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Study for periodontal pathogenesis and apoptosis in periodontal tissues induced by volatile sulfur compounds

挥发性硫化合物诱导牙周组织凋亡及牙周发病机制的研究

基本信息

批准号：
17390568
负责人：
YAEGAKI Ken
金额：
$ 10.15万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

Hydrogen sulfide (H_2S) is one of the main reasons of halitosis and periodontally pathogenic. H_2S causes apoptotic cell death in aorta smooth muscle cells and other tissues, and apoptosis plays an important role in the onset and progress of periodontitis. Therefore, we investigated if H_2S causes apoptosis in human gingival fibroblasts (HGF). Materials and Methods : Necrotic cell were determined using lactate dehydrogenase assay. Apoptosis was ascertained with detecting histone-complexed DNA fragments and a flow cytometry. Caspase 3, a key enzyme in apoptotic signaling, was assessed. Reactive oxygen species (ROS) were also detected and inhibition of superoxide dismutase (SOD) by H_2S was verified. DNA damage was examined with single-cell gel electrophoresis (SCGE). Results : Necrosis was found less than 10% of HGF during 72 hours incubation with 100 ng/ml H_2S, whereas significant increment of DNA fragmentation in cytoplasm was found (p < 0.05). Apoptotic HGF cells were also increased in a flow cytometry. Caspase 3 activity was significantly increased at 72 hours incubation (p < 0.01). Tail length, %DNA in tail and Tail moment obtained by SCGE increased in HGF exposed to H_2S for 48 and 72 hours. Moreover, ROS was significantly increased at 48 and 72 hour incubation with H_2S (p < 0.005 and p < 0.001, respectively), and SOD of HGF was strongly inhibited. Conclusion : H_2S caused apoptosis in HGF, and an inhibition of SOD by H_2S increased ROS, then induced apoptosis and DNA strand breaks.

硫化氢（H_2S）是引起口臭和牙周病的主要原因之一。H_2S可引起主动脉平滑肌细胞等组织细胞凋亡，细胞凋亡在牙周炎的发生、发展中起重要作用。因此，我们研究了H_2S是否引起人牙龈成纤维细胞（HGF）凋亡。材料与方法：采用乳酸脱氢酶法测定坏死细胞。通过检测组蛋白复合DNA片段和流式细胞术确定细胞凋亡。评估了凋亡信号传导中的关键酶半胱天冬酶3。活性氧（ROS）的测定和H_2S对超氧化物歧化酶（SOD）的抑制作用。用单细胞凝胶电泳（SCGE）检测DNA损伤。结果如下：100 ng/mlH_2S孵育72小时，HGF的坏死率低于10%，而胞浆DNA断裂率显著增加（p < 0.05）。流式细胞仪检测凋亡的HGF细胞也增加。Caspase 3活性在孵育72小时时显著升高（p < 0.01）。H_2S处理48和72小时后，SCGE结果显示HGF的尾长、尾中DNA含量和尾矩均增加。在H_2S作用48和72小时时，肝细胞内ROS明显增加（p < 0.005和p < 0.001），SOD活性受到明显抑制。结论：H_2S可引起HGF细胞凋亡，H_2S抑制SOD可增加ROS，进而引起细胞凋亡和DNA链断裂。