中文摘要：

目的探讨牙龈蛋白酶在诱导成骨细胞凋亡过程中对整合素α5、β1的调节机制，为牙周炎发病机制的研究提供理论依据。方法标准厌氧环境下培养牙龈卟啉单胞菌W83，分离提纯牙龈蛋白酶。用8．348U／L牙龈蛋白酶处理小鼠成骨细胞MC3T3-E148h，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法4’，6-二脒基．2一苯基吲哚[transferase—mediateddeoxyuridinetriphosphate—biotinnickendlabeling-（4-Amidinophenyl）-6-indolecarbamidinedihydrochloride，TUNEL—DAPI]双染色法检测细胞凋亡；蛋白质印迹法检测整合素α5、β1蛋白的表达水平。结果精氨酸．蛋白酶活性为（41．74±2．11）U／L，赖氨酸一蛋白酶活性为（1．02±0．25）U／L。TUNEL—DAPI染色显示牙龈蛋白酶作用于MC3T3一E1细胞48h后诱导细胞发生凋亡。在短时间内（≤72h）牙龈蛋白酶呈时间依赖性下调整合素α5、β1的表达水平，48h时整合素α5、β1相对表达量分别为（0．485±0．039）、（0．504±0．002），72h时分别为（0．398±0．058）、（0．179±0．001），与对照组（蛋白相对表达量分别为1．000±0．000、1．000±0．000）相比差异均有统计学意义（P〈0．05）；72h后，整合素α5表达水平与对照组相比差异无统计学意义，但整合素B1仍持续下调（96、120h时相对表达量分别为0．604±0．003、0．357±0．002），均显著低于对照组（1．000±0．000）（P〈0．05）。牙龈蛋白酶特异性抑制剂甲苯磺酰．L．赖氨酰．氯甲基酮（tosyl．CL—clysine-chloromethyl—ketone，TLCK）可有效抑制蛋白酶活性，使整合素α5、β1蛋白表达量分别从（0．398士0．058）、（0．179±0．001）显著上升至（0．7814-0．012）、（0．857±0．060）（P〈0．05），但TLCK本身不改变整合素α5、β1的表达水平（P〉0．05）。同时牙龈蛋白酶还呈剂量依赖性下调整合索α5、β1的表达水平，20．8700U／L牙龈?

英文摘要：

Objective To investigate the regulatory mechanisms of integrin a5 and B1 in osteoblast in the process of gingipains-induced apoptosis. Methods Gingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions. MC3T3-E1 was challenged with or without 8. 3480 U/L gingipains for 48 h and apoptosis was examined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-（4-Amidinophenyl）-6- indolecarbamidinc dihydrochloride （TUNEL-DAPI） staining. The expression of integrin ct5 and [M was analyzed by Western blotting after MC3T3-E1 was treated under different conditions. Results Arginine-specific proteinases （ Rgp ） activity was （ 41.74 ± 2. 11 ） U/L and lysine-specific proteinase （ Kgp ） was （ 1.02 ± 0. 25 ） U/L. Gingipains induced MC3T3-E1 cells apoptosis after 48 h. Compared with control group, expression of integrin a5 and B1 was down-regulated by gingipains in a time-dependent manner within short periods （ 〈72 h）, integrin ct5 and B1 relative expression was （0. 485 ± 0. 039 ）, （0. 504 ± 0. 002） at 48 h, （0. 398±0. 058）, （0. 179 ＋0. 001 ） at 72 h respectively （P 〈0.05）. After 72 h,integrin a5 expression in MC3T3-E1 cells was stable compared with control group while integrin B1 was still lower（ control group： 1. 000 ± 0. 000,96 h ： 0. 604 ± 0. 003,120 h ： 0. 357 -＋ 0. 002 ） （ P 〈 0. 05 ）. Proteinase inhibitor tosyl- L- lysine-chloromethyl-ketone（TLCK） effectively blocked the activity of gingipain and inhibited down-regulation of in tegrlna5 and B1 induced by gingipains from （0. 398 ± 0. 058,0. 179 ± 0. 001 ） to （0. 781 ± 0. 012, 0. 857 ± 0. 060 ） （ P 〈 0. 05 ）. TLCK alone did not have any effect on integrin a5 and B1 （ P 〉 0.05 ）. Gingipains also decreased integrin a5 and B1 in a dose-dependent manner. When cells were treated with 20. 8700 U/L gingipains, integrin A5 and B1 relative expression reached to the lowest （