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小干扰RNA沉默细胞外钙敏感受体抑制人脐静脉内皮细胞钙内流和NO生成

梁霄, 罗小林, 钟华, 胡清华, 何芳^*

新疆石河子大学医学院病理生理学教研室/新疆地方与民族高发病教育部重点实验室，石河子 832002；华中科技大学同济医学院病理生理学系/卫生部呼吸疾病重点实验室，武汉 430030

摘要

为了探讨细胞外钙敏感受体(extracellular Ca2+-sensing receptor, CaR)在人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVEC)介导钙内流和NO生成中的作用，本实验针对GenBank中人CaR的cDNA序列设计合成小干扰RNA (small interference RNA, siRNA)，并将其转染入HUVEC中，采用激光共聚焦显微镜观察细胞转染效果，Western blot检测CaR蛋白表达及转染后的干扰效率，Fura-2/AM负载检测细胞内Ca2+浓度(intracellular Ca2+ concentration, [Ca2+]i)变化，NO荧光探针DAF-FM DA负载检测细胞内NO的生成及内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)活性。结果显示，实验成功将CaR siRNA转入HUVEC，Western blot 检测结果显示转染48 h后，CaR蛋白表达降低(P < 0.05)；与未转染对照组和阴性对照组相比，在精胺 + Ca2+刺激下，CaR siRNA转染组的[Ca2+]i、eNOS活性和NO含量均显著降低(P < 0.05)。上述结果表明，CaR在精胺介导的HUVEC Ca2+内流和NO生成中发挥重要作用。

关键词： 内皮细胞; 钙敏感受体; 钙信号; NO; siRNA

分类号：R363

[Extracellular Ca(2+) influx and NO generation are inhibited by small interference RNA targeting extracellular Ca(2+)-sensing receptor in human umbilical vein endothelial cells.] [Article in Chinese]

LIANG Xiao, LUO Xiao-Lin, ZHONG Hua, HU Qing-Hua, HE Fang^*

Department of Pathophysiology/Key Laboratory of Education Ministry of Xinjiang Endemic and Ethnic Diseases, Medical College of Shihezi University, Shihezi 832002, China； Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology/Key Laboratory for Respiratory Diseases, Health Ministry of China, Wuhan 430030, China

Abstract

To investigate the effect of Ca2+-sensing receptor (CaR) on Spermine-induced extracellular Ca2+ influx and NO generation in human umbilical vein endothelial cells (HUVEC), the small interference RNA (siRNA) specifically targeting CaR gene was designed, synthesized and transfected into HUVEC according to the cDNA sequence of human CaR gene in GenBank. The transfection efficiency and the interference efficiency of CaR protein were determined by laser scanning confocal microscopy and Western blot, respectively. Intracellular Ca2+ concentration ([Ca2+]i) was measured by Fura-2/AM loading. The production of NO and the activity of endothelial nitric oxide synthase (eNOS) were determined by the DAF-FM diacetate (DAF-FM DA). Western blot results demonstrated that siRNA targeting the CaR specifically decreased the expression of CaR protein in CaR siRNA group 48 h after transfection (P < 0.05). At the same time, the Spermine-induced [Ca2+]i, eNOS activity and NO generation were also significantly reduced (P < 0.05) in CaR siRNA group compared with those in the untransfected or negative siRNA transfected group. In conclusion, the present study suggests that the CaR plays an important role in the Spermine-evoked process of extracellular Ca2+ influx and NO generation in HUVEC.

Key words: Endothelial cells; calcium-sensing receptors; calcium signaling; nitric oxide; small interference RNA

收稿日期：2011-12-03　　录用日期：2012-02-17

通讯作者：何芳　　E-mail: fangf2002shz@126.com

引用本文：

梁霄, 罗小林, 钟华, 胡清华, 何芳. 小干扰RNA沉默细胞外钙敏感受体抑制人脐静脉内皮细胞钙内流和NO生成[J]. 生理学报 2012; 64 (3): 289-295.

LIANG Xiao, LUO Xiao-Lin, ZHONG Hua, HU Qing-Hua, HE Fang. [Extracellular Ca(2+) influx and NO generation are inhibited by small interference RNA targeting extracellular Ca(2+)-sensing receptor in human umbilical vein endothelial cells.] [Article in Chinese]. Acta Physiol Sin 2012; 64 (3): 289-295 (in Chinese with English abstract).