to elucidate the inhibition of SIRT1 on the expression of SFRS10, Lipin 1 The effect of resuscitation. Methods: AML-12 cells containing 10% fetal bovine serum, 1% sodium pyruvate, 1 per thousand HEPES, 1% mycillin high glucose DMEM medium at 5%CO2,37 deg.c cultured to the logarithmic growth phase, gradient alcohol cultivation of AML-12 cells, according to the different concentration of alcohol, divided into 0m M group (normal control group 40m), M group, 80m M group, 120m M group; SIRT1 gene knockout, divided into control group (Blank), SIRT1si RNA negative control group (SIRT1si RNA NC), SIRT1si RNA group, SIRT1si RNA+Ethanol group.1 first experimental group were treated with 40m M, 80m M, 120m M, the concentration of alcohol cultivation AML-12 cells, the control group received routine training and add the equivalent volume of normal saline, 24h after culture, learn from the old culture medium and cells were collected respectively by real-time quantitative polymerase chain reaction (Real Time-quantitative Polymerase Chain Reaction, RT-q PCR and Western blot) detection method SIRT1, splicing factor SFRS1 0 and 1 Lipin (nuclear, plasma) expression levels of M RNA and protein; fixed by 4% paraformaldehyde to observe intracellular lipid deposition by oil red O staining; immunofluorescence staining, Lipin 1 was observed in cytoplasm or nucleolus distribution of.2 groups were SIRT1si RNA negative control sequence, SIRT1si RNA transfection to AML-12 cells by liposome only with the same volume as the blank control group, after 6h, the replacement for the fresh complete medium cultured for 24 hours, SIRT1si RNA+Ethanol were given 120m M concentration of alcohol, more than saline group with an equal volume of cultured 24h cells were collected to detect SIRT1 respectively using RT-q PCR and Western blot, SFRS10 and Lipin 1 shear factor (nuclear, plasma) expression levels of M RNA and protein; fixed by 4% paraformaldehyde to observe intracellular lipid deposition by oil red O staining; immunofluorescence staining, Lipin 1 was observed in cytoplasm or nucleolus distribution. Results: 1 Gradient alcohol (0m M, 40m M, 80m M, 120m M) stimulation of AML-12 cells, each index of M RNA and protein expression changes and cell pathological change detection of SIRT1 1.1 RT-q, PCR SFRS10, Lipin 1, Lipin1- and Lipin1- expression of beta alpha m RNA such as Fig.1 and shown in Table 2. The expression of SIRT1, AML-12 in SFRS10 cells are often more general Lipin 1 beta cytoplasmic Lipin1- and nuclear Lipin1- expression was less, with the increase of ethanol concentration, the expression of SIRT1 decreased, the expression of SFRS10 gradually decreased; volume gradually increased the expression of Lipin1- in the total Lipin 1 and cytoplasm, and the nucleus of Lipin1- alpha the expression decreased (F-measure respectively 35.64,16.59100.81,78.92,28.41, P 0.01). Such as Fig.2 and Table 3, with the increasing of the alcohol concentration, Lipin1- beta / alpha ratio increased gradually (F-measure 119.98, P 0.01).1.2 Western blot assay SIRT1, SFRS10, Lipin 1, Lipin1- beta The expression of Lipin1- and alpha: such as Fig.4 and Table 4, the expression of SIRT1 in normal AML-12 cells SFRS10 more, the total 1 Lipin, cytoplasmic Lipin1- beta and nuclear Lipin1- expression was less, with the increase of ethanol concentration, the expression of SIRT1 decreased, the expression of SFRS10 was gradually increased gradually; the expression of Lipin1- in the total Lipin 1 and in the cytoplasm and the nucleus, the expression of Lipin1- decreased gradually (F-measure respectively 64.79,74.46,97.11,71.74,57.62, P 0.01).1.3 oil red O staining of mouse AML-12 cells steatosis: as shown in Fig.5, normal control group (0m group M) AML-12 cells were not found in fatty degeneration 40m, M concentration of alcohol stimulation, AML-12 cells showed a small orange red lipid droplets, 120m concentration of M alcohol stimulation, observed Lipin mouse AML-12 cells 1 AML-12 cells showed more orange red lipid droplet.1.4 immunofluorescence staining method The cellular localization of proteins such as Fig.6 shown in the normal control group showed that Lipin 1 protein mainly located in cytoplasm, also expressed a small amount of Lipin 1 protein in the nucleus. With the increase of ethanol concentration in the cytoplasm, the expression of Lipin 1 protein gradually increased, the nucleus of the Lipin 1 protein expression decreased in.2 knockout mice SIRT1 gene in AML-12 cells. Each index of M RNA and protein expression changes and cell pathological changes of 2.1 SIRT1 gene knockout by detection of SIRT1, RT-q PCR SFRS10, Lipin 1, the expression of Lipin1- beta and Lipin1- alpha m RNA: Fig.7 and Table 5, the expression of SIRT1 in normal AML-12 cells SFRS10 more, the total Lipin 1. Cytoplasmic Lipin1- beta and nuclear Lipin1- expression was less, SIRT1 gene knockout, SIRT1si RNA group compared with the control group, the expression of SIRT1 decreased by about 42% while the expression of SFRS10 was significantly reduced, Lipin1 Lipin 1 and cytoplasm. Beta expression increased and nuclear expression of Lipin1- decreased significantly (P < 0.01); in the SIRT1 knockout and adding alcohol stimulation, SIRT1, SFRS10, Lipin1- expression decreased further, total 1 Lipin, the expression of cytoplasmic Lipin1- beta was further increased, the differences were statistically significant (P0.05 or P0.01); SIRT1si RNA negative control group and blank control group, the expression changes of each index, there were no significant differences (P < 0.05). Such as Fig.8 and Table 6, the ratio of Lipin1- / alpha beta, SIRT1 gene knockout, SIRT1si RNA group compared with the blank control group, the ratio of alpha / beta Lipin1- significantly, the difference was statistically significant (P0.01). In SIRT1 knockout and adding alcohol stimulation, Lipin1- beta / alpha ratio increased further, the SIRT1si RNA+Ethanol SIRT1si group compared with RNA group, the difference was statistically significant (P0.01).2.2 knockout SIRT1 gene by Western blot Detection of SIRT1, SFRS10, Lipin 1, the expression level of Lipin1- protein and Lipin1- alpha beta: Fig.10 and Table 7, the expression of SIRT1 in normal AML-12 cells SFRS10 more, 1 total Lipin, cytoplasmic Lipin1- beta and nuclear Lipin1- expression was less, SIRT1 gene knockout, SIRT1si RNA group and blank compared to the control group, the expression of SIRT1 decreased by 45% while the expression of SFRS10 was significantly reduced, significantly increased the expression of Lipin1- in the total Lipin 1 and in the cytoplasm and the nucleus, the expression of Lipin1- was significantly reduced (P < 0.01); in the SIRT1 knockout and adding alcohol stimulation, SIRT1, SFRS10, Lipin1- expression further reduce the total Lipin 1, the expression of cytoplasmic Lipin1- beta further increased in SIRT1si RNA+Ethanol group and SIRT1si RNA group, the differences were statistically significant (P < 0.01). Each index in SIRT1si RNA negative control group and blank control group, the difference There were no statistically significant (P 0.05).2.3 oil red O staining of mouse AML-12 cells steatosis: as shown in Fig.11, oil red O staining showed no fatty degeneration of AML-12 cells in the normal control group and negative control group, SIRT1 gene knockout, AML-12 cells showed more orange red lipid droplets knockdown of SIRT1 gene, and joined the 120m M concentration of alcohol stimulation, cellular localization of AML-12 cells showed a large number of orange red lipid droplets.2.4 immunofluorescence staining of Lipin mouse AML-12 cells in 1 proteins: as shown in Fig.12, normal control group and negative control group showed that Lipin 1 protein was mainly located in the cytoplasm, also expressed a small amount of Lipin 1 protein in nucleus. SIRT1 gene knockout, cytoplasmic Lipin 1 protein expression increased significantly in the nucleus of Lipin 1 protein expression was significantly reduced. SIRT1 gene knockout and joined the 120m M concentration of alcohol stimulation, cytoplasmic Lipin protein 1 The expression of Lipin 1 protein was further decreased in the nuclei. Conclusion: the upstream of SIRT1 regulates the expression of SFRS10 and Lipin 1, which may be one of the important mechanisms of the occurrence of alcoholic fatty liver disease.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575

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相關(guān)期刊論文 前1條

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本文編號(hào)：1375928