目的:观察高迁移率族蛋白1(high-mobility group box 1,HMGB1)对急性髓系白血病细胞株(human leukemiacell line,K562)自噬及化学治疗(化疗)耐药的影响,并探讨其相关的分子机制。方法:体外培养K562细胞,分为化疗药物处理组、化疗药物处...目的:观察高迁移率族蛋白1(high-mobility group box 1,HMGB1)对急性髓系白血病细胞株(human leukemiacell line,K562)自噬及化学治疗(化疗)耐药的影响,并探讨其相关的分子机制。方法:体外培养K562细胞,分为化疗药物处理组、化疗药物处理对照组、HMGB1纯化蛋白预处理组、HMGB1纯化蛋白预处理对照组、HMGB1si RNA转染组和HMGB1 si RNA转染对照组,其中化疗药物处理组又分为长春新碱(v incristine,VCR)、足叶乙甙(etoposide,VP-16)、阿糖胞苷(cytosine arabinoside,Ara-C)、阿霉素(adriamycin,ADM)和三氧化二砷(arsenic trioxide,As2O3)处理组。细胞计数试剂盒-8检测细胞活性;Western印迹检测HMGB1,微管相关蛋白1轻链3(microtubule-associate protein1light chain3,LC3),腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)和哺乳动物雷帕霉素靶点(mammalian target of rapamycin,m-TOR)磷酸化蛋白表达水平;ELISA法检测细胞上清液HMGB1蛋白含量;单丹磺酰尸胺染色和透射电镜观察细胞自噬状态。结果:与相应对照组比较,各化疗药物处理组(VCR,VP-16,Ara-C,ADM和As2O3)的细胞活性均显著降低,HMGB1蛋白表达均显著上调(均P<0.05)。与相应对照组比较,HMGB1纯化蛋白预处理组的细胞活性显著增加(P<0.05),HMGB1蛋白表达显著上调(P<0.05)。与HMGB si RNA转染对照组比较,HMGB1si RNA转染组的细胞活性显著降低(P<0.05),HMGB1蛋白表达显著下调(P<0.05);与相应对照组比较,HMGB1纯化蛋白处理组中LC3-II表达明显增强(P<0.05),光镜下观察到自噬小体和自噬泡数量明显增加。同时,与相应对照组比较,HMGB1纯化蛋白处理组p-AMPKa的蛋白表达明显增强,而p-m TOR表达明显减弱(均P<0.05)。结论:HMGB1可能通过AMPK/m-TOR信号通路增强K562细胞自噬发挥化疗耐药性。展开更多
Background Endothelial dysfunction is a key event in the onset and progression of atherosclerosis in diabetic patients. Apoptosis may lead to endothelial dysfunction and contribute to vascular complications. However, ...Background Endothelial dysfunction is a key event in the onset and progression of atherosclerosis in diabetic patients. Apoptosis may lead to endothelial dysfunction and contribute to vascular complications. However, no study has addressed apoptosis in human umbilical vein endothelial cells （HUVECs） induced by an intermittent high-glucose media and its association with adiponectin receptor 1 （adipoR1）, adipoR2, or adenosine monophosphate （AMP）-activated protein kinase （AMPK）. Methods HUVECs were cultured in continuous normal glucose （5.5 mmol/L）, continuous high glucose （25 mmol/L）, alternating normal and high glucose and mannitol. In the alternating normal and high-glucose media, HUVECs were treated under different conditions. First, cells were transfected with the adipoRl-specific small-interfering RNA （siRNA） and then stimulated with globular adiponectin （gAD）. Second, cells were cultured in both gAD and the AMPK activator 5-aminoimidazole-4-carboxamide-l-13-D-ribofuranoside （AICAR）. Third, cells were cultured in the AMPK inhibitor adenine-9-13-D-arabino-furanoside （araA）, gAD, and in AICAR. Results HUVEC apoptosis increased more significantly in an intermittent high-glucose medium than in a constant high-glucose medium. HUVEC apoptosis induced by an intermittent high-glucose medium was inhibited when the cells were pretreated with 3 pg/ml gAD, which rapidly activated AMPK and adipoR1 in HUVECs. However, adipoR2 was not activated. Conclusions We found that adipoR1, not adipoR2, is involved in mediating intermittent high-concentration glucose- evoked apoptosis in endothelial cells, gAD activated AMPK through adipoR1, leads to the partial inhibition of HUVEC apoptosis. A fluctuating glucose medium is more harmful than a constant high-glucose medium to endothelial cells.展开更多
文摘目的:观察高迁移率族蛋白1(high-mobility group box 1,HMGB1)对急性髓系白血病细胞株(human leukemiacell line,K562)自噬及化学治疗(化疗)耐药的影响,并探讨其相关的分子机制。方法:体外培养K562细胞,分为化疗药物处理组、化疗药物处理对照组、HMGB1纯化蛋白预处理组、HMGB1纯化蛋白预处理对照组、HMGB1si RNA转染组和HMGB1 si RNA转染对照组,其中化疗药物处理组又分为长春新碱(v incristine,VCR)、足叶乙甙(etoposide,VP-16)、阿糖胞苷(cytosine arabinoside,Ara-C)、阿霉素(adriamycin,ADM)和三氧化二砷(arsenic trioxide,As2O3)处理组。细胞计数试剂盒-8检测细胞活性;Western印迹检测HMGB1,微管相关蛋白1轻链3(microtubule-associate protein1light chain3,LC3),腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)和哺乳动物雷帕霉素靶点(mammalian target of rapamycin,m-TOR)磷酸化蛋白表达水平;ELISA法检测细胞上清液HMGB1蛋白含量;单丹磺酰尸胺染色和透射电镜观察细胞自噬状态。结果:与相应对照组比较,各化疗药物处理组(VCR,VP-16,Ara-C,ADM和As2O3)的细胞活性均显著降低,HMGB1蛋白表达均显著上调(均P<0.05)。与相应对照组比较,HMGB1纯化蛋白预处理组的细胞活性显著增加(P<0.05),HMGB1蛋白表达显著上调(P<0.05)。与HMGB si RNA转染对照组比较,HMGB1si RNA转染组的细胞活性显著降低(P<0.05),HMGB1蛋白表达显著下调(P<0.05);与相应对照组比较,HMGB1纯化蛋白处理组中LC3-II表达明显增强(P<0.05),光镜下观察到自噬小体和自噬泡数量明显增加。同时,与相应对照组比较,HMGB1纯化蛋白处理组p-AMPKa的蛋白表达明显增强,而p-m TOR表达明显减弱(均P<0.05)。结论:HMGB1可能通过AMPK/m-TOR信号通路增强K562细胞自噬发挥化疗耐药性。
文摘Background Endothelial dysfunction is a key event in the onset and progression of atherosclerosis in diabetic patients. Apoptosis may lead to endothelial dysfunction and contribute to vascular complications. However, no study has addressed apoptosis in human umbilical vein endothelial cells （HUVECs） induced by an intermittent high-glucose media and its association with adiponectin receptor 1 （adipoR1）, adipoR2, or adenosine monophosphate （AMP）-activated protein kinase （AMPK）. Methods HUVECs were cultured in continuous normal glucose （5.5 mmol/L）, continuous high glucose （25 mmol/L）, alternating normal and high glucose and mannitol. In the alternating normal and high-glucose media, HUVECs were treated under different conditions. First, cells were transfected with the adipoRl-specific small-interfering RNA （siRNA） and then stimulated with globular adiponectin （gAD）. Second, cells were cultured in both gAD and the AMPK activator 5-aminoimidazole-4-carboxamide-l-13-D-ribofuranoside （AICAR）. Third, cells were cultured in the AMPK inhibitor adenine-9-13-D-arabino-furanoside （araA）, gAD, and in AICAR. Results HUVEC apoptosis increased more significantly in an intermittent high-glucose medium than in a constant high-glucose medium. HUVEC apoptosis induced by an intermittent high-glucose medium was inhibited when the cells were pretreated with 3 pg/ml gAD, which rapidly activated AMPK and adipoR1 in HUVECs. However, adipoR2 was not activated. Conclusions We found that adipoR1, not adipoR2, is involved in mediating intermittent high-concentration glucose- evoked apoptosis in endothelial cells, gAD activated AMPK through adipoR1, leads to the partial inhibition of HUVEC apoptosis. A fluctuating glucose medium is more harmful than a constant high-glucose medium to endothelial cells.
