摘要
目的建立牙龈蛋白酶诱导成骨细胞凋亡模型，检测成骨细胞凋亡过程中与Bc｜-2蛋白相互作用的细胞死亡调解子（Bcl-2 interacting mediator，Bim）、Bcl-2蛋白相关X蛋白（Bcl-2associated X protein，Bax）和Bcl-2蛋白拮抗剂（Bcl-2 antagonist／killer，Bak）的表达，探寻保护成骨细胞的方法，为牙周炎的发病机制研究提供依据。方法提取牙龈蛋白酶并测定其活性；将0．453、0．906、1．812U／L牙龈蛋白酶分别与成骨细胞共培养0、16、24和48h，4’，6-二脒基-2-苯基吲哚（4’，6-diamidino-2-phenylindole，DAPI）染色及膜联蛋白V-碘化丙啶双染色检测牙龈蛋白酶诱导成骨细胞（小鼠颅顶前骨细胞亚克隆14）凋亡，建立牙龈蛋白酶诱导成骨细胞凋亡模型；1．812U／L牙龈蛋白酶与成骨细胞共培养0、4、8、16、24和48h，蛋白质印迹法确定成骨细胞凋亡过程中Bim、Bax、Bak的蛋白表达；胰蛋白酶抑制剂抑制1．812U／L牙龈蛋白酶活性后与成骨细胞共培养24h，蛋白质印迹法检测Bim蛋白表达与成骨细胞凋亡率的改变。结果精氨酸-牙龈蛋白酶活性为（18．11±2．11）U／L，赖氨酸-牙龈蛋白酶活性为（1．02±0．25）U／L；DAPI染色显示1．812U／L牙龈蛋白酶可诱导成骨细胞凋亡，16h凋亡率为（6．31±0．37）％，24h达（11．20±0．35）％，48h为（10．80±0．46）％；膜联蛋白V-碘化丙啶染色结果与DAPI染色结果基本-致。成骨细胞凋亡过程伴随促凋亡蛋白Bim的表达升高，4h时Bim相对蛋白表达量为（0．31±0．03），约为对照组（0．17±0．03）的2倍，24h时Bim相对蛋白达峰值（0．57±0．05），为对照组的3～4倍；胰蛋白酶抑制剂可有效抑制牙龈蛋白酶活性，使Bim蛋白表达量由（0．58±0．04）降至（0．14±0．03）；同时DAPI染色显示胰蛋白酶抑制剂可逆转牙龈蛋白酶诱导的细胞凋亡，24h成骨细胞凋亡率由（11．20±0．35）％降至（4．31±0．38）％。成骨细胞高表达Bax（相对蛋白表达量为0．85±0．05），在成骨细胞凋亡过程中，Bax的相埘蛋白表达总量无明显改变；蛋白质印迹法未检测到成骨细胞表达Bak。结论1．812U／L牙龈蛋向酶可诱导成骨细胞凋亡，凋亡过程中伴随Bim表达升高．抑制牙龈蛋白酶活性可有效降低Bim的蛋白表达，提示促凋亡蛋白Bim参与牙龈蛋白酶诱导成骨细胞凋亡过程，抑制Bim的表达可使成骨细胞免于凋亡。
Objective To establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator（Bim） , Bcl-2 associated X protein （Bax） and Bcl-2 antagonist/killer（Bak）. Methods Gingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0. 453, 0. 906, 1. 812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4＇,6-diamidino-2-phenylindole （DAPI） staining or annexin V/propidine iodide（PI） staining. Protein expression of Bim, Bax and Bak wasdetermined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1. 812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone（TLCK）. Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis. Results Arginine-specific proteinases （Rgp） activity was （ 18. 11 ± 2. 11 ） U/L and specific proteinases （ Kgp ） was （ 1.02 ± 0. 25 ） U/L. Percentage of osteoblast apoptosis induced by 1.812 U/Lgingipainroseto （6.31 ±0.37）% after 16 h, and reached （11.20±0.35）% at 24 h and （10. 80±0. 46）% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining. Bim protein level increased during osteoblast apoptosis, the relative fold rose to （0. 31 ± 0. 03） after 4 h（about 2 fold compared to control）, peaking at 24 h（0. 57 ±0.05, 3-4 fold compared to control）. Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from （ 0. 58 ±0. 04 ） to （ 0. 14± 0. 03 ）. The percentage of osteoblast apoptosis decreased from （ 11.20±0. 35 ）% to （4. 31 ±0. 38 ）% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1. Conclusions 1. 812 U/Lgingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was clown-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2013年第5期272-277,共6页
Chinese Journal of Stomatology
基金
基金项目：国家自然科学基金（81170970）
留学回国人员科研启动基金（教启2009-134号）
广东省科技计划（20118031800259）
