伴放线聚集杆菌细胞致死膨胀毒素诱导Jurkat细胞凋亡的机制

The mechanism of Jurkat cells apoptosis induced by Aggregatibacter actinomycetemcomitans cytolethal distending toxin.

作者信息

Chen Hui-Ping, Li Lu, Chen Xu, Yang Mi-Fang, Ye Yu, Wang Xiao-Qian, Xu Yan

机构信息

Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, Jiangsu, China.

Department of Periodontics, Affiliated Hospital of Stomatology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, Jiangsu, China.

出版信息

Apoptosis. 2017 Jun;22(6):841-851. doi: 10.1007/s10495-017-1357-3.

DOI:10.1007/s10495-017-1357-3

PMID:28247205

Abstract

Cytolethal distending toxin (CDT) which is produced by Aggregatibacter actinomycetemcomitans causes apoptosis in lymphocytes. But the specific mechanism is not clear. The aim of our research was to investigate the effect and mechanism during this process. The wild-type CdtA, CdtB, CdtC (CdtA, CdtB, CdtC) and mutant CdtB (CdtB) were expressed and purified respectively and the purity of each subunit was examined by BandScan software. And the type I deoxyribonuclease and PI-3,4,5-triphosphate (PI-3,4,5-P3, PIP3) phosphatase activity were detected by DNA agarose gel electrophoresis and enzyme-linked immunosorbent assay respectively. The cell apoptosis rates were analyzed by flow cytometry. The morphological changes of apoptosis cells were observed by confocal laser scanning microscopy. The protein expression of Bax and Bcl-2 was examined by western blot. Differentially expressed apoptosis-related proteins were identified based on isobaric tags for relative and absolute quantitation technology. In the present study we found that: (i) recombinant wild-type CdtA, CdtB and CdtC (CdtA, CdtB, CdtC) and mutant CdtB (CdtB) were correctly expressed and the purity of each protein was higher than 80%, (ii) the average apoptosis rate in wild-type CDT (CDT) treated groups was 50.54%, which was significantly higher than the controls (4.71%) and mutant CDT (CDT) treated groups (5.58%) (p < 0.05), (iii) morphological changes of apoptosis were observed in CDT treated cells, (iv) the expression of Bax protein was significantly increased in CDT treated cells, while Bcl-2 protein expression was significantly decreased, (v) 17 apoptosis-related proteins were expressed differentially, among which 10 proteins (SMNDC1, TNFRSF10B, UBE2I, ITM2A, CASP3, P53, EIF1, TCF3, HMGN5, CASP8) were up-regulated and 7 proteins (RRM2, TPX2, KIF11, NUCKS1, TOP2A, XRCC1, PTPLAD1, RRM2) were down-regulated, (vi) one possible apoptotic pathway [Ubc9 (UBE2I)/P53/DR5 (TNFRSF10B)/Caspase-8 (CASP8)/ Caspase-3 (CASP3)] was selected and partially proved.

摘要

伴放线聚集杆菌产生的细胞致死性膨胀毒素（CDT）可导致淋巴细胞凋亡。但其具体机制尚不清楚。我们研究的目的是探讨这一过程中的作用及机制。分别表达并纯化野生型CdtA、CdtB、CdtC（CdtA、CdtB、CdtC）和突变型CdtB（CdtB），并通过BandScan软件检测各亚基的纯度。分别通过DNA琼脂糖凝胶电泳和酶联免疫吸附测定检测I型脱氧核糖核酸酶和磷脂酰肌醇-3,4,5-三磷酸（PI-3,4,5-P3，PIP3）磷酸酶活性。通过流式细胞术分析细胞凋亡率。通过共聚焦激光扫描显微镜观察凋亡细胞的形态变化。通过蛋白质免疫印迹法检测Bax和Bcl-2的蛋白表达。基于相对和绝对定量的等压标签技术鉴定差异表达的凋亡相关蛋白。在本研究中我们发现：（i）重组野生型CdtA、CdtB和CdtC（CdtA、CdtB、CdtC）以及突变型CdtB（CdtB）均正确表达，且各蛋白纯度均高于80%；（ii）野生型CDT（CDT）处理组的平均凋亡率为50.54%，显著高于对照组（4.71%）和突变型CDT（CDT）处理组（5.58%）（p＜0.05）；（iii）在CDT处理的细胞中观察到凋亡的形态学变化；（iv）CDT处理的细胞中Bax蛋白表达显著增加，而Bcl-2蛋白表达显著降低；（v）17种凋亡相关蛋白差异表达，其中10种蛋白（SMNDC1、TNFRSF10B、UBE2I、ITM2A、CASP3、P53、EIF1、TCF3、HMGN5、CASP8）上调，7种蛋白（RRM2、TPX2、KIF11、NUCKS1、TOP2A、XRCC1、PTPLAD1、RRM2）下调；（vi）选择并部分证实了一条可能的凋亡途径[Ubc9（UBE2I）/P53/DR5（TNFRSF10B）/半胱天冬酶-8（CASP8）/半胱天冬酶-3（CASP3）]。