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Regulation of activation and cell death of neutrophils infiltrated into periapical lesions

浸润根尖周病变的中性粒细胞活化和细胞死亡的调节

基本信息

批准号：
15592029
负责人：
TAKAHASHI Keiso
金额：
$ 1.92万
依托单位：
MEIKAI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15592029/
关键词：
periapical lesion oral PMNs reactive oxygen species signal transduction antioxidants apoptosis 唾液好中球 flow cytometry グルタチオン

项目摘要

Although the cellular function and their physiology of peripheral neutrophils have been widely investigated, those of local neutrophils in oral cavity, intestine and mucosa have not well been known. It is crucial to investigate the mechanism by which regulates the cellular function of oral polymorphonuclear leukocytes (OPMN) in order to manipulate host defensive ability in the oral cavity. The aim of this study was to assess the status of cell activation and cell death of OPMN. OPMN were prepared by mouth rinse, filtration and Mono-Poly density gradient centrifugation method. Cellular activity of OPMN was assessed by superoxide production using NBT reagent. The cell death of OPMN was evaluated by flow cytometry using annexin V-FITC/propidium iodide staining. One to two millions OPMN was collected from each of healthy volunteers and 90% of freshly prepared OPMN were viable, as judged by trypan blue dye exclusion. OPMN produced significantly higher amounts of superoxide than peripheral PMN (p<0.05). The superoxide production by OPMN was significantly enhanced by PMA and Zymosan stimulation (p<0.05) and was not inhibited by H-7. OPMN died significantly faster at 37℃ than at 4℃ (p<0.05). Cell death of OPMN was partially inhibited by antioxidants, but was promoted by TNF-α. Caspase inhibitors and H-7 showed little effect. OPMN died via apoptosis and/or necrosis, and its ratio was varied by the types of stimulators. These results suggest that oral PMN are not activated via protein kinase C and that oxidative stress enhances apoptosis of OPMN. Further research will be required to investigate the underlying mechanism relating with cell activation and cell death of OPMN.

虽然外周血中性粒细胞的功能和生理已被广泛研究，但口腔、肠和粘膜局部中性粒细胞的功能和生理尚不清楚。研究口腔多形核白细胞（oral polymorphisms leukocytes，OPMN）的细胞功能调控机制对调控宿主口腔防御能力具有重要意义。本研究的目的是评估OPMN的细胞活化和细胞死亡的状态。通过漱口、过滤和单聚密度梯度离心法制备OPMN。OPMN的细胞活性通过使用NBT试剂的超氧化物产生来评估。采用Annexin V-FITC/碘化丙啶染色流式细胞术检测OPMN的细胞死亡情况。从每个健康志愿者中收集了一到两百万个OPMN，通过台盼蓝染料排除法判断，90%的新鲜制备的OPMN是活的。OPMN产生的超氧化物显著高于外周PMN（p<0.05）。PMA和酵母多糖刺激OPMN产生超氧化物歧化酶显著增强（p<0.05），H-7不抑制OPMN产生超氧化物歧化酶。OPMN在37℃时死亡速度明显快于4℃（p<0.05）。抗氧化剂可部分抑制OPMN的细胞死亡，而TNF-α可促进OPMN的细胞死亡。半胱天冬酶抑制剂和H-7几乎没有效果。OPMN通过凋亡和/或坏死死亡，其比例因刺激物的类型而异。这些结果表明，口腔中性粒细胞没有激活通过蛋白激酶C和氧化应激增强OPMN的凋亡。OPMN的细胞活化和细胞死亡机制有待进一步研究。