MYOC基因C1009del突变对人小梁网细胞功能的影响

doi:10.3969/j.issn.1673-8640.2017.010.017

检验医学 ›› 2017, Vol. 32 ›› Issue (10): 904-910.DOI: 10.3969/j.issn.1673-8640.2017.010.017

MYOC基因C1009del突变对人小梁网细胞功能的影响

李萍¹, 许涛², 谢小兵¹, 张贞¹, 胡燕¹, 苏敏¹, 那婧婧¹

1. 湖南中医药大学第一附属医院医学检验与病理中心,湖南长沙 410007
2. 湖南中医药大学第一附属医院急诊科,湖南长沙 410007

收稿日期:2017-04-27 出版日期:2017-10-20 发布日期:2017-11-20
作者简介:null
作者简介：李萍,女,1981年生,博士,副主任技师,主要从事遗传代谢性疾病的基因诊断工作。

通信作者：谢小兵,联系电话：0731-89669350。
基金资助:
湖南省科学技术厅科学计划项目（2012FJ4289）;湖南省卫生厅科研项目（B2011-068）;湖南省教育厅优秀青年科研项目（12B094）

Influence of MYOC gene C1009del mutation on the function of human trabecular meshwork cell

LI Ping¹, XU Tao², XIE Xiaobing¹, ZHANG Zhen¹, HU Yan¹, SU Min¹, NA Jingjing¹

1. Medical Laboratory Center,the First Hospital of Hunan University of Chinese Medicine,Changsha 410007,Hunan,China
2. Department of Emergency Medicine,the First Hospital of Hunan University of Chinese Medicine,Changsha 410007,Hunan,China

Received:2017-04-27 Online:2017-10-20 Published:2017-11-20

摘要/Abstract

摘要：

目的探讨小梁网糖皮质激素诱导反应蛋白（MYOC）基因C1009缺失突变（MYOC-C1009del）对体外人小梁网细胞（HTMC）功能及核因子κB（NF-κB）通路的影响。方法针对MYOC基因野生型及C1009del突变型分别构建慢病毒质粒载体pLVX-3FLAG-MYOC-EGFP（简称rLV-MYOC）和pLVX-3FLAG-MYOC-mut-EGFP（简称rLV-MYOC-mut）。慢病毒质粒经包装和纯化后体外感染HTMC,以空白HTMC及感染空质粒病毒的HTMC作为2组空白对照,感染48 h后用于后续功能实验。采用流式细胞术检测HTMC的凋亡率及细胞周期;采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（MTT）比色法检测细胞存活情况;采用荧光定量聚合酶链反应（PCR）检测NF-κB及其抑制因子（IκB）的mRNA表达情况;采用免疫印迹法检测NF-κB和IκB的蛋白质表达水平。结果与感染rLV-MYOC的HTMC相比,感染rLV-MYOC-mut的HTMC呈现细胞G1期缩短、S期延长（P<0.05）,而细胞凋亡率和细胞存活率二者之间差异均无统计学意义（P>0.05）。感染rLV-MYOC-mut的HTMC的NF-κB mRNA和IκB mRNA表达水平均明显低于感染rLV-MYOC的HTMC（P<0.05）,而在蛋白质表达水平上,感染rLV-MYOC-mut的HTMC的NF-κB表达水平与感染rLV-MYOC的HTMC持平,而IκB的表达水平在感染rLV-MYOC-mut的HTMC中呈下降趋势（P<0.05）。结论 MYOC基因C1009del突变可通过影响HTMC周期,使细胞阻滞于S期,进而影响HTMC的正常功能。C1009del突变尽管可使IκB表达下调,但并不是通过活化NF-κB通路这个途径导致原发性开角型青光眼的发生。

关键词: MYOC基因, 核因子κB, 核因子κB抑制因子, 原发性开角型青光眼

Abstract:

Objective To study the influence of myocilin（MYOC） gene C1009del mutation（MYOC-C1009del） on the function of human trabecular meshwork cells（HTMC） in vitro and the pathway of nuclear factor-kappa B（NF-κB）.Methods Lentiviral vector pLVX-3FLAG-MYOC-EGFP（rLV-MYOC） and pLVX-3FLAG-MYOC-mut-EGFP（rLV-MYOC-mut） were constructed according to MYOC gene wild type and MYOC-C1009del. The lentiviral vectors were packaged and purified,and then were infected into HTMC. The blank HTMC and HTMC infected with empty plasmid were used as 2 groups of blank control. After 48 h of infection,the follow-up cell function assay was performed. Flow cytometry was used to determine HTMC apoptosis rate and cell cycle. Cell viability was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay. The mRNA expressions of NF-κB and its inhibitor of NF-κB（IκB） were determined by fluorescence quantitation polymerase chain reaction（PCR）. Western blotting was used to determine the expressions of NF-κB and IκB at protein levels. Results HTMC infected with rLV-MYOC-mut showed a shortened G1 phase and prolonged S phase compared with those infected with rLV-MYOC（P<0.05）. However,there was no statistical significance in apoptosis and viability（P>0.05）. The mRNA expressions of NF-κB and IκB in HTMC infected with rLV-MYOC-mut were lower than those infected with rLV-MYOC（P<0.05）. At protein levels,the expressions of NF-κB in HTMC infected with rLV-MYOC-mut and with rLV-MYOC were the same,while the expression of IκB in HTMC infected with rLV-MYOC-mut showed downward trend（P<0.05）. ConclusionsMYOC-C1009del can affect HTMC cycle that may arrest in S phase and affect the normal function of HTMC. Although MYOC-C1009del can down regulate the expression of IκB,it does not lead to the occurrence of primary open-angle glaucoma by activating NF-κB pathway.

Key words: Myocilin gene, Nuclear factor-kappa B, Inhibitor of nuclear factor-kappa B, Primary open-angle glaucoma

中图分类号:

R446.1

李萍, 许涛, 谢小兵, 张贞, 胡燕, 苏敏, 那婧婧. MYOC基因C1009del突变对人小梁网细胞功能的影响[J]. 检验医学, 2017, 32(10): 904-910.

LI Ping, XU Tao, XIE Xiaobing, ZHANG Zhen, HU Yan, SU Min, NA Jingjing. Influence of MYOC gene C1009del mutation on the function of human trabecular meshwork cell[J]. Laboratory Medicine, 2017, 32(10): 904-910.

图/表 7

图1 慢病毒质粒转染HTMC细胞

表1 各组各细胞周期HTMC百分比（%）

组别	G1期	S期	G2期
HTMC组	80.72±1.29	7.47±1.23	11.51±0.65
rLV组	64.98±1.95^*	24.73±1.44^*	10.29±0.65
rLV-MYOC组	63.56±1.05	25.75±0.79	10.70±0.29
rLV-MYOC-mut组	60.63±0.72^#	28.26±0.80^#	11.11±0.14

图2 各组各细胞周期HTMC的变化情况

图3 各组细胞凋亡率的比较

图4 各组细胞存活率的比较

图5 各组HTMC NF-κB mRNA和IκB mRNA的表达水平

图6 各组HTMC NF-κB蛋白和IκB蛋白的表达水平

参考文献 21

[1]	WIGGS J L,PASQUALE L R.Genetics of glaucoma[J]. Hum Mol Genet,2017,26(R1):R21-R27.
[2]	KUMAR S,MALIK M A,GOSWAMI S,et al.Candidate genes involved in the susceptibility of primary open angle glaucoma[J]. Gene,2016,577(2):119-131.
[3]	唐冰花,曹文俊. 原发性开角型青光眼易感基因研究中GWAS的应用[J]. 检验医学,2016,31(4):329-333.
[4]	JUNGLAS B,KUESPERT S,SELEEM A A,et al.Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork[J]. Am J Pathol,2012,180(6):2386-2403.
[5]	VOHRA R,TSAI J C,KOLKO M.The role of inflammation in the pathogenesis of glaucoma[J]. Surv Ophthalmol,2013,58(4):311-320.
[6]	谢小兵,周新,田艳丽,等. 原发性开角型青光眼家系的MYOC基因突变研究[J]. 中华检验医学杂志,2009,32(2):157-161.
[7]	XIE X,ZHOU X,QU X,et al.Two novel myocilin mutations in a Chinese family with primary open-angle glaucoma[J]. Mol Vis,2008,14:1666-1672.
[8]	匡多秀,李萍,周新,等. C1009del突变质粒载体的构建及MYOC基因的有效干扰[J]. 国际检验医学杂志,2011,32(5):529-530.
[9]	STONE E M,FINGERT J H,ALWARD W L,et al.Identification of a gene that causes primary open angle glaucoma[J]. Science,1997,275(5300):668-670.
[10]	GOLDWICH A,SCHOLZ M,TAMM E R.Myocilin promotes substrate adhesion,spreading and formation of focal contacts in podocytes and mesangial cells[J]. Histochem Cell Biol,2009,131(2):167-180.
[11]	KWON H S,TOMAREV S I.Myocilin,a glaucoma-associated protein,promotes cell migration through activation of integrin-focal adhesion kinase-serine/threonine kinase signaling pathway[J]. J Cell Physiol,2011,226(12):3392-3402.
[12]	SAKAI H,SHEN X,KOGA T,et al.Mitochondrial association of myocilin,product of a glaucoma gene,in human trabecular meshwork cells[J]. J Cell Physiol,2007,213(3):775-784.
[13]	NGUYEN T D,CHEN P,HUANG W D,et al.Gene structure and properties of TIGR,an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells[J]. J Biol Chem,1998,273(11):6341-6350.
[14]	KUBOTA R,NODA S,WANG Y,et al.A novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor:molecular cloning,tissue expression,and chromosomal mapping[J]. Genomics,1997,41(3):360-369.
[15]	ZHANG Q,LENARDO M J,BALTIMORE D.30 Year of NF-κb:a blossoming of relevance to human pathobiology[J]. Cell,2017,168(1-2):37-57.
[16]	CHI W,CHEN H,LI F,et al.HMGB1 promotes the activation of NLRP3 and caspase-8 inflammasomes via NF-κB pathway in acute glaucoma[J]. J Neuroinflammation,2015,12:137.
[17]	WU M,BIAN Q,LIU Y,et al.Sustained oxidative stress inhibits NF-kappaB activation partially via inactivating the proteasome[J]. Free Radic Biol Med,2009,46(1):62-69.
[18]	HSU S M,YANG C H,SHEN F H,et al.Proteasome inhibitor bortezomib suppresses nuclear factor-kappa B activation and ameliorates eye inflammation in experimental autoimmune uveitis[J]. Mediators Inflamm,2015,847373.
[19]	KIRSTEIN L,CVEKL A,CHAUHAN B K,et al.Regulation of human myocilin/TIGR gene transcription in trabecular meshwork cells and astrocytes:role of upstream stimulatory factor[J]. Genes Cells,2000,5(8):661-676.
[20]	LAN Y Q,ZHANG C,XIAO J H,et al.Suppression of IkappaBalpha increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells[J]. Mol Vis,2009,15:1977-1987.
[21]	ITAKURA T,PETERS D M,FINI M E.Glaucomatous MYOC mutations activate the IL-1/NF-κB inflammatory stress response and the glaucoma marker SELE in trabecular meshwork cells[J]. Mol Vis,2015,21:1071-1084.

MYOC基因C1009del突变对人小梁网细胞功能的影响

Influence of MYOC gene C1009del mutation on the function of human trabecular meshwork cell

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 21

相关文章 2

编辑推荐

Metrics

[1]	刘勇刚, 施立煌, 张子杨, 贾忠. 牙周炎患者病原菌感染状况及RANKL/OPG变化[J]. 检验医学, 2018, 33(12): 1123-1126.
[2]	唐冰花, 曹文俊. 原发性开角型青光眼易感基因研究中GWAS的应用[J]. 检验医学, 2016, 31(4): 329-333.