敲除SIRT1基因对小鼠AML-12细胞中SFRS10、Lipin 1表达的影响

发布时间：2018-01-03 22:35

本文关键词：敲除SIRT1基因对小鼠AML-12细胞中SFRS10、Lipin 1表达的影响　出处：《河北医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:酒精性肝病(alcoholic liver disease,ALD)是因长期过量饮酒引起的中毒性肝脏疾病,其中包括轻症ALD、酒精性脂肪肝(alcohol fatty liver disease,AFLD)、酒精性肝炎、酒精性肝纤维化和酒精性肝硬化等类型。严重酗酒时可诱发广泛肝细胞坏死,甚至肝功能衰竭。ALD病因明确,但其发病机制目前尚不完全清楚,可能与酒精及其代谢产物对肝脏的毒性作用、氧化应激、脂质过氧化、免疫反应与内毒素、细胞因子等密切相关。我们应用酒精灌胃建立的大鼠ALD模型发现,肝组织SIRT1 m RNA和蛋白表达逐渐减少的同时SFRS10 m RNA和蛋白的表达逐渐减弱,总Lipin 1及细胞质中Lipin1-β的表达逐渐增多而细胞核中的Lipin1-α表达逐渐减少。Pihlajam?ki等研究发现在肥胖患者和高脂饮食小鼠肝组织中SFRS10表达减弱,且SFRS10的表达量与选择性剪接表达Lipin 1的功能亚型有关。Yin等报道酒精以浓度依赖的方式抑制小鼠肝细胞株SIRT1表达的同时SFRS10表达减弱。因此,抑制SIRT1的表达对SFRS10以及Lipin 1表达的影响值得进一步研究。本研究选用能代谢酒精的小鼠AML-12细胞,给予梯度酒精培养及si RNA干扰敲除SIRT1基因,应用RT-q PCR和Western blot法检测SFRS10,Lipin 1,Lipin1-β和Lipin1-α的m RNA和蛋白表达;应用油红O染色观察细胞内脂质沉积;应用免疫荧光染色观察Lipin 1的细胞定位。在细胞水平阐明抑制SIRT1的表达对SFRS10、Lipin 1表达的影响。方法:复苏AML-12细胞用含10%胎牛血清、1%丙酮酸钠、1‰的HEPES、1%青链霉素的高糖DMEM培养基在5%CO2,37℃条件下培养至对数生长期,梯度酒精培育AML-12细胞,根据不同酒精浓度,分为0m M组(正常对照组)、40m M组、80m M组、120m M组;敲除SIRT1基因,分为空白对照组(Blank)、SIRT1si RNA阴性对照组(SIRT1si RNA NC)、SIRT1si RNA组、SIRT1si RNA+Ethanol组。1首先实验组分别用40m M、80m M、120m M浓度的酒精培育AML-12细胞,对照组常规培养并加入等体积无菌生理盐水,培养24h后,吸取旧培养液,收集细胞,分别应用实时荧光定量聚合酶连反应(Real Time-quantitative Polymerase Chain Reaction,RT-q PCR)和Western blot法检测SIRT1、剪切因子SFRS10和Lipin 1(核、浆)的m RNA和蛋白表达水平;用4%多聚甲醛固定,行油红O染色观察细胞内脂质沉积;行免疫荧光染色,观察Lipin 1的胞浆或胞核分布。2实验组分别将SIRT1si RNA阴性对照序列、SIRT1si RNA转染至AML-12细胞,以仅加相同体积的脂质体作为空白对照组,培养6h后,更换为新鲜完全培养液继续培养至24小时,SIRT1si RNA+Ethanol组给予120m M浓度的酒精,余组加等体积的生理盐水,继续培养24h,收集细胞,分别应用RT-q PCR和Western blot法检测SIRT1、剪切因子SFRS10和Lipin 1(核、浆)的m RNA和蛋白表达水平;用4%多聚甲醛固定,行油红O染色观察细胞内脂质沉积;行免疫荧光染色,观察Lipin 1的胞浆或胞核分布。结果:1梯度酒精(0m M、40m M、80m M、120m M)刺激AML-12细胞,各个指标m RNA和蛋白表达变化及细胞病理学改变1.1 RT-q PCR法检测SIRT1、SFRS10、总Lipin 1、Lipin1-β及Lipin1-αm RNA的表达变化:如Fig.1及Table 2所示,正常AML-12细胞中表达SIRT1,SFRS10较多,总的Lipin 1,细胞质Lipin1-β及细胞核Lipin1-α表达均较少,随着酒精浓度的增加,SIRT1表达逐渐减少,SFRS10的表达逐渐减弱;总的Lipin 1和细胞质中Lipin1-β表达量均逐渐增多,而细胞核中Lipin1-α表达逐渐减少(F值分别为35.64,16.59,100.81,78.92,28.41,P均0.01)。如Fig.2及Table 3所示,随着酒精浓度增加,Lipin1-β/α比值逐渐升高(F值为119.98,P均0.01)。1.2 Western blot法检测SIRT1、SFRS10、总Lipin 1、Lipin1-β及Lipin1-α的蛋白水平表达变化:如Fig.4及Table 4所示,正常AML-12细胞中表达SIRT1,SFRS10较多,总的Lipin 1,细胞质Lipin1-β及细胞核Lipin1-α表达均较少,随着酒精浓度的增加,SIRT1表达逐渐减少,SFRS10的表达逐渐减弱;总的Lipin 1和细胞质中Lipin1-β表达量均逐渐增多,而细胞核中Lipin1-α表达逐渐减少(F值分别为64.79,74.46,97.11,71.74,57.62,P均0.01)。1.3油红O染色法观察小鼠AML-12细胞内的脂变:如Fig.5所示,正常对照组(0m M组)AML-12细胞内未见脂肪变性,40m M浓度酒精刺激时,AML-12细胞内可见少量橘红色脂滴,120m M浓度酒精刺激时,AML-12细胞内可见较多的橘红色脂滴。1.4免疫荧光染色法观察小鼠AML-12细胞中Lipin 1蛋白的细胞定位:如Fig.6所示,正常对照组示Lipin 1蛋白主要位于细胞质,细胞核也表达少量的Lipin 1蛋白。随着酒精浓度的增加,细胞质中Lipin 1蛋白表达逐渐增多,细胞核中的Lipin 1蛋白表达逐渐减少。2敲除小鼠AML-12细胞中SIRT1基因,各个指标m RNA和蛋白表达变化及细胞病理学改变2.1敲除SIRT1基因通过RT-q PCR法检测SIRT1、SFRS10、总Lipin 1、Lipin1-β及Lipin1-αm RNA的表达:如Fig.7及Table 5所示,正常AML-12细胞中表达SIRT1、SFRS10较多,总的Lipin 1、细胞质Lipin1-β及细胞核Lipin1-α表达均较少,敲除SIRT1基因,SIRT1si RNA组与空白对照组比较,SIRT1的表达降低约42%的同时SFRS10的表达明显减少,总的Lipin 1和细胞质中Lipin1-β表达量明显增多,而细胞核中Lipin1-α表达明显减少(P均0.01);在敲除SIRT1基因并加入酒精刺激后,SIRT1、SFRS10、Lipin1-α表达进一步减少,总Lipin 1、细胞质Lipin1-β的表达进一步增加,差异均有统计学意义(P0.05或P0.01);SIRT1si RNA阴性对照组和空白对照组比较,各个指标表达的变化,差异均无统计学意义(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组与空白对照组比较,Lipin1-β/α的比值明显增高,差异有统计学意义(P0.01)。在敲除SIRT1基因并加入酒精刺激后,Lipin1-β/α的比值进一步升高,SIRT1si RNA+Ethanol组与SIRT1si RNA组比较,差异有统计学意义(P0.01)。2.2敲除SIRT1基因通过Western blot法检测SIRT1、SFRS10、总Lipin 1、Lipin1-β及Lipin1-α的蛋白水平表达变化:如Fig.10及Table 7所示,正常AML-12细胞中表达SIRT1、SFRS10较多,总Lipin 1、细胞质Lipin1-β及细胞核Lipin1-α表达均较少,敲除SIRT1基因,SIRT1si RNA组与空白对照组比较,SIRT1的表达降低45%的同时SFRS10的表达明显减少,总的Lipin 1和细胞质中Lipin1-β表达明显增多,而细胞核中Lipin1-α表达明显减少(P均0.01);在敲除SIRT1基因并加入酒精刺激后,SIRT1、SFRS10、Lipin1-α表达进一步减少,总Lipin 1、细胞质Lipin1-β的表达进一步增加,SIRT1si RNA+Ethanol组与SIRT1si RNA组比较,差异均有统计学意义(P均0.01)。而各个指标中SIRT1si RNA阴性对照组和空白对照组比较,差异均无统计学意义(P均0.05)。2.3油红O染色法观察小鼠AML-12细胞内的脂变:如Fig.11所示,油红O染色示正常对照组及阴性对照组中AML-12细胞内未见脂肪变性,敲除SIRT1基因,AML-12细胞内可见较多的橘红色脂滴,敲除SIRT1基因并加入120m M浓度酒精刺激,AML-12细胞内可见大量的橘红色脂滴。2.4免疫荧光染色法观察小鼠AML-12细胞中Lipin 1蛋白的细胞定位:如Fig.12所示,正常对照组及阴性对照组示Lipin 1蛋白主要位于细胞质,细胞核也表达少量Lipin 1蛋白。敲除SIRT1基因,细胞质中的Lipin 1蛋白表达明显增多,细胞核中的Lipin 1蛋白表达明显减少。敲除SIRT1基因并加入120m M浓度酒精刺激,细胞质中的Lipin 1蛋白表达进一步增多,细胞核中的Lipin 1蛋白表达进一步减少。结论:SIRT1上游调节着SFRS10、Lipin 1的表达,这可能为酒精性脂肪性肝病发生的重要机制之一。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575

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