目的观察高氧对人肺泡上皮细胞(HPAEC)凋亡的影响,并探讨其机制。方法取对数生长期HPAEC细胞,随机分为高氧组和对照组。对照组细胞不作处理,放置于50 m L/L CO2培养箱中培养;高氧组细胞换液1次后,以3 L/min速度通入含900 m L/L O2和50 m...目的观察高氧对人肺泡上皮细胞(HPAEC)凋亡的影响,并探讨其机制。方法取对数生长期HPAEC细胞,随机分为高氧组和对照组。对照组细胞不作处理,放置于50 m L/L CO2培养箱中培养;高氧组细胞换液1次后,以3 L/min速度通入含900 m L/L O2和50 m L/L CO2高纯混合气,通入时间10 min;两组细胞均培养24 h。采用流式细胞仪检测细胞凋亡率,倒置相差显微镜下观察细胞形态学变化,免疫共聚焦检测细胞组蛋白去乙酰化酶(SIRT1)转位情况,Western blotting法检测细胞SENP1蛋白、乙酰化p53蛋白。结果高氧组和对照组细胞凋亡率分别为24.77%±2.17%、5.33%±2.60%,两组比较,P<0.05。高氧组细胞从正常的长梭形、多角形变成圆形、椭圆形,细胞间的间隙增宽,悬浮的细胞较多;对照组细胞大多为长梭形、多角形,贴壁较好,细胞间的间隙较小,悬浮的细胞较少。高氧组和对照组细胞SIRT1蛋白转位率分别为88.89%、16.23%,两组比较,P<0.05。高氧组和对照组细胞SENP1蛋白相对表达量分别为0.76±0.12、0.67±0.02,乙酰化p53蛋白相对表达量分别为0.81±0.07、0.52±0.03,两组比较,P均<0.05。结论高氧可促进HPAEC细胞凋亡,其机制可能是高氧诱导HPAEC细胞SENP1蛋白表达增加,进而SIRT1蛋白从胞核转位到胞质,使乙酰化p53蛋白表达增加。展开更多
目的探讨miRNA-20a在脂多糖(lipopolysaccharide,LPS)诱导的A549细胞焦亡与炎症反应中的作用与调控机制。方法培养人肺泡上皮A549细胞,以LPS为刺激物建立细胞焦亡及炎症反应模型,给予LPS+三磷酸腺苷刺激为LPS组,未给予刺激为空白对照组...目的探讨miRNA-20a在脂多糖(lipopolysaccharide,LPS)诱导的A549细胞焦亡与炎症反应中的作用与调控机制。方法培养人肺泡上皮A549细胞,以LPS为刺激物建立细胞焦亡及炎症反应模型,给予LPS+三磷酸腺苷刺激为LPS组,未给予刺激为空白对照组,验证模型是否成功。将A549细胞根据转染物质分为miRNA-20a模拟物(mimics)组、mimics-阴性对照(negative control,NC)组、miRNA-20a抑制物(inhibitor)组和inhibitor-NC组,构建过表达及沉默miRNA-20a的A549细胞模型,各组细胞在转染24 h后给予LPS+三磷酸腺苷刺激。构建TLR4-3'UTR双荧光素酶报告基因载体质粒,包括野生型(WT)载体质粒TLR4-3'UTR-WT1、TLR4-3'UTR-WT2及相应的变异型(MUT)载体质粒TLR4-3'UTR-MUT1、TLR4-3'UTR-MUT2,用双荧光素酶报告基因实验验证miRNA-20a的靶基因。采用实时荧光定量聚合酶链反应、免疫印迹试验及酶联免疫吸附试验检测各组焦亡标志因子消皮素D(gsdermin D,GSDMD),炎症因子凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、半胱氨酸天冬氨酸蛋白酶1(cysteinyl aspartate specific proteinase-1,Caspase-1)、白细胞介素1β(interleukin-1β,IL-1β),以及信号分子Toll样受体4(Toll-like receptor-4,TLR4)、核因子κB(nuclear factor-κB,NF-κB)mRNA及蛋白表达。免疫荧光检测各组细胞TLR4表达和NF-κB入核情况。结果LPS组A549细胞GSDMD、ASC、NLRP3、Caspase-1、IL-1βmRNA和蛋白表达量均高于空白对照组(P<0.05),模型建立成功。mimics组miRNA-20a表达量高于mimic-NC组(P<0.05),inhibitor组miRNA-20a表达量低于inhibitor-NC组(P<0.01)。双荧光素酶报告基因实验显示,TLR4-3'UTR-WT与miRNA-20a mimics共转染组的相对荧光值低于TLR4-3'UTR-WT与mimics-NC共转染组(P<0.05)。mimics组LPS介导的A549细胞GSDMD、ASC、NLRP3、Caspase-1、IL-1β、TLR4、NF-κB 展开更多
Objective:To investigate the effects of mechanical stretching and lipopolysaccharide(LPS) on the early apoptosis and IL-8 production of alveolar epithelial type II cells A549.Methods:The experimental matrix consisted ...Objective:To investigate the effects of mechanical stretching and lipopolysaccharide(LPS) on the early apoptosis and IL-8 production of alveolar epithelial type II cells A549.Methods:The experimental matrix consisted of three integrated studies.In the first study,A549 cells were subjected to different stretching strain frequency and duration time to see the effects on the early apoptosis.In the second study,A549 cells were subjected to mechanical stretch(13%4 h, 0.3 Hz) and LPS(1 or 100 ng/mL) to see whether mechanical strain and LPS also have an addictive effect on the early apoptosis.In the third study to investigate whether this addictive effect could be induced by LPS and mechanical stretch on IL-8 production,A549 cells were subjected to LPS(100 ng/mL) and mechanical strain(13%.0.3 Hz,4 h).Real time PCR and enzyme linked immunosorbent assay were used to measure mRN A and protein level of IL-8.The early apoptosis was detected by flow cytometry.Results:Mechanical stretch induced the early apoptosis in a force and frequency and time-dependent manner.In the presence of LPS,mechanical stretch enhanced LPS-induced early apoptosis,especially in 100 ng/mL IPS group compared with 1 ng/ mL LPS and the control group.Mechanical stretch increased IL-8 production and enhanced LPS-induced IL-8 screation both in mRNA and protein levels.Conclusions:Mechanical stretch can induce the early apoptosis and IL-8 secretion.Mechanical stretch and LPS have an addictive effect on the early apoptosis and IL-8 production in alveolar type 2 cells,which is one of the mechanisms of ventilator-induced lung injury.展开更多
文摘目的观察高氧对人肺泡上皮细胞(HPAEC)凋亡的影响,并探讨其机制。方法取对数生长期HPAEC细胞,随机分为高氧组和对照组。对照组细胞不作处理,放置于50 m L/L CO2培养箱中培养;高氧组细胞换液1次后,以3 L/min速度通入含900 m L/L O2和50 m L/L CO2高纯混合气,通入时间10 min;两组细胞均培养24 h。采用流式细胞仪检测细胞凋亡率,倒置相差显微镜下观察细胞形态学变化,免疫共聚焦检测细胞组蛋白去乙酰化酶(SIRT1)转位情况,Western blotting法检测细胞SENP1蛋白、乙酰化p53蛋白。结果高氧组和对照组细胞凋亡率分别为24.77%±2.17%、5.33%±2.60%,两组比较,P<0.05。高氧组细胞从正常的长梭形、多角形变成圆形、椭圆形,细胞间的间隙增宽,悬浮的细胞较多;对照组细胞大多为长梭形、多角形,贴壁较好,细胞间的间隙较小,悬浮的细胞较少。高氧组和对照组细胞SIRT1蛋白转位率分别为88.89%、16.23%,两组比较,P<0.05。高氧组和对照组细胞SENP1蛋白相对表达量分别为0.76±0.12、0.67±0.02,乙酰化p53蛋白相对表达量分别为0.81±0.07、0.52±0.03,两组比较,P均<0.05。结论高氧可促进HPAEC细胞凋亡,其机制可能是高氧诱导HPAEC细胞SENP1蛋白表达增加,进而SIRT1蛋白从胞核转位到胞质,使乙酰化p53蛋白表达增加。
文摘目的探讨miRNA-20a在脂多糖(lipopolysaccharide,LPS)诱导的A549细胞焦亡与炎症反应中的作用与调控机制。方法培养人肺泡上皮A549细胞,以LPS为刺激物建立细胞焦亡及炎症反应模型,给予LPS+三磷酸腺苷刺激为LPS组,未给予刺激为空白对照组,验证模型是否成功。将A549细胞根据转染物质分为miRNA-20a模拟物(mimics)组、mimics-阴性对照(negative control,NC)组、miRNA-20a抑制物(inhibitor)组和inhibitor-NC组,构建过表达及沉默miRNA-20a的A549细胞模型,各组细胞在转染24 h后给予LPS+三磷酸腺苷刺激。构建TLR4-3'UTR双荧光素酶报告基因载体质粒,包括野生型(WT)载体质粒TLR4-3'UTR-WT1、TLR4-3'UTR-WT2及相应的变异型(MUT)载体质粒TLR4-3'UTR-MUT1、TLR4-3'UTR-MUT2,用双荧光素酶报告基因实验验证miRNA-20a的靶基因。采用实时荧光定量聚合酶链反应、免疫印迹试验及酶联免疫吸附试验检测各组焦亡标志因子消皮素D(gsdermin D,GSDMD),炎症因子凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、半胱氨酸天冬氨酸蛋白酶1(cysteinyl aspartate specific proteinase-1,Caspase-1)、白细胞介素1β(interleukin-1β,IL-1β),以及信号分子Toll样受体4(Toll-like receptor-4,TLR4)、核因子κB(nuclear factor-κB,NF-κB)mRNA及蛋白表达。免疫荧光检测各组细胞TLR4表达和NF-κB入核情况。结果LPS组A549细胞GSDMD、ASC、NLRP3、Caspase-1、IL-1βmRNA和蛋白表达量均高于空白对照组(P<0.05),模型建立成功。mimics组miRNA-20a表达量高于mimic-NC组(P<0.05),inhibitor组miRNA-20a表达量低于inhibitor-NC组(P<0.01)。双荧光素酶报告基因实验显示,TLR4-3'UTR-WT与miRNA-20a mimics共转染组的相对荧光值低于TLR4-3'UTR-WT与mimics-NC共转染组(P<0.05)。mimics组LPS介导的A549细胞GSDMD、ASC、NLRP3、Caspase-1、IL-1β、TLR4、NF-κB
文摘Objective:To investigate the effects of mechanical stretching and lipopolysaccharide(LPS) on the early apoptosis and IL-8 production of alveolar epithelial type II cells A549.Methods:The experimental matrix consisted of three integrated studies.In the first study,A549 cells were subjected to different stretching strain frequency and duration time to see the effects on the early apoptosis.In the second study,A549 cells were subjected to mechanical stretch(13%4 h, 0.3 Hz) and LPS(1 or 100 ng/mL) to see whether mechanical strain and LPS also have an addictive effect on the early apoptosis.In the third study to investigate whether this addictive effect could be induced by LPS and mechanical stretch on IL-8 production,A549 cells were subjected to LPS(100 ng/mL) and mechanical strain(13%.0.3 Hz,4 h).Real time PCR and enzyme linked immunosorbent assay were used to measure mRN A and protein level of IL-8.The early apoptosis was detected by flow cytometry.Results:Mechanical stretch induced the early apoptosis in a force and frequency and time-dependent manner.In the presence of LPS,mechanical stretch enhanced LPS-induced early apoptosis,especially in 100 ng/mL IPS group compared with 1 ng/ mL LPS and the control group.Mechanical stretch increased IL-8 production and enhanced LPS-induced IL-8 screation both in mRNA and protein levels.Conclusions:Mechanical stretch can induce the early apoptosis and IL-8 secretion.Mechanical stretch and LPS have an addictive effect on the early apoptosis and IL-8 production in alveolar type 2 cells,which is one of the mechanisms of ventilator-induced lung injury.
