血管内皮生长因子165对HEK293细胞KCNQ1/KCNE1电流的影响及其机制研究
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摘要
目的探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)165对KCNQ1/KCNE1电流的影响及其可能机制。方法 HEK293细胞分为对照组、KCNQ1/KCNE1/KDR组和KCNQ1/KDR组,对照组细胞正常培养、不做处理,KCNQ1/KCNE1/KDR组细胞转染含KCNQ1、KCNE1和KDR基因的质粒,KCNQ1/KDR组细胞转染含KCNQ1和KDR基因的质粒,转染后48h,采用PCR法检测转染是否成功。KCNQ1/KCNE1/KDR组和KCNQ1/KDR组HEK293细胞加入100μg/L VEGF165处理10min,采用全细胞膜片钳技术记录处理前、后2组标准化激活电流和标准化尾电流的变化情况,并进行比较。结果 KCNQ1/KCNE1/KDR组和KCNQ1/KDR组细胞均成功转染;-20、0、20、40、60mV电压下,KCNQ1/KCNE1/KDR组处理后标准化激活电流(0.045±0.050、0.166±0.060、0.342±0.050、0.551±0.080、0.742±0.100)和标准化尾电流(0.049±0.040、0.154±0.090、0.346±0.090、0.572±0.080、0.751±0.100)均明显低于处理前(0.081±0.070、0.238±0.100、0.459±0.090、0.758±0.580、1.000±0.000,0.069±0.040、0.245±0.080、0.509±0.090、0.787±0.060、1.000±0.000)(P<0.05);-20、0、20、40、60mV电压下,KCNQ1/KDR组处理后标准化激活电流和尾电流与处理前比较差异均无统计学意义(P>0.05)。结论VEGF165能直接抑制KCNQ1/KCNE1电流,该作用通过β亚基发挥作用,由VEGF受体-2介导。
Objective To explore the effect of vascular endothelial growth factor(VEGE)165 on KCNQ1/KCNE1 current of HEK293 cells and its possible mechanism.Methods HEK293 cells were divided into control group,KCNQ1/KCNE1/KDR group and KCNQ1/KDR group.Control group was cultured normally without treatment.KCNQ1/KCNE1/KDR group was transfected with plasmids containing KCNQ1,KCNE1 and KDR genes.KCNQ1/KDR group was transfected with plasmids containing KCNQ1 and KDR genes.PCR was used to determine whether the transfection was successful after 48 h.HEK293 cells in KCNQ1/KCNE1/KDR and KCNQ1/KDR groups were treated with 100μg/L VEGF165 for 10 min,and the whole cell patch clamp technique was used to record and compare the normalized activated current and the normalized tail current before and after treatment.Results HEK293 cells in KCNQ1/KCNE1/KDR and KCNQ1/KDR groups were transfected successfully.At-20,0,20,40 and 60 mV,the normalized activated currents(0.045±0.050,0.166±0.060,0.342±0.050,0.551±0.080,0.742±0.100)and the normalized tail currents(0.049±0.040,0.154±0.090,0.346±0.090,0.572±0.080,0.751±0.100)were significantly lower after treatment than those before treatment(0.081±0.070,0.238±0.100,0.459±0.090,0.758±0.580,1.000±0.000;0.069±0.040,0.245±0.080,0.509±0.090,0.787±0.060,1.000±0.000)in KCNQ1/KCNE1/KDR group(P< 0.05),and showed no significant differences before and after treatment in KCNQ1/KDR group(P>0.05).Conclusion VEGF165 can directly inhibit KCNQ1/KCNE1 current byβsubunit mediated by VEGF receptor-2.
Objective To explore the effect of vascular endothelial growth factor(VEGE)165 on KCNQ1/KCNE1 current of HEK293 cells and its possible mechanism.Methods HEK293 cells were divided into control group,KCNQ1/KCNE1/KDR group and KCNQ1/KDR group.Control group was cultured normally without treatment.KCNQ1/KCNE1/KDR group was transfected with plasmids containing KCNQ1,KCNE1 and KDR genes.KCNQ1/KDR group was transfected with plasmids containing KCNQ1 and KDR genes.PCR was used to determine whether the transfection was successful after 48 h.HEK293 cells in KCNQ1/KCNE1/KDR and KCNQ1/KDR groups were treated with 100μg/L VEGF165 for 10 min,and the whole cell patch clamp technique was used to record and compare the normalized activated current and the normalized tail current before and after treatment.Results HEK293 cells in KCNQ1/KCNE1/KDR and KCNQ1/KDR groups were transfected successfully.At-20,0,20,40 and 60 mV,the normalized activated currents(0.045±0.050,0.166±0.060,0.342±0.050,0.551±0.080,0.742±0.100)and the normalized tail currents(0.049±0.040,0.154±0.090,0.346±0.090,0.572±0.080,0.751±0.100)were significantly lower after treatment than those before treatment(0.081±0.070,0.238±0.100,0.459±0.090,0.758±0.580,1.000±0.000;0.069±0.040,0.245±0.080,0.509±0.090,0.787±0.060,1.000±0.000)in KCNQ1/KCNE1/KDR group(P< 0.05),and showed no significant differences before and after treatment in KCNQ1/KDR group(P>0.05).Conclusion VEGF165 can directly inhibit KCNQ1/KCNE1 current byβsubunit mediated by VEGF receptor-2.
引文
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[16]鞠晨晖,张双霞,王宪沛,等.KCa3.1离子通道及相关疾病研究进展[J].中华实用诊断与治疗杂志,2015,29(11):1041-1043.
[17]陈庆隆,朱咏瑶,周敏华.东莞地区汉族人群KCNJ11基因E23K位点多态性与2型糖尿病关系[J].中华实用诊断与治疗杂志,2015,29(12):1173-1175.
[2]姚娟,王清传,韩瑞梅,等.KCNE1基因多态性在新疆维吾尔族及汉族人群中分布特征[J].中华实用诊断与治疗杂志,2013,27(8):747-749.
[3]李寅辉,张婷婷,刘浩.KCNQ1、SRR基因多态性与维吾尔族及汉族2型糖尿病的相关性研究[J].中国糖尿病杂志,2018,26(7):534-542.
[4]LIN Z,XING W,GAO C,et al.Inhibitory effect of vascular endothelial growth factor on the slowly activating delayed rectifier potassium current in guinea pig ventricular myocytes[J].J Am Heart Assoc,2018,7(3):e007730.
[5]李小青,郭瑞娜,李晓丽,等.KCNE1胞外N段功能的研究[J].南通大学学报(医学版),2014,34(5):352-354.
[6]张赛圣,程丽霞,杨志宏.低剂量X线照射促进大鼠缺血皮瓣成活及与血管内皮生长因子和基质金属蛋白酶-9表达的关系[J].中华实用诊断与治疗杂志,2018,32(8):747-750.
[7]陈烨,李薇薇,于小蕊,等.渗出型年龄相关性黄斑变性患者血清新生血管调控细胞因子水平变化及意义[J].中华实用诊断与治疗杂志,2015,29(6):568-570.
[8]何皓,曲益萱.冠心病患者血管内皮生长因子+450C/G基因多态性研究[J].中华实用诊断与治疗杂志,2017,31(8):757-759.
[9]AZIMINEZHAD M.Vascular endothelial growth factor from embryonic status to cardiovascular pathology[J].Rep Biochem Mol Biol,2014,2(2):59-69.
[10]YANG J G,WANG L L,MA D C.Effects of vascular endothelial growth factors and their receptors on megakaryocytes and platelets and related diseases[J].Br JHaematol,2018,180(3):321-334.
[11]唐乾利,郭满,吴标良.血管内皮生长因子的研究现状与进展[J].中国烧伤创疡杂志,2017,29(2):77-87.
[12]王世进,李玉洁,申素芳,等.孕激素对子宫内膜电切术后EGFR、VEGF及KDR表达的影响[J].中华全科医学,2008,6(3):226-227.
[13]GARCIA L F,MATAVELI F D,MADER A M,et al.Cells involved in extracellular matrix remodeling after acute myocardial infarction[J].Einstein(Sao Paulo),2015,13(1):89-95.
[14]ZHU H,JIANG X,LI X,et al.Intramyocardial delivery of VEGF165 via a novel biodegradable hydrogel induces angiogenesis and improves cardiac function after rat myocardial infarction[J].Heart Vessels,2016,31(6):963-975.
[15]WU M,OBARA Y,NOROTA I,et al.Insulin suppresses IKs(KCNQ1/KCNE1)currents,which requireβ-subunit KCNE1[J].Pflugers Arch,2014,466(5):937-946.
[16]鞠晨晖,张双霞,王宪沛,等.KCa3.1离子通道及相关疾病研究进展[J].中华实用诊断与治疗杂志,2015,29(11):1041-1043.
[17]陈庆隆,朱咏瑶,周敏华.东莞地区汉族人群KCNJ11基因E23K位点多态性与2型糖尿病关系[J].中华实用诊断与治疗杂志,2015,29(12):1173-1175.