[1]陈中林,聂敏海,贾颖,等.HSG细胞中MBD2对AQP5表达的调控作用[J].第三军医大学学报,2019,41(10):955-960.
 CHEN Zhonglin,NIE Minhai,JIA Ying,et al.Role of methyl-CpG-binding domain protein 2 in regulating aquaporin-5 expression in human submandibular gland cells in vitro [J].J Third Mil Med Univ,2019,41(10):955-960.
点击复制

HSG细胞中MBD2对AQP5表达的调控作用(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第10期
页码:
955-960
栏目:
基础医学
出版日期:
2019-05-30

文章信息/Info

Title:
Role of methyl-CpG-binding domain protein 2 in regulating aquaporin-5 expression in human submandibular gland cells in vitro
 
作者:
陈中林聂敏海贾颖叶艳艳邓舒婷黄葳刘旭倩
西南医科大学附属口腔医院牙周黏膜科;陆军军医大学(第三军医大学)第一附属医院口腔科
Author(s):
CHEN Zhonglin NIE Minhai JIA Ying YE Yanyan DENG Shuting HUANG Wei LIU Xuqian  

Department of Periodontal Medicine, Affiliated Hospital of Stomatology, Southwest Medical University, Luzhou, Sichuan Province, 646000; Department of Stomatology, First Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400038, China

 

关键词:
干燥综合征MBD2AQP5基因治疗
Keywords:
Sjogren&rsquos syndrome methyl-CpG-binding domain protein 2 aquaporin-5 gene therapy
分类号:
R329.2;R394.2;R781.7
文献标志码:
A
摘要:

目的 探讨人下颌下腺细胞(human submandibular gland,HSG)中,甲基化CpG结合域蛋白2(menthyl-CpG-binding domain protein 2,MBD2)是否影响水通道蛋白调控-5(aquaporin 5,AQP5)的表达,为干燥综合征(Sjgren’s syndrome,SS)的治疗提供新的思路。方法 体外培养HSG细胞,通过AQP5的启动子质粒与MBD2siRNA或MBD2过表达质粒共转染HSG细胞,利用双荧光素酶报告基因系统检测AQP5启动子的活性;再用MBD2-siRNA或MBD2过表达质粒转染HSG细胞,利用RT-PCR及Western blot技术检测MBD2和AQP5在mRNA水平及蛋白水平上表达的改变。结果①MBD2-siRNA转染HSG细胞后,AQP5 pro活性分别上调49.30%和58.35%(P<0.05), AQP5在mRNA 水平上表达上调35.40%和471.19%(P<0.05),蛋白水平上调56.16%和82.78% (P<0.05)。结果 表明:MBD2-siRNA转染HSG细胞后,AQP5启动子活性增加,AQP5表达上调。②MBD2过表达质粒转染HSG细胞后,AQP5 pro 活性分别下调25.36%、30.52%及34.78%(P<0.05), AQP5在mRNA 水平上表达下调8.06%和87.60%(过表达质粒1 μg组与对照组相比无统计学意义,P>0.05;过表达质粒2 μg组与对照组相比差异有统计学意义P<0.05),在蛋白水平上表达下调44.50%和53.18%(P<0.05)。实验结果表明:MBD2转染ASG细胞后,AQP5启动子活性降低,AQP5的表达下降(P<0.05)。结论 MBD2可以下调AQP5的表达,这与SS患者的AQP5表达趋势一致,因此MBD2可能成为干燥综合征基因治疗的新靶点.

Abstract:

Objective To study whether methyl-CpG-binding domain protein 2 (MBD2) affects the expression of aquaporin-5 (AQP5) in human submandibular gland (HSG) cells, thereby exploring a new approach to treatment of Sjogren’s syndrome (SS). Methods Cultured HSG cells were co-transfected with the promoter plasmid of AQP5 and the plasmids for MBD2 silencing (MBD2-siRNA) or overexpression. Luciferase report gene assay was used to detect the transcription level of AQP5 in the transfected cells. In HSG cells transfected with the MBD2-siRNA or the overexpression plasmids, RT-qPCR and Western blotting were performed to analyze the changes in MBD2 and AQP5 expression at both the mRNA and protein levels. Results Transfection of the cells with the 2 MBD2-siRNAs respectively increased AQP5 promoter activity by 49.30% and 58.35% (P<0.05) and obviously upregulated the expression of AQP5 by 35.40% and 471.19% at the mRNA level (P<0.05) and by 56.16% and 82.78% at the protein level (P<0.05). Transfection of the cells with the three MBD2 overexpression plasmids resulted in significantly lowered AQP5 promoter activity by 25.36%, 30.52% and 34.78% (P<0.05); while 1 plasmid produced no significant changes in AQP5 mRNA level, the other 2 plasmids significantly down-regulated AQP5 expression by 8.06% and 87.60% at the mRNA level (P<0.05) and by 44.50% and 53.18% at the protein level (P<0.05). Conclusion MBD2 could down-regulats the expression of AQP5 in HSG cells in vitro, which consistent with the AQP5 expression about patients with SS,and this finding indentifies MBD2 as a potential target in gene therapy for the syndrome.
 

参考文献/References:

[1]Fox RI. Sjogren’s syndrome[J]. Lancet,2005,366(9482): 321-331. DOI: 10.1016/S01406736(05)669905
[2]Nezos A,Mavragani C P. Contribution of genetic factors to Sjgren’s Syndrome and Sjgren’s Syndrome related lymphomagenesis[J]. J Immunol Res,2015: 754-825. DOI: 10.1155/2015/754825. Epub 2015 Oct 15.
[3]KULKARNI K, SELESNIEMI K, BROWN T L. Interferon-gamma sensitizes the human salivary gland cell line, HSG, to tumor necrosis factor-alpha induced activation of dual apoptotic pathways[J]. Apoptosis, 2006, 11(12): 2205-2215. DOI: 10.1007/s10495-006-0281-8.
[4]WANG F, FENG X C, LI Y M, et al. Aquaporins as potential drug targets1[J]. Acta Pharmacologica Sinica, 2006, 27(4): 395-401. DOI: 10.1111/j.1745-7254.2006.00318.x.
[5]阮光峰, 汤建平. 水通道蛋白5在干燥综合征发病机制中的作用研究进展[J]. 同济大学学报(医学版), 2016, 37(2): 113-117. DOI: 10.16118/j.10080392.2016.02.026.
RUAN G F, TANG J P. The function of aquaporin 5 in the pathogenesis of Sjgren’s syndrome[J]. J Tongji Univ(Med Sci), 2016, 37(2): 113-117. DOI: 10.16118/j.10080392.2016.02.026.
[6]DELPORTE C, STEINFELD S. Distribution and roles of aquaporins in salivary glands[J]. BBABiomembranes, 2006, 1758(8): 1061-1070. DOI: 10.1016/j.bbamem.2006.01.022.
[7]AURE M H, LARSEN H S, RUUS A K, et al. Aquaporin 5 distribution pattern during development of the mouse sublingual salivary gland[J]. J Mol Hist, 2011, 42(5): 401-408. DOI: 10.1007/s10735-011-9343-5.
[8]张朋, 赵莹, 杨舒婷, 等. 舍格伦综合征病因和发病机制及AQP5在其中的作用[J]. 海南医学, 2016, 27(13): 2171-2173. DOI: 10.3969/j.issn.1003-6350.2016.13.036.
ZHANG P, ZHAO Y, YANG S T, et al. Etiology and pathogenesis of Sj-gren’s syndrome and the role of AQP5 in the disease[J]. Hainan Medi J, 2016, 27(13): 2171-2173. DOI: 10.3969/j.issn.1003-6350.2016.13.036.
[9]MATHEWS P M, HAHN S, HESSEN M, et al. Ocular complications of primary Sj-gren syndrome in men[J]. Am J Ophthalmol, 2015, 160(3): 447-452.e1. DOI: 10.1016/j.ajo.2015.06.004.
[10]ALAMANOS Y, TSIFETAKI N, VOULGARI P V, et al. Epidemiology of primary Sj-gren’s syndrome in north-west Greece, 1982-2003[J]. Rheumatology (Oxford), 2006, 45(2): 187-191. DOI: 10.1093/rheumatology/kei107.
[11]VON BLTZINGSLWEN I, SOLLECITO T P, FOX P C, et al. Salivary dysfunction associated with systemic diseases: systematic review and clinical management recommendations[J]. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2007, 103(Suppl): S57.e1-S57.15. DOI: 10.1016/j.tripleo.2006.11.010.
[12]STEINFELD S, COGAN E, KING L S, et al. Abnormal distribution of aquaporin-5 water channel protein in salivary glands from Sj-gren’s syndrome patients[J]. Lab Invest, 2001, 81(2): 143-148. DOI: 10.1038/labinvest.3780221.
[13]WU G L, PU X H, YU G Y, et al. Effects of total glucosides of peony on AQP-5 and its mRNA expression in submandibular glands of NOD mice with Sj-gren’s syndrome[J]. Eur Rev Med Pharmacol Sci, 2015, 19(1): 173-178.
[14]李方凯, 谷新怡, 崔民英, 等. 活血解毒方对干燥综合征小鼠颌下腺AQP5表达的影响[J]. 西部中医药, 2016, 29(9): 19-22. DOI: 10.3969/j.issn.1004-6852.2016.09.007.
LI F K, GU X Y, CUI M Y, et al. The effects of Huo Xue Jie Du prescription on AQP5 expressions in the salivary gland of SS mice[J]. Western J Tradit Chin Med, 2016, 29(9): 19-22. DOI: 10.3969/j.issn.10046852.2016.09.007.
[15]刘鹏, 孙一丹, 王梓霖, 等. 中药麦冬对干燥综合征作用的研究进展[J]. 吉林医学, 2017, 38(4): 773-775. DOI: 10.3969/j.issn.1004-0412.2017.04.080.
LIU P, SUN Y D, WANG Z L, et al. Research progress about the effect of Ophiopogon japonicas on Sj-gren’s syndrome [J]. Jilin Med J, 2017, 38(4): 773-775. DOI: 10.3969/j.issn.1004-0412.2017.04.080.
[16]LEE B H, GAUNA A E, PEREZ G, et al. Autoantibodies against muscarinic type 3 receptor in Sj-gren’s syndrome inhibit aquaporin 5 trafficking[J]. PLoS ONE, 2013, 8(1): e53113. DOI: 10.1371/journal.pone.0053113.
[17]MOTEGI K, AZUMA M, TAMATANI T, et al. Expression of aquaporin-5 in and fluid secretion from immortalized human salivary gland ductal cells by treatment with 5-aza-2′-deoxycytidine: A possibility for improvement of xerostomia in patients with Sj-gren’s syndrome[J]. Lab Invest, 2005, 85(3): 342-353. DOI: 10.1038/labinvest.3700234.
[18]DEFOSSEZ P A, STANCHEVA I. Biological Functions of Methyl-CpG-Binding Proteins[M]//DEFOSSEZ P A, STANCHEVA I. Eds. Progress in molecular biology and translational science. Elsevier, 2011: 377-398. DOI: 10.1016/b978-0-12-387685-0.00012-3.
[19]BIRD A P, WOLFFE A P. Methylationinduced repression: belts, braces, and chromatin[J]. Cell, 1999, 99(5): 451-454. DOI: 10.1016/s0092-8674(00)81532-9.
[20]闫伟伟, 黄昂, 戴广海. MBD蛋白家族研究进展[J]. 传染病信息, 2017, 30(3): 181-185. DOI: 10.3969/j.issn.1007-8134.2017.03.015.
YAN W W, HUANG A, DAI G H. Research progress of MBD protein family[J]. Infect Dis Inform, 2017, 30(3): 181-185. DOI: 10.3969/j.issn.1007-8134.2017.03.015.
 

更新日期/Last Update: 2019-05-24