摘要
目的:观察肌内皮缝隙连接(MEGJ)是否通过传递环磷酸腺苷(cAMP)介导血管生成素2(Ang2)对血管平滑肌细胞(VSMCs)中诱导型一氧化氮合酶(iNOS)的表达和血管低反应性的调节。方法:在双面共培养系统中培养大鼠血管内皮细胞(VECs)和VSMCs,并予以RNA干扰和缺氧等处理;采用Western blot技术检测VSMCs中iNOS的表达;通过检测FITC标记的牛血清白蛋白的荧光透过率反映VSMCs收缩反应性;采用Alexa Fluor 488-c AMP为示踪剂,观察cAMP从VECs到VSMCs的跨MEGJ转运。结果:在单独培养的VECs和VSMCs中,缺氧及Ang2处理后cAMP浓度均显著增高(P<0.05),但在VECs和VSMCs中的水平并不相同,在VECs中的水平显著高于VSMCs中的水平(P<0.05)。在双面共培养系统中,VECs和VSMCs中cAMP的浓度差则明显降低,且VSMCs中增高的c AMP水平可被缝隙连接蛋白43(Cx43)小干扰RNA(siRNA)显著抑制(P<0.05)。外源性Ang2作用下缺氧后荧光标记的Alexa Fluor 488-cAMP发生明显的从VECs到VSMCs的转运,且可被Cx43 siRNA显著抑制(P<0.05)。在双面共培养系统的VECs和VSMCs侧给予c AMP抑制剂均可显著抑制外源性Ang2作用下缺氧VSMCs的i NOS高表达,改善VSMCs的收缩低反应性(P<0.05)。结论:缺氧后Ang2可能通过Cx43,促进c AMP经MEGJ传递进入VSMCs,导致VSMCs的iNOS高表达和收缩低反应性。
AIM:To observe the cyclic adenosine monophosphate(cAMP)transfer across myoendothelial gap junctions(MEGJ)in the regulatory effect of angiopoietin-2(Ang2)on hyporeactivity after hypoxia in vascular smooth muscle cells(VSMCs).METHODS:The double-sided cell co-culture model of vascular endothelial cells(VECs)and VSMCs was set up.The protein expression of inducible nitric oxide synthase(iNOS)was determined by Western blot.The contraction of VSMCs was detected via the leakage of FITC-labeled bovine serum albumin.Alexa Fluor488-cAMP was used as the tracer to observe the cAMP transfer across MEGJ from VECs to VSMCs.RESULTS:In cultured VECs and VSMCs alone,the cAMP concentrations were both significantly increased after exogenous Ang2treatment and hypoxia,and more in VECs than that in VSMCs(P<0.05).In the double-sided cell co-culture model,the difference was weakened,and the increase in cAMP concentration in VSMCs after exogenous Ang2treatment and hypoxia was antagonized by connexin43(Cx43)small interfering RNA(siRNA)(P<0.05).Alexa Fluor488-cAMP in VECs transfered into VSMCs after exogenous Ang2treatment and hypoxia,which was also antagonized by Cx43siRNA(P<0.05).The cAMP antagonist inhibited the protein expression of iNOS in the VSMCs and the hyporeactivity of the VSMCs after exogenous Ang2treatment and hypoxia(P<0.05).CONCLUSION:Ang2may regulate the protein expression of iNOS in VSMCs and the hyporeactivity of VSMCs after hypoxia through the cAMP transfer across MEGJ.
作者
徐竞
杨光明
李涛
刘良明
XU Jing;YANG Guangming;LI Tao;LIU Liangming(State Key Laboratory of Trauma, Burns and Combined Injury, Department 2, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042 , China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第1期41-46,共6页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81370426)
重庆市自然科学基金资助项目(No.stc2013jcyjA10128)
