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首页> 外文期刊>Digestive Diseases and Sciences >Helicobacter pylori-Induced Oxidative Stress and DNA Damage in a Primary Culture of Human Gastric Mucosal Cells
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Helicobacter pylori-Induced Oxidative Stress and DNA Damage in a Primary Culture of Human Gastric Mucosal Cells

机译：幽门螺杆菌诱导的人类胃粘膜细胞原代培养中的氧化应激和DNA损伤。

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Helicobacter pylori has been identified in the pathogenesis of chronic active gastritis and peptic ulcer disease and is epidemiologically linked to gastric cancer and lymphoma. Our previous studies have demonstrated enhanced production of reactive oxygen species (ROS) in cultured gastric adenocarcinoma cells (ATCC CRL/1739) in association with H. pylori. Recently, we have isolated and cultured normal human gastric mucosal cells (GMC) from H. pylori-negative endoscopic biopsies. The integrity of these mucosal cells was determined by periodic acid–Schiff staining. We assessed the effects of various H. pylori strains including 60190 (a 87-kDa cytotoxin producing strain), ATCC 43504, and 60190-v1 (in which the cytotoxin gene has been disrupted) on the primary culture of human gastric mucosal cells. The induction of ROS and DNA fragmentation in the mucosal cells in association with these H. pylori strains were assessed by cytochrome c reduction (an index of superoxide anion production), hydroxyl radical production, and DNA fragmentation. Following incubation of the mucosal cells with 1:0.5 and 1:1 ratios of H. pylori strain 60190, approximately 6.2- and 9.9-fold increases were observed in cytochrome c reduction, respectively, as compared to mucosal cells in the absence of H. pylori, demonstrating the production of superoxide anion. The detection of hydroxyl radicals based on the formation of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was determined by using a high-performance liquid chromatograph equipped with an electrochemical detector. Approximately 3.5- and 7.7-fold increases in hydroxyl radical production were observed following incubation of the mucosal cells with 1:0.5 and 1:1 ratios of H. pylori, respectively. Approximately 3.6- and 4.5-fold increases in DNA fragmentation were observed in gastric mucosal cells following incubation with 1:0.5 and 1:1 ratios of H. pylori, respectively. The effects of culture supernatant preparations from H. pylori strains 60190 and 60190-v1 on the enhanced production of ROS and increased DNA fragmentation in mucosal cells were also investigated. Culture supernatant preparations, the prime source of the 87-kDa cytotoxin, from both H. pylori strains 60190 and 60190-v1 were extracted under identical conditions to determine the role of 87-kDa cytotoxin on the enhanced production of ROS and DNA fragmentation. The cytotoxin rich-H. pylori strain 60190 induced greater production of ROS and DNA fragmentation in mucosal cells as compared to the supernatant preparation from H. pylori strain 60190-v1, in which the cytotoxin gene has been disrupted. This study demonstrates that H. pylori induces enhanced production of ROS and DNA damage in association with human gastric mucosal cells andthat the 87-kDa cytotoxin protein plays a prime role in the induction of oxidative stress and DNA damage.
机译：幽门螺杆菌已在慢性活动性胃炎和消化性溃疡疾病的发病机理中得到鉴定，并在流行病学上与胃癌和淋巴瘤有关。我们以前的研究表明，与幽门螺杆菌相关的培养的胃腺癌细胞（ATCC CRL / 1739）中活性氧（ROS）的产生增加。最近，我们从幽门螺杆菌阴性内镜活检组织中分离并培养了正常的人胃粘膜细胞（GMC）。这些粘膜细胞的完整性通过高碘酸-席夫氏染色确定。我们评估了各种幽门螺杆菌菌株对人胃粘膜细胞原代培养的影响，其中包括60190（产生87-kDa细胞毒素的菌株），ATCC 43504和60190-v1（其中细胞毒素基因已被破坏）。与这些幽门螺杆菌菌株相关的粘膜细胞中ROS的诱导和DNA片段化通过细胞色素c还原（超氧化物阴离子产生的指数），羟基自由基产生和DNA片段化来评估。与不存在H的粘膜细胞相比，以1：0.5和1：1的H.pylori菌株60190比例孵育粘膜细胞后，细胞色素C的减少分别观察到分别约6.2和9.9倍的增加。幽门螺杆菌，证明超氧阴离子的产生。通过使用配备有电化学检测器的高效液相色谱仪，基于2,3-二羟基苯甲酸和2,5-二羟基苯甲酸的形成来检测羟基自由基。分别以1：0.5和1：1的幽门螺杆菌温育粘膜细胞后，观察到羟基自由基产生分别增加了约3.5倍和7.7倍。在分别以1：0.5和1：1的幽门螺杆菌温育后，在胃粘膜细胞中观察到DNA片段增加了约3.6倍和4.5倍。还研究了幽门螺杆菌60190和60190-v1的培养上清液制剂对黏膜细胞中ROS产生增加和DNA片段增加的影响。在相同条件下提取幽门螺杆菌60190和60190-v1的培养上清液制备物，这是87-kDa细胞毒素的主要来源，以确定87-kDa细胞毒素在ROS产生和DNA片段化中的作用。富含细胞毒素的H。与来自幽门螺杆菌菌株60190-v1的上清液制备相比，幽门螺杆菌菌株60190在粘膜细胞中诱导产生更多的ROS和DNA片段化，其中细胞毒素基因已被破坏。这项研究表明幽门螺杆菌诱导与人胃粘膜细胞相关的ROS和DNA损伤的产生增加，并且87 kDa的细胞毒素蛋白在氧化应激和DNA损伤的诱导中起主要作用。

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