银杏叶提取物对同型半胱氨酸诱导HUVECs凋亡中JNK表达的影响(1)
摘 要:目的:研究银杏叶提取物(EGb)对同型半胱氨酸诱导人脐静脉内皮细胞(HUVECs)JNK表达的影响。方法:在人脐静脉内皮细胞培养基中加入不同浓度Hcy培养24h,以及加入EGb预处理再加入10.0mmol/L的Hcy作用2h。用TUNEL法检测HUVECs凋亡情况。用Western-Blot法检测不同组HUVECs表达P-JNK的蛋白含量。结果:用0.3mmol/L的Hcy干预后,HUVECs凋亡和JNK表达开始高于对照组(P<0.01),10.0mmol/L最显著(P<0.01),呈浓度依赖关系;而EGb两种浓度对HUVECs凋亡和JNK表达均有抑制作用,其中25.0μg/mL更显著。结论:Hcy能够诱导HUVECs凋亡和上调JNK的表达,而EGb具有明显抑制JNK的表达,这可能是Hcy促进动脉粥样硬化形成的重要机制之一,JNK信号通路可能是EGb保护作用机制之一。
关键词:同型半胱氨酸;内皮细胞;c-Jun氨基末端激酶;银杏叶提取物
中图分类号:R285.5 文献标识码:A 文章编号:1673-7717(2010)01-0176-04
, 百拇医药
The Effects of EGb in the Expression of JNK on Apoptosis of HUVECs Induced by Homocysteine
YE Xiaojun1,FENG Yan1,WANG Xiaotong2,HE Zhiyong2,HUANG Hanjin2
(1.Affiliated Hospital of Hangzhou Normal University,Hangzhou 310015,Zhejiang,China;
2.Second Affiliated Hospital of Wenzhou Medical College,Wenzhou 325027,Zhejiang,China)
Abstract:Objective: To explore the effects of EGb in the expression of JNK on apoptosis of HUVECs induced by homocysteine.Methods: The HUVECs were treated with the different concentration of Hcy for 24 hours. Before HUVECs were treated with 10.0mmol/L Hcy,they were treated with the concentrations of EGb mentioned above for 2 hours. Cell apoptosis was detected by TUNEL method.P-JNK was detected by western blot in different HUVECs.Results:After exporsure of theHUVECs to Hcy at the concentration of 0.3mmol/L(P<0.01),TUNEL-positive cells and the serection of P-JNK were higher than that of the control group. Those of 10.0mmol/L were the most significant group. They increased with a dose-dependent manner(P<0.01).Addition of two concentration of EGb can reduced the apoptosic cells induced by 10.0mmol/L Hcy (P<0.01),that was the phosphorylation levels of JNK.They were more significant at the concentration of 25.0μg/mL group(P<0.01).Conclusion:Homocysteine can induce apoptosis of HUVECs in vitro culture,upregulate the expression of JNK in HUVECs induced by Hcy,which may be associated with the pathogenesis of atherosclerosis. JNK signal pathway may be one of the protection roles by EGb.
, 百拇医药
Key words:homocysteine;endothelial cell;c-Jun-N-terminal kinase;atherosclerosis; extract of ginkgo biloba
近来研究表明血浆Hcy水平升高是冠心病、脑血管疾病以及外周阻塞性血管疾病等心脑血管疾病的一个重要独立危险因素[1]。有研究认为Hcy可以损伤血管内皮促进动脉粥样斑块形成[2];体外细胞培养显示Hcy能诱导内皮细胞凋亡[3],而目前有关Hcy诱导人脐静脉内皮细胞(HUVECs)凋亡的具体机理尚不明确。银杏叶提取物(extract of ginkgo biloba,EGb)是我国传统中药,具有较强的清除自由基、降低血液黏度、延长凝血时间等和抗凋亡的作用,目前关于EGb抗凋亡分子研究报道较少。本实验通过对HUVECs的培养,进一步验证Hcy诱导HUVECs的凋亡以及JNK的表达,并且应用EGb进行干预以及JNK表达的检测,初步探讨JNK信号通路是否为EGb抑制Hcy诱导HUVECs凋亡可能分子作用机制之一。
, http://www.100md.com
1 材料与方法
1.1 材料
人脐静脉内皮细胞细胞株(Human Umbilical Veins Endothelial Cells Line,HUVECs)购于上海坤肯特生物技术有限公司。同型半胱氨酸(美国Sigma公司)和RPMI1640培养基(美国GBICO公司),EGb(美国Biomol公司)与胰蛋白酶和EDTA混合液(美国GBICO公司),胎牛血清(杭州四季青生物工程材料有限公司),Tunel法凋亡检测试剂盒(美国Roche公司),兔抗人P-JNK1/2、JNK1/2多克隆抗体、辣根过氧化物酶(HRP)标记的羊抗兔二抗(美国Cell Singnal Technology公司),BCA蛋白定量试剂盒、增强的化学发光检测试剂盒(美国Piece公司)。
1.2 实验方法
人脐静脉内皮细胞的培养和传代方法见文献[4],实验采用3～5代的细胞。
将HUVECs按1×105/mL的密度接种于含有盖玻片的6孔板内进行爬片培养,用含10%FBS的RPMI1640培养液培养,待细胞长至盖玻片50%的时候,改换成用无血清的培养基继续培养24h,细胞饥饿24h,使细胞同步化于G0 期(细胞同步化)后,加入下列实验组:A组(正常对照组)、B组(Hcy 0.1mmol/L)、C组(Hcy 0.3mmol/L)、D组(Hcy 1.0mmol/L)、E组(Hcy 3.0mmol/L)、F组(Hcy 10.0mmol/L)、EGb1(Hcy 10.0mmol/L+EGb12.5μg/mL)组和EGb2(Hcy 10.0mmol/L+EGb25.0μg/mL)组,各组细胞设立3个复孔。, http://www.100md.com(叶小军 冯 燕 王小同 何志勇 黄汉津)
关键词:同型半胱氨酸;内皮细胞;c-Jun氨基末端激酶;银杏叶提取物
中图分类号:R285.5 文献标识码:A 文章编号:1673-7717(2010)01-0176-04
, 百拇医药
The Effects of EGb in the Expression of JNK on Apoptosis of HUVECs Induced by Homocysteine
YE Xiaojun1,FENG Yan1,WANG Xiaotong2,HE Zhiyong2,HUANG Hanjin2
(1.Affiliated Hospital of Hangzhou Normal University,Hangzhou 310015,Zhejiang,China;
2.Second Affiliated Hospital of Wenzhou Medical College,Wenzhou 325027,Zhejiang,China)
Abstract:Objective: To explore the effects of EGb in the expression of JNK on apoptosis of HUVECs induced by homocysteine.Methods: The HUVECs were treated with the different concentration of Hcy for 24 hours. Before HUVECs were treated with 10.0mmol/L Hcy,they were treated with the concentrations of EGb mentioned above for 2 hours. Cell apoptosis was detected by TUNEL method.P-JNK was detected by western blot in different HUVECs.Results:After exporsure of theHUVECs to Hcy at the concentration of 0.3mmol/L(P<0.01),TUNEL-positive cells and the serection of P-JNK were higher than that of the control group. Those of 10.0mmol/L were the most significant group. They increased with a dose-dependent manner(P<0.01).Addition of two concentration of EGb can reduced the apoptosic cells induced by 10.0mmol/L Hcy (P<0.01),that was the phosphorylation levels of JNK.They were more significant at the concentration of 25.0μg/mL group(P<0.01).Conclusion:Homocysteine can induce apoptosis of HUVECs in vitro culture,upregulate the expression of JNK in HUVECs induced by Hcy,which may be associated with the pathogenesis of atherosclerosis. JNK signal pathway may be one of the protection roles by EGb.
, 百拇医药
Key words:homocysteine;endothelial cell;c-Jun-N-terminal kinase;atherosclerosis; extract of ginkgo biloba
近来研究表明血浆Hcy水平升高是冠心病、脑血管疾病以及外周阻塞性血管疾病等心脑血管疾病的一个重要独立危险因素[1]。有研究认为Hcy可以损伤血管内皮促进动脉粥样斑块形成[2];体外细胞培养显示Hcy能诱导内皮细胞凋亡[3],而目前有关Hcy诱导人脐静脉内皮细胞(HUVECs)凋亡的具体机理尚不明确。银杏叶提取物(extract of ginkgo biloba,EGb)是我国传统中药,具有较强的清除自由基、降低血液黏度、延长凝血时间等和抗凋亡的作用,目前关于EGb抗凋亡分子研究报道较少。本实验通过对HUVECs的培养,进一步验证Hcy诱导HUVECs的凋亡以及JNK的表达,并且应用EGb进行干预以及JNK表达的检测,初步探讨JNK信号通路是否为EGb抑制Hcy诱导HUVECs凋亡可能分子作用机制之一。
, http://www.100md.com
1 材料与方法
1.1 材料
人脐静脉内皮细胞细胞株(Human Umbilical Veins Endothelial Cells Line,HUVECs)购于上海坤肯特生物技术有限公司。同型半胱氨酸(美国Sigma公司)和RPMI1640培养基(美国GBICO公司),EGb(美国Biomol公司)与胰蛋白酶和EDTA混合液(美国GBICO公司),胎牛血清(杭州四季青生物工程材料有限公司),Tunel法凋亡检测试剂盒(美国Roche公司),兔抗人P-JNK1/2、JNK1/2多克隆抗体、辣根过氧化物酶(HRP)标记的羊抗兔二抗(美国Cell Singnal Technology公司),BCA蛋白定量试剂盒、增强的化学发光检测试剂盒(美国Piece公司)。
1.2 实验方法
人脐静脉内皮细胞的培养和传代方法见文献[4],实验采用3～5代的细胞。
将HUVECs按1×105/mL的密度接种于含有盖玻片的6孔板内进行爬片培养,用含10%FBS的RPMI1640培养液培养,待细胞长至盖玻片50%的时候,改换成用无血清的培养基继续培养24h,细胞饥饿24h,使细胞同步化于G0 期(细胞同步化)后,加入下列实验组:A组(正常对照组)、B组(Hcy 0.1mmol/L)、C组(Hcy 0.3mmol/L)、D组(Hcy 1.0mmol/L)、E组(Hcy 3.0mmol/L)、F组(Hcy 10.0mmol/L)、EGb1(Hcy 10.0mmol/L+EGb12.5μg/mL)组和EGb2(Hcy 10.0mmol/L+EGb25.0μg/mL)组,各组细胞设立3个复孔。, http://www.100md.com(叶小军 冯 燕 王小同 何志勇 黄汉津)