蛋白质氧连糖基化修饰对肿瘤坏死因子α诱导的小鼠心肌成纤维细胞增殖的影响  

The effect of protein O-GlcNAc modification on proliferation of mice cardiac fibroblasts induced by tumor necrosis factor-α

作　　者：李如君[1] 龚开政[1] 张振刚[1] 

机构地区：[1]扬州大学第二临床医学院心血管内科,江苏扬州225012

出　　处：《中华高血压杂志》2017年第4期370-376,共7页Chinese Journal of Hypertension

基　　金：国家自然科学基金(81270198)

摘　　要：目的采用肿瘤坏死因子α(TNF-α)诱导心肌成纤维细胞(CF)增殖,探讨快速提高细胞内氧连糖基化(O-GlcNAc)水平对CF功能及P65、核因子κB信号通路负向调控因子TNFAIP3蛋白表达的影响。方法胰酶联合Ⅳ型胶原酶消化成年小鼠心脏组织培养CF,差速贴壁法分离纯化原代CF,波形蛋白免疫荧光染色鉴定细胞类型。将CF分为空白对照组;TNF-α(10μg/L)组;Thiamet-G(150μmol/L)预处理+空白对照组;Thiamet-G(150μmol/L)预处理+TNF-α(10μg/L)组。活细胞计数试剂盒(CCK-8)检测CF增殖,细胞划痕实验评价迁移能力,荧光定量聚合酶链反应(PCR)检测TNF-α靶基因血管细胞黏附分子1(VCAM-1)、单核细胞趋化蛋白1(MCP-1)mRNA表达,Western blot检测磷酸化P65(p-P65)、总P65(t-P65)、TNFAIP3蛋白水平。结果原代培养的心脏细胞经波形蛋白免疫荧光染色鉴定为CF,纯度>95%。Thiamet-G预处理CF 2h即可观察到全细胞蛋白O-GlcNAc水平显著升高(3.22±0.32比1.00±0.22,P<0.05);与空白对照组相比,TNF-α处理组可激活核因子κB通路(p-P65增高:3.13±0.27比1.00±0.13,P<0.05),诱导靶基因VCAM-1、MCP-1mRNA表达(4.21±0.37比1.00±0.21,11.26±0.98比1.00±0.32,均P<0.05),明显提高CF增殖及迁移能力(1.74±0.21比0.48±0.11,P<0.05);与TNF-α处理组相比,Thiamet-G预处理后TNF-α诱导的CF增殖及迁移被抑制(1.04±0.07比1.74±0.21,P<0.05),靶基因VCAM-1、MCP-1mRNA表达减少(2.09±0.13比4.21±0.37,3.13±0.85比11.26±0.98,均P<0.05),P65蛋白总水平在12h无明显变化(1.08±0.13比1.00±0.24,P>0.05),但p-P65水平下降(1.82±0.08比3.13±0.21,P<0.05),TNFAIP3蛋白表达显著上调(5.86±0.61比2.95±0.33,P<0.05)。结论快速提高细胞内O-GlcNAc水平对TNF-α诱导的成年小鼠CF增殖和迁移具有抑制作用,其作用机制可能与上调TNFAIP3的表达有关。Objective To investigate effects of increasing protein Odinked-N-acetylglucosamine （O-GleNAc） levels with Thiamet-G on proliferation of mice cardiac {ibroblasts （CFs） induced by tumor necrosis factor-or and expression of P65, the negative regulator of nuclear factor-κB（NF-κB） signaling pathway （TNFAIP3）. Methods CFs from adult mice were digested with trypsin and collagenase IV, purified with differential adhesion, and identified by vim- entin immunofluorescence staining. The cells were divided into control group, TNF-α （10 μg/L） group, Thiamet- G （150 μmol/L） pretreated＋control group, Thiamet-G（150 μmol/L） pretreated＋TNF-α （10 μg/L） group. The proliferation and migration of CF were analyzed by cell counting kit 8 （CCK-8） assay and scratch-wound assay respec tively. The mRNA of TNF-α target genes, vascular cell adhesion molecule-1 （VCAM-1）, monocyte chemoatractant protein-1 （MCP-1）, were detected by real-time polymerase chain reaction （PCR） and phosphorylated P65 （p P65）, total P65 （t-P65） and TNFAIP3 proteins were detected by Western blot. Results The purity of CF was more than 95% under fluorescent microscope. Thiamet-G treatment can quickly increase O-GlcNAc levels significantly in CF at 2 hours（3.22±0.32 vs 1.00±0.22, P〈0.05）. Compared with control group, NF-κB signaling pathway was significantly activated in TNF-α group（the level of p-P65 ： 3.13±0.27 vs 1.00±0.13, P〈0.05）, and the mRNA levels of TNF-α target gene（VCAM-1 ： 4. 21±0.37 vs 1. 00±0.21, MCP-1 ： 11. 26±0.98 vs 1. 00±0.32, P〈0.05） were remarkably increased; the proliferation and migration of CF were enhanced（ 1.74±0.21 vs 0.48±0.11, P〈0.05）. After Thiamet-G pretreatment, the proliferation and migration of CF induced by TNF-α were inhibited（ 1. 04±0.07 vs 1.74±0.21, P〈0.05）. Furthermore, the level of p-P65 （ 3.13±0. 27 vs 1.00±0.13, P〈0.05） and the mRNA levels of VCAM-1, MCP-1 were decreased（2. 094±0.13 vs 4. 21±0.37, 3.

关 键 词：氧连糖基化修饰 心肌成纤维细胞 核因子ΚB 肿瘤坏死因子Α 

分 类 号：R542.2[医药卫生—心血管疾病]