摘要
目的:探讨缺血缺氧条件下1-磷酸鞘胺醇(S1P)在人主动脉血管平滑肌细胞(T/G HA-VSMC)收缩中的作用机制。方法:将传代培养的T/G HA-VSMC分为正常对照组和缺血缺氧组。缺血缺氧组采用低氧条件培养T/G HA-VSMC细胞来模拟蛛网膜下腔出血(SAH)所致的缺血缺氧环境;采用QT-PCR检测人鞘胺醇激酶1(SphK1)基因的m RNA表达;Western-blot法检测人SphK1基因的蛋白表达;ELISA法测定T/G HAVSMC细胞上清液和细胞内S1P浓度;慢病毒载体shRNA介导的T/G HA-VSMC细胞内SphK1基因表达沉默;采用钙离子荧光探针Fluo-3/AM检测T/G HA-VSMC细胞内Ca2+浓度。结果:与正常对照组相比,缺血缺氧条件下人T/G HA-VSMC细胞内SphK1基因表达上调,且缺血缺氧组细胞培养液和细胞裂解液中S1P浓度均显著升高。缺血缺氧可诱导T/G HA-VSMC细胞内Ca2+离子浓度增加,并且外源性S1P显著上调T/G HA-VSMC细胞内Ca2+浓度;与正常对照组相比,shRNA干扰技术沉默SphK1基因表达和SphK1特异性抑制剂二甲基鞘胺醇(DMS)均显著抑制低氧诱导的T/G HA-VSMC细胞内S1P和Ca2+浓度上调。结论:缺血缺氧可导致T/G HA-VSMC细胞内SphK1基因表达上调,进而促进S1P的合成代谢,而高浓度的S1P通过某种机制导致细胞内Ca2+浓度水平增加导致血管收缩。
Objective:To investigate the mechanism of sphingosine-1-phosphate(S1P)in contraction of human aortic vascular smooth muscle cells(T/G HA-VSMC)under hypoxic/ischemic stress.Methods:The cultured T/G HA-VSMC were divided into normal control group and ischemia-hypoxia model group.In the ischemia-hypoxia group,T/G HA-VSMC cells were cultured under hypoxic conditions to simulate the ischemic-hypoxic environment induced by subarachnoid hemorrhage(SAH).The m RNA expression of sphingosine kinase 1(SphK1)was detected by QT-PCR,and the protein levels of SphK1 were measured by western blot.The levels of S1P in the supernatant of the cell culture medium were measured by ELISA.Lentivirus vector shRNA mediated SphK1 gene silencing was applied as a reverse study.The calcium fluorescent probe Fluo-3/AM was used to measure intracellular Ca2+concentration.Results:Compared with that of normal control group,the protein expression of SphK1 in T/G HA-VSMC was significantly increased in the ischemia-hypoxia group,and the concentration of S1P in the supernatant of the cell culture medium was increased significantly in the ischemia-hypoxia group.The concentration of intracellular Ca2+increased significantly in T/G HA-VSMC under ischemia and hypoxia stress,and exogenous S1P significantly increased the intracellular Ca2+concentration.Compared with normal control group,shRNA interference SphK1 gene expression and SphK1 specific inhibitors dimethylsphingosine(DMS)significantly inhibited the concentrations of both S1P and Ca2+in T/G HA-VSMC in the ischemia-hypoxia group.Conclusion:SphK1 gene expression is up-regulated in T/G HA-VSMC cells under ischemic/hypoxic stress,which promotes the synthesis and metabolism of S1P.High concentration of S1P leads to vasoconstriction by increasing the level of intracellular Ca2+in cells.
作者
汤华
陈少军
赵东刚
闫俊
TANG Hua;CHEN Shao-jun;ZHAO Dong-gang;YAN Jun(Department of Neurosurgery,Renmin Hospital of The Three Gorges University,The First Pepole’s Hospital of Yichang,Hubei 443000,China)
出处
《神经损伤与功能重建》
2020年第3期129-133,共5页
Neural Injury and Functional Reconstruction
关键词
1-磷酸鞘胺醇
缺氧
人主动脉平滑肌细胞
sphingosine-1-phosphate
hypoxia
human aortic vascular smooth muscle cells
作者简介
通讯作者:汤华,tlb173@163.com。
