四川动物  2016, Vol. 35 Issue (4): 517-522

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文章信息

刘浩然, 范新荣, 李鹏云, 李涛, 曾博
LIU Haoran, FAN Xinrong, LI Pengyun, LI Tao, ZENG Bo
温度敏感型离子通道基因在哺乳动物内脏器官和血管中的表达研究
Expression Profiles of Genes Encoding Temperature-Sensitive Ion Channels in Mammalian Internal Organs and Blood Vessels
四川动物, 2016, 35(4): 517-522
Sichuan Journal of Zoology, 2016, 35(4): 517-522
10.11984/j.issn.1000-7083.20160052

文章历史

收稿日期: 2016-03-14
接受日期: 2016-05-11
温度敏感型离子通道基因在哺乳动物内脏器官和血管中的表达研究
刘浩然, 范新荣, 李鹏云, 李涛, 曾博*     
西南医科大学心血管医学研究所, 医学电生理教育部重点实验室, 四川泸州 646000
摘要: 温度敏感型离子通道广泛存在于哺乳动物内脏器官和血管组织,在多种生理功能的调节中起重要作用。本研究以雄性比格犬和雄性SD大鼠的心脏、肝、脾、肾、肺、主动脉、肺动脉、肺静脉、股动脉、股静脉及颈总动脉为材料提取RNA,经逆转录PCR以及半定量方法获得这些通道基因的表达情况。结果显示:TRPV1在2种动物的上述组织中均有表达,而TRPV2和TRPM5在所有组织中均无表达;TRPV3仅表达在大鼠的肝、肺及肺动脉中,在狗的所有组织中均无表达;TRPV4在狗的肺中无表达,在2种动物的其他组织中均有表达;TRPM2在大鼠的主动脉、心脏、肺动脉中无表达,在2种动物的其他组织中均有表达;TRPM3在狗的肺、肺动脉及大鼠的肺、心脏、主动脉中无表达,在2种动物的其他组织中均有表达;TRPM4在狗的肺动脉以及大鼠的脾、肾、心脏中没有检测到表达,在2种动物的其他组织中均有表达;TRPM8在狗的肝、脾、肾以及大鼠的肾中无表达,在2种动物的其他组织中均有表达;TRPA1仅在大鼠的肺和脾中有表达。本研究比较全面地展示了温度敏感型离子通道基因在哺乳动物内脏器官和血管中的表达情况,为进一步探究其在哺乳动物生理调节中的功能奠定基础。
关键词温度敏感     瞬时电位受体通道     哺乳动物     基因表达     内脏器官     血管    
Expression Profiles of Genes Encoding Temperature-Sensitive Ion Channels in Mammalian Internal Organs and Blood Vessels
LIU Haoran, FAN Xinrong, LI Pengyun, LI Tao, ZENG Bo*     
Key Laboratory of Medical Electrophysiology, Ministry of Education, Insitute of Cardiovascular Research, Southwest Medical University, Luzhou, Sichuan Province 646000, China
Abstract: Temperature-sensitive ion channels widely exist in internal organs and blood vessels of mammals and play important roles in the regulation of various physiological functions. In this study, we collected tissues including the heart, liver, spleen, kidney, lung, aorta, pulmonary artery/vein, femoral artery/vein, and carotid artery from dogs and rats, respectively. Subsequently, the expression of genes encoding temperature-sensitive ion channels were detected using reverse transcription PCR and agarose gel electrophoresis. The results showed that TRPV1 existed in all the tissues of the two species examined, while TRPV2 and TRPM5 were undetectable in all the examined tissues. TRPV3 was expressed in the liver, lung and pulmonary artery of rat but absent in all canine tissues. TRPV4 existed in all the tissues of both species except in the lung of dog. TRPM2 was expressed in all the tissues of both species except in the aorta, heart and pulmonary artery of rat. TRPM3 expression was absent in the lung and pulmonary artery of dog and the lung, heart and aorta of rat, but presented in all other tissues. TRPM4 existed in all the tissues of both species except the pulmonary artery of dog and spleen, kidney and heart of rat. TRPM8 existed in all the tissues of both species except the liver, spleen and kidney of dog and kidney of rat. TRPA1 expressoin was found only in the lung and spleen of rat. These results provided detailed information for the expression of genes encoding temperature-sensitive ion channels in mammalian internal organs and blood vessels, which were essential for further exploration of physiological regulation by these channels.
Key words: temperature-sensitive     transient receptor potential channels     mammal     gene expression     internal organ     blood vessel    

瞬时电位受体通道(transient receptor potential channels,TRP channels)是一类具有多种调控方式的非选择性阳离子通道,这是一个拥有7大亚科(bTRPC、TRPV、TRPA、TRPM、TRPML、TRPP、TRPN)共30余种通道蛋白的总家族(TRPC、TRPV、TRPA、TRPM、TRPML、TRPP、TRPN)共30余种通道蛋白的总家族(Venkatachalam & Montell,2007),最早于1979年在黑腹果蝇Drosophila melanogaster的视觉传导系统中被发现,此类离子通道广泛地参与到机体生理和病理水平调控中,如介导免疫(Zhao et al.,2015)、异常的痛觉感应(Julius,2013)、肾脏疾病(Tomilin et al.,2016)及神经水平调控(Tomilin et al.,2015)、先天性心脏疾病(Yue & Xie,2015)、癌症(Fiorio et al.,2012)等的发生。在TRP家族离子通道中含有1组分别可以感受热刺激的TRPV1、TRPV2、TRPV3、TRPV4、TRPM2(Togashi et al.,2006)、TRPM3(Vriens et al.,2011)、TRPM4(Vennekens & Nilius,2007)、TRPM5(Talavera et al.,2005)离子通道和感受冷刺激的TRPM8、TRPA1(Madrid & Pertusa,2014)离子通道。此类蛋白在受到环境温度变化等刺激后会开放通道并导致钙离子内流,进而调控细胞内钙离子稳态平衡从而广泛地参与到哺乳动物生理及病理水平的调控中,如TRPV1参与介导急性结肠炎后的内脏疼痛(Lapointe et al.,2015),TRPV2参与维持心肌细胞的结构及功能(Katanosaka et al.,2014),TRPV3参与介导口腔上皮创伤后灼热感觉的形成(Aijima et al.,2015),TRPV4介导调控大脑微血管在机械牵张条件下相关酶的表达(蒲九君等,2015),TRPM2参与介导肾脏缺血后的氧化应激及损伤(Gao et al.,2014),TRPM3参与感受伤害性热刺激(Vriens et al.,2011),TRPM4和TRPM5参与介导胰岛素分泌(Shigeto et al.,2015),TRPM8参与介导肿瘤发生机制(Liu et al.,2016)等。 温度敏感型离子通道在生理水平调控及疾病发生发展中均起到重要的作用,但在目前国内外的相关文献报道中,对于此类温度敏感型离子通道基因在哺乳动物内脏器官和血管中的具体表达情况尚未有系统性的研究。因此,本文较为系统地检测了温度敏感型离子通道基因在哺乳动物内脏器官和部分血管中的表达情况,为此类通道在哺乳动物体内的功能以及在人体内的后续研究做铺垫。

1 材料与方法 1.1 材料

实验使用的3只未成年雄性比格犬和3只成年雄性SD大鼠均购自西南医科大学实验动物中心,取狗和大鼠的心脏、脾、肝、肾、肺、主动脉、肺动脉、肺静脉、股动脉、股静脉、颈总动脉,并存入RNALater(Qiagen)后-80 ℃保存;PCR中所使用的Goldstar Best MasterMix高保真聚合酶(CW0960A)、总RNA提取所用的超纯RNA提取试剂盒(CW0581)以及逆转录所用HiFi-script cDNA第一链合成试剂盒(CW2569-100)均购自康为世纪公司(北京);引物由成都擎科梓熙生物技术公司合成。

1.2 方法 1.2.1 RNA提取

取狗和大鼠相关组织约500 μg,按照超纯RNA提取试剂盒说明方法进行,收集RNA溶液,-80 ℃保存。

1.2.2 反转录

以各组织总RNA为模板,反转录合成cDNA序列。按照HiFi-script cDNA第一链合成试剂盒说明方法合成cDNA,-20 ℃保存。

1.2.3 引物设计

根据GenBank中狗和大鼠的TRPV1、TRPV2、TRPV3、TRPV4、TRPM2、TRPM3、TRPM4、TRPM5、TRPM8、TRPA1基因序列,设计可以扩增2种动物相应基因的跨物种通用引物(表 1)。

表 1 温度敏感型离子通道基因的引物 Table 1 Primers of genes encoding temperature-sensitive ion channel
基因引物序列(5’-3’)退火温度/℃产物长度/bp
TRPV1F:GTCAAGCGCATCTTCTACTTCA R:ATCTCACTGTAGCTGTCCACAAA57251
TRPV2F:AGACGTGCCTGATGAAGGC R:TGCACCAAGCAGTGGGAT57320
TRPV3F:AAGAAGAGTGCACACTTCTTCCTG R:TTCATCAGGCAGGTCTTCCC59410
TRPV4F:GACCTGGAGATGCTGAGCA R:TGTCCAGGATGGTGGTGG56197
TRPM2F:TACGTGCTCATGGTGGACTT R:TCCTTCATCATCCGCTTCA56356
TRPM3F:CAAAGACCCCCATAGGTGTT R:ATGTCATTTCGGGAGAGGC56117
TRPM4F:GGGAGGGACTGGAATTGACAT R:TTCCAAGCCACAGCCAAAC59420
TRPM5F:TACTTCTGGGCCATGGGC R:TGGGCAAAGAAGGCCTTG58272
TRPM8F:TGGGGCATGGTCTCCAA R:AGCCTTCCACCACCACACA58346
TRPA1F:GTGGAATTTTCATTGTTATGTTGGAGG R:TGGTATGAAGTTCCACCTGCATAGC64387
1.2.4 温度敏感型离子通道基因的PCR扩增

温度敏感型离子通道基因的逆转录PCR(RT-PCR)以狗和大鼠相关组织的cDNA为模板进行扩增,PCR体系为:10 μL Goldstar Best MasterMix,1 μL cDNA模板,1 μL 5’端引物以及1 μL 3’端引物,加水补足至20 μL体系。将PCR体系混合均匀后,95 ℃预变性10 min;94 ℃变性30 s,在各基因的特有退火温度下退火30 s,72 ℃延伸2 min,35个循环;72 ℃最后延伸5 min。反应完成后,取3 μL PCR产物用1%琼脂糖凝胶进行电泳检测。

1.2.5 表达水平的计算

参照表 1中各目的基因的扩增产物长度,鉴别琼脂糖凝胶电泳中的正确条带(图 1),用Quantity One测量目的基因和内参基因的条带亮度,用二者的比值作为目的基因的相对表达量,比值≥1为高表达,1>比值≥0.5为中等表达,比值<0.5为低表达。

图 1 温度敏感型离子通道基因在狗和大鼠内脏器官及血管中的PCR检测结果 Fig. 1 PCR results of genes encoding temperature-sensitive ion channels in internal organs and blood vessels of dog and rat V1. TRPV1,V2. TRPV2,V3. TRPV3,V4. TRPV4,M2. TRPM2,M3. TRPM3,M4. TRPM4,M5. TRPM5,M8. TRPM8,A1. TRPA1,Act. β-actin.
2 结果

TRPV1在狗的相关组织中均有表达,且在肺静脉和股静脉中表达水平较高,在股动脉中有中等水平的表达,在肺、肝、脾、主动脉、颈总动脉、肾、心脏和肺动脉中的表达水平较低;TRPV2、TRPV3、TRPM5和TRPA1在以上组织中均没有表达;TRPV4在肺中没有表达,在肺静脉、股动脉中的表达水平较高,在肝、脾、颈总动脉、股静脉中有中等水平的表达,在主动脉、肾、心脏和肺动脉中的表达水平较低;TRPM2在肺动脉、股动脉、颈总动脉、股静脉中有较高水平的表达,在肝、主动脉中有中等水平的表达,在肺、肾、心脏和肺动脉中的表达水平较低;TRPM3除了在肺和肺动脉中没有表达以外,在其他组织中均有表达,且在肺静脉、股动脉中的表达水平较高,在肝、脾、颈总动脉、股静脉、肾中有中等水平的表达,在主动脉和心脏中的表达水平较低;TRPM4在肺动脉中没有检测到表达,在脾、肺静脉、股动脉、股静脉中有中等水平的表达,在肺、肝、主动脉、颈总动脉、肾、心脏中的表达水平较低;TRPM8在肝、脾、肾中没有检测到表达,在肺静脉、股动脉和股静脉中有中等水平的表达,在肺、主动脉、颈总动脉、心脏和肺动脉中的表达较低(表 2)。

表 2 温度敏感型离子通道基因在狗内脏器官及血管中的表达水平(xn=3) Table 2 Relative expression of genes encoding temperature-sensitive ion channels in internal organs and blood vessels of dog(xn=3)
基因肺静脉股动脉主动脉颈总动脉股静脉心脏肺动脉
TRPV10.3000.3180.4461.1870.9420.2430.4021.1790.1790.4640.166
TRPV2-----------
TRPV3-----------
TRPV4-0.5010.9031.8701.4420.4180.6100.6080.3690.2250.224
TRPM20.2930.8092.0953.7804.2340.7751.0831.9580.3280.2510.250
TRPM3-0.5020.9552.1761.2160.4110.6430.9770.7210.177-
TRPM40.2930.1840.5070.8820.8530.2820.4720.6030.0910.204-
TRPM5-----------
TRPM80.296--0.7820.8520.2530.3980.507-0.2160.216
TRPA1-----------

TRPV1在大鼠的上述组织中均有表达且在肝和心脏中的表达水平较高,在肺、颈总动脉和肺动脉中有中等水平的表达,在脾、肺静脉、股动脉、主动脉、股静脉和肾中的表达水平较低;TRPV2和TRPM5在所有组织中均没有检测到表达;TRPV3在肺、肝和肺动脉中检测到有表达但表达水平较低,在脾、肺静脉、股动脉、主动脉、颈总动脉、股静脉、肾和心脏中未检测到表达;TRPV4在所有组织中均有表达,在肺、股动脉和颈总动脉中的表达水平较高,在肝、肺静脉、主动脉、股静脉、肾、心脏和肺动脉中有中等水平的表达,在脾中的表达水平较低;TRPM2在肺、肝、脾、肾中有高水平的表达,在股动脉、颈总动脉和股静脉中有中等水平的表达,在肺静脉中的表达水平较低,而在主动脉、心脏和肺动脉中没有检测到表达;TRPM3在肺、主动脉和心脏中没有检测到表达,在其他组织中均有表达且在颈总动脉中有高水平表达,在肝、脾、肾中有中等水平的表达,在肺静脉、股动脉、股静脉和肺动脉中的表达水平较低;TRPM4在脾、肾和心脏中没有检测到表达,在其他组织中均有表达,且在肺、肝、股动脉、主动脉、颈总动脉中的表达水平较高,在股静脉中有中等水平的表达,在肺静脉和肺动脉的表达水平较低;TRPM8

在肾中没有检测到表达,在其他组织中均有表达且在股动脉及颈总动脉中的表达水平较高,在股静脉中有中等水平的表达,在肺、肝、脾、肺静脉、主动脉、心脏、肺动脉中的表达水平较低;TRPA1在肺和脾中有表达但是水平较低,在肝、肺静脉、股动脉、主动脉、颈总动脉、股静脉、肾、心脏和肺动脉中均未检测到表达(表 3)。

表 3 温度敏感型离子通道基因在大鼠内脏器官及血管中的表达水平(xn=3) Table 3 Relative expression of genes encoding temperature-sensitive ion channels in internal organs and blood vessels of rat(xn=3)
基因肺静脉股动脉主动脉颈总动脉股静脉心脏肺动脉
TRPV10.6021.0310.2610.3060.2890.4900.6080.4250.4041.1430.718
TRPV2-----------
TRPV30.3040.258--------0.411
TRPV41.2120.9660.3920.5301.5550.5531.6290.7570.7190.6130.503
TRPM21.2891.3451.0020.2970.910-0.7990.6451.387--
TRPM3-0.6560.5360.3310.288-1.2900.4930.595-0.469
TRPM41.3001.096-0.4191.0931.3261.6660.998--0.384
TRPM5-----------
TRPM80.4670.3700.2900.469-1.2942.2250.803-0.2730.294
TRPA10.475-0.360--------
3 讨论

TRP通道家族尤其是对温度敏感的离子通道在哺乳动物体内广泛分布,本文对温度敏感型TRP通道基因在以比格犬和大鼠为代表的较大型和小型哺乳动物内脏器官和部分血管的表达情况进行了较全面的研究。其中,TRPV1被证实参与大鼠心脏急性缺血的血管调节(Gao et al.,2015),本实验检测到TRPV1在大鼠和狗的心脏和主动脉中都有表达,表明这一离子通道同样也参与到狗心脏急性缺血时的血管调节中;而对于缺血后灌注的心肌有保护作用的TRPM2被发现在狗心脏中有表达而在大鼠心脏中几乎没有表达(Zhan & Yu,2016),表明这一通道不会参与到大鼠心肌缺血后的灌注保护作用中;TRPV4和TRPV3在大鼠体内参与肺动脉的收缩(Yoo et al.,2012; Suresh et al.,2015),但在狗的肺动脉中几乎没有表达,表明狗肺动脉压的调控中没有TRPV3和TRPV4的参与;TRPM4在大鼠的心脏中对心肌肥厚有抑制作用(Kecskés et al.,2015),其被检测到在狗心脏中有表达表明TRPM4亦参与到狗的心肌保护机制中;TRPM8被证实参与到降低大鼠肾缺血后灌注损伤作用中(Dusmez et al.,2014),其在狗肾中几乎没有表达,表明其不会参与狗肾缺血后的保护机制。

不仅如此,在诸如人类视网膜这样的器官中都能够检测到TRPV4等基因的表达(Zhao et al.,2015),对于这一系列通道功能的探索一直以来都是研究热点,在细胞层面和个体层面均得到广泛开展,在其他器官及血管的生理调控机制研究中,研究人员都发现了温度敏感型离子通道的参与,如:TRPA1被发现参与听力及疼痛的形成(Wu et al.,2016),TRPV1被发现对于预防阿尔兹海默症有保护作用(Jayant et al.,2016)等,这些结果对于了解温度敏感型离子通道对于生理和疾病的影响以及探索疾病治疗的新方法都有非常大的研究和应用前景(Santoni & Farfariello,2011)。

以上部分温度敏感型离子通道基因在狗和大鼠体内不同部位表达情况的差异或许揭示了体型大小不同的哺乳动物在温度感受和体温调控上的区别,总体看来,在大鼠各种组织中表达的温度敏感型离子通道基因种类更多,或许表明相对于比格犬这样体型较大的动物,大鼠的生理活动更容易受到温度的影响。同时,对于这一系列基因的表达强弱情况与功能的相关性还有待进一步探索。

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