过刊浏览

TRPC6在低氧高二氧化碳肺动脉平滑肌细胞增殖、凋亡中的作用

贾旭广, 郑梦晓, 张晶晶, 张聪聪, 赵美平, 吴益明, 陈锡文, 王万铁

四川省宜宾卫生学校内科教研室，宜宾 644000；黄河科技学院医学院，郑州 450063；温州医科大学病理生理学教研室，温州 325035；爱荷华大学卡佛医学院心血管医学系，爱荷华 52242，美国；温州医科大学实验动物中心，温州 325035

摘要

本文旨在探讨TRPC6在低氧高二氧化碳性肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells, PASMCs)增殖和凋亡中的作用。原代培养Sprague-Dawley (SD)雄性大鼠PASMCs，采用平滑肌α-肌动蛋白免疫荧光染色法鉴定。选用4~6代的PASMCs，加入无血清DMEM饥饿24 h，随机分为5组：常氧组、低氧高二氧化碳组、DMSO组、TRPC6抑制剂SKF-96365组和TRPC6激动剂OAG组。常氧组置于37 °C常氧培养箱(21% O2, 5% CO2)中培养24 h，其余4组置于37 °C低氧高二氧化碳培养箱(5% O2, 6% CO2)中，用不同药物处理24 h。用逆转录聚合酶链式反应和Western blot分别检测TRPC6 mRNA和蛋白的表达；用CCK-8法检测细胞增殖情况；用原位末端标记法检测细胞凋亡；用Fura 2-AM双波长法检测细胞内Ca2+浓度([Ca2+]i)。结果显示，低氧高二氧化碳条件下，PASMCs的TRPC6 mRNA和蛋白表达上调，[Ca2+]i升高，细胞增殖增加，而凋亡减少；OAG可促进低氧高二氧化碳的上述作用，而SKF-96365能够逆转这些作用。以上结果提示，TRPC6参与了低氧高二氧化碳对PASMCs增殖和凋亡的调节。

关键词： 动脉平滑肌细胞; 肺动脉高压 ; C型瞬时受体电位 ; 低氧高二氧化碳 ; Ca2+

分类号：R363

Role of TRPC6 in pulmonary artery smooth muscle cells proliferation and apoptosis under hypoxia and hypercapnia

JIA Xu-Guang, ZHENG Meng-Xiao, ZHANG Jing-Jing, ZHANG Cong-Cong, ZHAO Mei-Ping, WU Yi-Ming, CHEN Xi-Wen, WANG Wan-Tie

Department of Internal Medicine, Yibin Health School, Yibin 644000, China； School of Medicine, Huanghe Science and Technology College, Zhengzhou 450063, China； Department of Pathophysiology, Wenzhou Medical University, Wenzhou 325035, China； Division of Cardiovascular Medicine, University of Iowa Carver College of Medicine, Iowa City 52242, USA； Experimental Animal Center, Wenzhou Medical University, Wenzhou 325035, China

Abstract

The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4–6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO2, 21% O2, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO2, 5% O2, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca2+]i in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca2+]i were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca2+]i.

Key words: pulmonary artery smooth muscle cells; pulmonary hypertension ; canonical transient receptor potential ; hypoxia and hypercapnia ; calcium

收稿日期：2016-04-17　　录用日期：2016-10-28

通讯作者：王万铁　　E-mail: wwt@wmu.edu.cn

引用本文：

贾旭广, 郑梦晓, 张晶晶, 张聪聪, 赵美平, 吴益明, 陈锡文, 王万铁. TRPC6在低氧高二氧化碳肺动脉平滑肌细胞增殖、凋亡中的作用[J]. 生理学报 2017; 69 (1): 47-54.

JIA Xu-Guang, ZHENG Meng-Xiao, ZHANG Jing-Jing, ZHANG Cong-Cong, ZHAO Mei-Ping, WU Yi-Ming, CHEN Xi-Wen, WANG Wan-Tie. Role of TRPC6 in pulmonary artery smooth muscle cells proliferation and apoptosis under hypoxia and hypercapnia. Acta Physiol Sin 2017; 69 (1): 47-54 (in Chinese with English abstract).