目的:探讨转录因子腺病毒E4启动子结合蛋白(adenovirus E4 promoter-binding protein,E4BP4)通过腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)-转化生长因子(transforming growth factor,TGF)-β1/SMAD...目的:探讨转录因子腺病毒E4启动子结合蛋白(adenovirus E4 promoter-binding protein,E4BP4)通过腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)-转化生长因子(transforming growth factor,TGF)-β1/SMAD同源物3(Smad homolog 3,SMAD3)通路在调控病理性心肌纤维化的作用。方法:建立小鼠心脏纤维化模型,分别于模型组和假手术组中检测E4BP4的表达差异。分离和培养原代心脏成纤维细胞,血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)刺激增殖活化,分别转染E4BP4过表达质粒(Ang Ⅱ+E4BP4组)、E4BP4干扰质粒(Ang Ⅱ+siE4BP4组)、Ang Ⅱ组和未经Ang Ⅱ处理的对照组。免疫荧光检测α-肌动蛋白(α-smooth muscle actin,α-SMA)荧光强度,细胞增殖检测试剂盒测定细胞活力,聚合酶链式反应检测E4BP4、α-SMA、Ⅰ型胶原蛋白(collagen type Ⅰ,Collagen Ⅰ)和Ⅲ型胶原蛋白(collagen type Ⅲ,Collagen Ⅲ)的表达,蛋白质印迹检测TGF-β1、AMPK和SMAD3的蛋白表达。结果:与假手术组比较,模型组心肌纤维化程度(38.46±1.21 vs. 3.39±0.39,t=-78.564,P=0.000)、E4BP4蛋白表达量(0.96±0.03 vs. 0.75±0.03,t=-11.480,P=0.000)均明显增加。体外实验发现,与Ang Ⅱ+E4BP4组比较,Ang Ⅱ+siE4BP4组在平均荧光强度(0.05±0.01 vs. 0.42±0.03,F=677.591,P=0.000)、细胞活力(91.30±2.39vs.123.74±2.60,F=132.696,P=0.000)、α-SMA(1.26±0.09vs.3.59±0.86,F=52.274,P=0.000)、Collagen Ⅰ(1.16±0.11vs.3.79±0.89,F=55.336,P=0.000)、Collagen Ⅲ(1.23±0.13 vs. 2.92±0.36,F=119.929,P=0.000)、TGF-β1(0.66±0.04 vs. 0.96±0.02,F=142.954,P=0.000)和p-SMAD3/SMAD3(0.81±0.03 vs. 1.37±0.02,F=739.609,P=0.000)的水平明显降低,而p-AMPK/AMPK的表达量在Ang Ⅱ+siE4BP4组明显高于Ang Ⅱ+E4BP4组(0.89±0.01 vs. 0.58±0.02,F=284.541,P=0.000)。结论:E4BP4是纤维化调控的关键因子,抑制其表达可通过激活AMPK进而抑制TGF-β1/SMAD3通路发挥抗纤维化作用。展开更多
目的:观察P38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)抑制剂SB203580对TNF-α诱导的原代培养大鼠小胶质细胞高迁移率族蛋白B1(high mobility group protein B1,HMGB1)表达的影响。方法:采用原代培养大鼠小...目的:观察P38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)抑制剂SB203580对TNF-α诱导的原代培养大鼠小胶质细胞高迁移率族蛋白B1(high mobility group protein B1,HMGB1)表达的影响。方法:采用原代培养大鼠小胶质细胞,设立对照组、TNF-α组(25 ng/m L)、TNF-α(25 ng/m L)+SB 203580组(10μmol/L)、SB203580组(10μmol/L),各组细胞经药物处理16 h后分别用ELISA法检测细胞培养上清中HMGB1的表达情况,Western印迹检测细胞HMGB1和磷酸化p38MAPK表达情况,用反转录PCR检测HMGB1 m RNA表达。结果:经TNF-α刺激后,大鼠小胶质细胞及其培养上清液中HMGB1蛋白表达增高;SB 203580可抑制TNF-α诱导的磷酸化p38 MAPK表达(P<0.01),同时下调HMGB1m RNA和HMGB1蛋白的表达。结论:TNF-α可诱导小胶质细胞晚期炎症因子HMGB1的表达,并且其诱导机制与p38 MAPK被快速激活相关。展开更多
Atherosclerosis is a chronic inflammatory disease in arteries. Nuclear factor-kappa B (NF-κB),a redox-sensitive transcription factor,has been indicated to play a critical role in both initiation and expansion of athe...Atherosclerosis is a chronic inflammatory disease in arteries. Nuclear factor-kappa B (NF-κB),a redox-sensitive transcription factor,has been indicated to play a critical role in both initiation and expansion of atherosclerosis. Activation of NF-κB activates a variety of target genes that promote inflammation,such as leukocyte adhesion molecules,cytokines,and chemokines,as well as genes that regulate cell proliferation and differentiation. Multiple risk factors relevant to atherosclerosis including hypertension,diabetes mellitus,smoking and lipoprotein disorders also activate NF-κB. Thus the activation of NF-κB may be linked to the onset of atherosclerosis.展开更多
探讨信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)在体内是否存在相互作用,并观察其作用如何影响肿瘤坏死因子-α(tumor necrosis...探讨信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)在体内是否存在相互作用,并观察其作用如何影响肿瘤坏死因子-α(tumor necrosis factor,TNF-α)的转录活性.采用聚合酶链反应技术,从人Flag-p38和Flag-细胞外信号蛋白调节激酶2(extracellular-signal regulated protein kinase 2,ERK2)中扩增出p38和ERK2基因,将其插入载体pcDNA3-HA中;用Westernblot方法在293T细胞中检测其表达后应用免疫共沉淀技术检测STAT3蛋白与p38/ERK2蛋白之间是否存在相互作用.然后应用报告基因技术检测这种相互作用如何影响TNF-α的转录表达,并在应用RNA干扰技术将STAT3通路抑制后,观察TNF-α启动子转录活性如何变化.酶切和测序结果表明,扩增的p38和ERK2基因序列正确,大小为1080bp,在293T细胞中正确表达大小约40ku的p38和ERK2蛋白.免疫共沉淀实验结果证实,p38和STAT3蛋白以及ERK2和STAT3蛋白在体内相互作用.下游基因TNF-α荧光素酶活性实验显示,p38和STAT3蛋白以及ERK2和STAT3蛋白复合物协同升高TNF-α的活性,应用STAT3的干扰RNA后其活性则明显下降.该研究表明,STAT3和p38/ERK2蛋白在体内存在相互作用,STAT3与p38、STAT3与ERK2均对TNF-α的表达发挥协同效应,在阻断STAT3通路后,STAT3与p38、STAT3与ERK2对TNF-α表达的协同效应将明显降低.展开更多
文摘目的:探讨转录因子腺病毒E4启动子结合蛋白(adenovirus E4 promoter-binding protein,E4BP4)通过腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)-转化生长因子(transforming growth factor,TGF)-β1/SMAD同源物3(Smad homolog 3,SMAD3)通路在调控病理性心肌纤维化的作用。方法:建立小鼠心脏纤维化模型,分别于模型组和假手术组中检测E4BP4的表达差异。分离和培养原代心脏成纤维细胞,血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)刺激增殖活化,分别转染E4BP4过表达质粒(Ang Ⅱ+E4BP4组)、E4BP4干扰质粒(Ang Ⅱ+siE4BP4组)、Ang Ⅱ组和未经Ang Ⅱ处理的对照组。免疫荧光检测α-肌动蛋白(α-smooth muscle actin,α-SMA)荧光强度,细胞增殖检测试剂盒测定细胞活力,聚合酶链式反应检测E4BP4、α-SMA、Ⅰ型胶原蛋白(collagen type Ⅰ,Collagen Ⅰ)和Ⅲ型胶原蛋白(collagen type Ⅲ,Collagen Ⅲ)的表达,蛋白质印迹检测TGF-β1、AMPK和SMAD3的蛋白表达。结果:与假手术组比较,模型组心肌纤维化程度(38.46±1.21 vs. 3.39±0.39,t=-78.564,P=0.000)、E4BP4蛋白表达量(0.96±0.03 vs. 0.75±0.03,t=-11.480,P=0.000)均明显增加。体外实验发现,与Ang Ⅱ+E4BP4组比较,Ang Ⅱ+siE4BP4组在平均荧光强度(0.05±0.01 vs. 0.42±0.03,F=677.591,P=0.000)、细胞活力(91.30±2.39vs.123.74±2.60,F=132.696,P=0.000)、α-SMA(1.26±0.09vs.3.59±0.86,F=52.274,P=0.000)、Collagen Ⅰ(1.16±0.11vs.3.79±0.89,F=55.336,P=0.000)、Collagen Ⅲ(1.23±0.13 vs. 2.92±0.36,F=119.929,P=0.000)、TGF-β1(0.66±0.04 vs. 0.96±0.02,F=142.954,P=0.000)和p-SMAD3/SMAD3(0.81±0.03 vs. 1.37±0.02,F=739.609,P=0.000)的水平明显降低,而p-AMPK/AMPK的表达量在Ang Ⅱ+siE4BP4组明显高于Ang Ⅱ+E4BP4组(0.89±0.01 vs. 0.58±0.02,F=284.541,P=0.000)。结论:E4BP4是纤维化调控的关键因子,抑制其表达可通过激活AMPK进而抑制TGF-β1/SMAD3通路发挥抗纤维化作用。
文摘Atherosclerosis is a chronic inflammatory disease in arteries. Nuclear factor-kappa B (NF-κB),a redox-sensitive transcription factor,has been indicated to play a critical role in both initiation and expansion of atherosclerosis. Activation of NF-κB activates a variety of target genes that promote inflammation,such as leukocyte adhesion molecules,cytokines,and chemokines,as well as genes that regulate cell proliferation and differentiation. Multiple risk factors relevant to atherosclerosis including hypertension,diabetes mellitus,smoking and lipoprotein disorders also activate NF-κB. Thus the activation of NF-κB may be linked to the onset of atherosclerosis.
文摘探讨信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)在体内是否存在相互作用,并观察其作用如何影响肿瘤坏死因子-α(tumor necrosis factor,TNF-α)的转录活性.采用聚合酶链反应技术,从人Flag-p38和Flag-细胞外信号蛋白调节激酶2(extracellular-signal regulated protein kinase 2,ERK2)中扩增出p38和ERK2基因,将其插入载体pcDNA3-HA中;用Westernblot方法在293T细胞中检测其表达后应用免疫共沉淀技术检测STAT3蛋白与p38/ERK2蛋白之间是否存在相互作用.然后应用报告基因技术检测这种相互作用如何影响TNF-α的转录表达,并在应用RNA干扰技术将STAT3通路抑制后,观察TNF-α启动子转录活性如何变化.酶切和测序结果表明,扩增的p38和ERK2基因序列正确,大小为1080bp,在293T细胞中正确表达大小约40ku的p38和ERK2蛋白.免疫共沉淀实验结果证实,p38和STAT3蛋白以及ERK2和STAT3蛋白在体内相互作用.下游基因TNF-α荧光素酶活性实验显示,p38和STAT3蛋白以及ERK2和STAT3蛋白复合物协同升高TNF-α的活性,应用STAT3的干扰RNA后其活性则明显下降.该研究表明,STAT3和p38/ERK2蛋白在体内存在相互作用,STAT3与p38、STAT3与ERK2均对TNF-α的表达发挥协同效应,在阻断STAT3通路后,STAT3与p38、STAT3与ERK2对TNF-α表达的协同效应将明显降低.
