钙离子信号蛋白TRPC1及Orai1参与了HUVEC经SOC和ROC介导的钙内流和NO生成
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(新疆地方病与民族高发病教育部重点实验室 石河子大学医学院病理生理教研室,新疆石河子市 832002)

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王腊梅,硕士,实验师,研究方向为心血管病理生理学,E-mail为375770358@qq.com。

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国家自然科学基金资助项目(31160239;81160018)


Calcium signaling proteins TRPC1 and Orai1 participate in Ca2+ entry and NO generation mediated by SOC and ROC in human umbilical vein endothelial cell
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Ministry of Education Key Laboratory of Xinjiang Endemic and Ethnic Diseases and Centre of Medical Functional Experiments, Medical College of Shihezi University, Shihezi, Xinjiang 832002, China)

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    摘要:

    目的 研究瞬时受体电位通道1(TRPC1)及钙释放激活钙通道调节分子1(Orai1)在人脐静脉内皮细胞(HUVEC)经钙池操纵性钙通道(SOC)和受体操纵性钙通道(ROC)介导的Ca2+内流和一氧化氮(NO)生成中的作用。 方法 取2~3代HUVEC进行随机分组,将构建的TRPC1、Orai1干扰质粒(shTRPC1、shOrai1)分别转染入HUVEC,采用real-time PCR和Western blot检测TRPC1、Orai1 mRNA和蛋白的表达及转染抑制效率。将各组细胞分别与钙敏感受体(CaR)激动剂精胺、ROC模拟剂(TPA)+CaR负性变构调节剂Calhex231、蛋白激酶C(PKC)抑制剂Ro31-8220与经典型PKCs和PKCμ抑制剂Go6967孵育后,荧光探针Fura-2/AM及DAF-FM负载方法同步检测 [Ca2+]i和NO生成的变化。 结果 与对照组比较,shTRPC1组和shOrai1组mRNA表达(抑制率分别为84.50%、76.10%)和蛋白的表达(抑制率分别为83.98%、71.73%)明显降低(P<0.05)。在4种不同药物作用下,shTRPC1组和shOrai1组[Ca2+]i△ratio值和NO净荧光强度值均明显降低(P<0.05)。 结论 TRPC1、Orai1参与了人脐静脉内皮细胞SOC和ROC介导的钙内流和NO生成。

    Abstract:

    Aim To study the function of TRPC1 and Orai1 in store and receptor-operated Ca2+ entry and nitric oxide generation by SOC and ROC in human umbilical vein endothelial cell. Methods HUVEC were collected and cultured to the second-third passage. We silenced the expression of their genes in HUVEC by transfection constructed TRPC1 or Orai1 RNA interference plasmids. The interference efficiency of their proteins and mRNA levels were determined by Western blot and real-time PCR, respectively. The cell were incubated with CaR agonist spermine, CaR negative allosteric modulator Calhex231 and ROC analogue TPA, protein kinase C (PKC) inhibitor Ro31-8220, PKCs and PKCμ inhibitor Go6967. Intracellular Ca2+ concentration ( [Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, the production of NO was determined by DAF-FM of every group in HUVEC. Results Compared with control group, shRNA targeted to the TRPC1 or Orai1 genes decreased their mRNA and protein levels, respectively (P<0.05); The results of their mRNA levels by 84.50% and 76.10% and proteins levels were decreased by 83.98% and 71.73%; In four different treatment under the action of factors, the [Ca2+]i and the net NO fluorescence intensity ratio values of transfection of TRPC1shRNA and Orai1shRNA group were significantly reduced (P<0.05). Conclusion TRPC1 and Orai1 participated in CaR-mediated Ca2+ influx and NO production activation mediated by SOC and ROC in HUVEC.

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王腊梅,钟华,唐娜,庞丽娟,孙志萍,何芳.钙离子信号蛋白TRPC1及Orai1参与了HUVEC经SOC和ROC介导的钙内流和NO生成[J].中国动脉硬化杂志,2017,25(12):1189~1195.

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  • 收稿日期:2017-04-12
  • 最后修改日期:2017-09-07
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  • 在线发布日期: 2017-12-28