初级纤毛细胞外氧感受器功能和机制研究

doi:10.3785/j.issn.1008-9292.2017.12.07

浙江大学学报（医学版）

2017, Vol. 46

Issue (6): 618-624 DOI: 10.3785/j.issn.1008-9292.2017.12.07

原著

初级纤毛细胞外氧感受器功能和机制研究

高玉海1(

),漆欣筑2,周建1,王昕2,陈克明1,马慧萍2,*(

)

1. 兰州军区兰州总医院全军骨科中心骨科研究所, 甘肃兰州 730050
2. 兰州军区兰州总医院全军高原损伤防治重点实验室, 甘肃兰州 730050

Primary cilium acts as an oxygen sensor in PC12 cells

GAO Yuhai1(

),QI Xinzhu2,ZHOU Jian1,WANG Xin2,CHEN Keming1,MA Huiping2,*(

)

1. Institute of Orthopaedics, Lanzhou General Hospital of PLA, Lanzhou 730050, China
2. Key Laboratory of Prevention and Treatment for the Plateau Environmental Damage of PLA, Lanzhou General Hospital of PLA, Lanzhou 730050, China

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摘要：

目的: 探索初级纤毛是否为PC12细胞的细胞外氧感受器。方法: 通过小干扰RNA（siRNA）干扰细胞纤毛内转运蛋白88的表达抑制体外培养PC12细胞初级纤毛的发生；免疫荧光染色法观察缺氧诱导因子1α（HIF-1α）、核因子E2相关因子2（Nrf2）核转位和PC12细胞初级纤毛生长情况；实时定量RT-PCR法检测HIF-1α、Nrf2、血管内皮生长因子（VEGF）和超氧化物歧化酶（SOD）基因表达。结果: siRNA干扰组细胞的初级纤毛被抑制。与常氧对照组比较，缺氧对照组和阴性对照组细胞的HIF-1α和Nrf2发生了明显的核转位，细胞内HIF-1α、Nrf2、VEGF基因表达量增加，SOD表达量下降；siRNA干扰组细胞在缺氧状态下HIF-1α和Nrf2未发生核转位，细胞内HIF-1α、Nrf2、VEGF基因表达受到抑制，而SOD表达量升高。结论: PC12细胞的初级纤毛可能具有细胞外氧感受器的作用，可以感知细胞外环境中的缺氧和氧化应激状态，并通过一定途径将缺氧信号传导至细胞内，进而启动细胞抗缺氧机制。

关键词： PC12细胞; 纤毛; 感受器, 感觉; 缺氧/病理生理学; 动力传导, 细胞; RNA, 小分子干扰

Abstract:

Objective: To investigate the function of primary cilium as an oxygen sensor in PC12 cells. Methods: The PC12 cells were transfected with IFT88 siRNA. The nuclear translocation of hypoxia inducible factor-1α (HIF-1α), nuclear factor erythroid-2 related factor 2 (Nrf2), and ciliogenesis were observed by immunofluorescence staining; and the mRNA expressions of HIF-1α, Nrf2, vascular endothelial growth factor (VEGF) and superoxide dismutase (SOD) were detected by real-time RT-PCR. Results: The ciliogenesis was inhibited in PC12 cells transfected with IFT88 siRNA. In hypoxia group and scramble control group, nuclear translocations of HIF-1α and Nrf2 were observed and mRNA expressions of HIF-1α, Nrf2, VEGF were increased, and those of SOD were decreased. While in PC12 cells transfected with IFT88 siRNA, nuclear translocations of HIF-1α and Nrf2 were not observed, and mRNA expressions of HIF-1α, Nrf2, VEGF were inhibited, and mRNA expression of SOD was increased. Conclusion: Primary cilia may act as an oxygen sensor to transfer the information related to hypoxia and oxidative stress into cells, activating intracellular defense mechanism against the hypoxic injuries.

Key words: PC12 cells Cilia Receptors, sensory Anoxia/physiopathology Mechanotransduction, cellular RNA, small interfering

收稿日期: 2017-03-05 出版日期: 2017-12-25

CLC:

Q74

基金资助: 国家自然科学基金(81571847, 81402848);全军医药卫生科研基金(CLZ14JA01);兰州军区高原科研攻关项目(LZ13GY07)

通讯作者: 马慧萍 E-mail: gyh1389@126.com;mahuipingcxr@aliyun.com

作者简介: 高玉海(1988-), 男, 硕士, 主管技师, 主要从事骨代谢研究; E-mail:gyh1389@126.com; https://oricd.org/0000-0001-7845-3796

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引用本文:

高玉海,漆欣筑,周建,王昕,陈克明,马慧萍. 初级纤毛细胞外氧感受器功能和机制研究[J]. 浙江大学学报（医学版）, 2017, 46(6): 618-624.

GAO Yuhai,QI Xinzhu,ZHOU Jian,WANG Xin,CHEN Keming,MA Huiping. Primary cilium acts as an oxygen sensor in PC12 cells. J Zhejiang Univ (Med Sci), 2017, 46(6): 618-624.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2017.12.07 或 http://www.zjujournals.com/med/CN/Y2017/V46/I6/618

表 1 实时定量RT-PCR引物序列

图 1 缺氧前后PC12细胞形态学变化

图 2 转染24 h荧光倒置相差显微镜下观察PC12细胞siRNA干扰结果

图 3 siRNA干扰后荧光倒置相差显微镜下观察PC12细胞初级纤毛生长情况

图 4 缺氧培养18 h各组细胞HIF1α和Nrf2核转位情况

图 5 各组细胞HIF-1α、Nrf2、VEGF和SOD基因表达量比较

1	ASCENZI M G , LENOX M , FARNUM C . Analysis of the orientation of primary cilia in growth plate cartilage:a mathematical method based on multiphoton microscopical images[J]. J Struct Biol, 2007, 158 (3): 293- 306 doi: 10.1016/j.jsb.2006.11.004
2	PRAETORIUS H A . The primary cilium as sensor of fluid flow:new building blocks to the model. A review in the theme:cell signaling:proteins, pathways and mechanisms[J]. Am J Physiol Cell Physiol, 2015, 308 (3): C198- C208 doi: 10.1152/ajpcell.00336.2014
3	TRENDELEVA T A , ALIVERDIEVA D A , ZVYAGILSKAYA R A . Mechanisms of sensing and adaptive responses to low oxygen conditions in mammals and yeasts[J]. Biochemistry(Mosc), 2014, 79 (8): 750- 760
4	MIYATA T , TAKIZAWA S , VAN YPERSELE DE STRIHOU C . Hypoxia. 1. Intracellular sensors for oxygen and oxidative stress:novel therapeutic targets[J]. Am J Physiol Cell Physiol, 2011, 300 (2): C226- C231 doi: 10.1152/ajpcell.00430.2010
5	漆欣筑, 马慧萍, 王昕 et al. 缺氧环境对PC12细胞初级纤毛长度的影响[J]. 解放军医药杂志, 2016, 28 (7): 10- 12 QI Xinzhu , MA Huiping , WANG Xin et al. Effect of hypoxia environment on primary cilia length of PC12 cells[J]. Medical & Pharmaceutical Journal of Chinese People's Liberation Army, 2016, 28 (7): 10- 12
6	VERGHESE E , ZHUANG J , SAITI D et al. In vitro investigation of renal epithelial injury suggests that primary cilium length is regulated by hypoxia-inducible mechanisms[J]. Cell Biol Int, 2011, 35 (9): 909- 913 doi: 10.1042/CBI20090154
7	PROULX-BONNEAU S , ANNABI B . The primary cilium as a biomarker in the hypoxic adaptation of bone marrow-derived mesenchymal stromal cells:a role for the secreted frizzled-related proteins[J]. Biomark Insights, 2011, 6 107- 118
8	BROWN J A , SANTRA T , OWENS P et al. Primary cilium-associated genes mediate bone marrow stromal cell response to hypoxia[J]. Stem Cell Res, 2014, 13 (2): 284- 299 doi: 10.1016/j.scr.2014.06.006
9	YAN J L , ZHOU J , MA H P et al. Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia[J]. Mol Cell Endocrinol, 2015, 404 132- 140 doi: 10.1016/j.mce.2015.01.031
10	MOSER S C , BENSADDEK D , ORTMANN B et al. PHD1 links cell-cycle progression to oxygen sensing through hydroxylation of the centrosomal protein Cep192[J]. Dev Cell, 2013, 26 (4): 381- 392 doi: 10.1016/j.devcel.2013.06.014
11	LIU W , XIN H , ECKERT D T et al. Hypoxia and cell cycle regulation of the von Hippel-Lindau tumor suppressor[J]. Oncogene, 2011, 30 (1): 21- 31 doi: 10.1038/onc.2010.395
12	ORTMANN B , DRUKER J , ROCHA S . Cell cycle progression in response to oxygen levels[J]. Cell Mol Life Sci, 2014, 71 (18): 3569- 3582 doi: 10.1007/s00018-014-1645-9
13	ORTMANN B , DRUKER J , ROCHA S . Cell cycle progression in response to oxygen levels[J]. Cell Mol Life Sci, 2014, 71 (18): 3569- 3582 doi: 10.1007/s00018-014-1645-9
14	MOVAFAGH S , CROOK S , VO K . Regulation of hypoxia-inducible factor-1α by reactive oxygen species:new developments in an old debate[J]. J Cell Biochem, 2015, 116 (5): 696- 703 doi: 10.1002/jcb.25074
15	BHULLAR K S , RUPASINGHE H P . Polyphenols:multipotent therapeutic agents in neurodegenerative diseases[J]. Oxid Med Cell Longev, 2013, 2013 891748
16	LISK C , MCCORD J , BOSE S et al. Nrf2 activation:a potential strategy for the prevention of acute mountain sickness[J]. Free Radic Biol Med, 2013, 63 264- 273 doi: 10.1016/j.freeradbiomed.2013.05.024
17	TROILO A , ALEXANDER I , MUEHL S et al. HIF1α deubiquitination by USP8 is essential for ciliogenesis in normoxia[J]. EMBO Rep, 2014, 15 77- 85 doi: 10.1002/embr.v15.1
18	PAZOUR G J . The role of intraflagellar transport proteins in ciliary assembly and function[J]. Cilia, 2012, 1 (Suppl 1): O9 doi: 10.1186/2046-2530-1-S1-O9

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