亚甲基蓝介导的光动力疗法诱导牙周炎中巨噬细胞凋亡的体内外研究

In vitro and in vivo study of apoptotic macrophages induced by methylene-blue-mediated photodynamic therapy in periodontitis

目的·探讨亚甲基蓝介导的光动力疗法(methylene blue-mediated photodynamic therapy,MB-PDT)能否诱导牙周炎组织中巨噬细胞的凋亡并抑制牙槽骨吸收。方法·体外实验中,将巨噬细胞分为3组:对照组(Control组),不接受治疗;MB组,只受MB处理;MB-PDT组,MB联合激光处理;检测各组细胞的凋亡情况及凋亡相关基因的表达。体内实验中,通过正畸钢丝结扎SD大鼠上颌双侧第一磨牙并接种牙周致病菌诱导牙周炎,6周后12只大鼠去除结扎丝并全部接受龈下刮治术和根面平整术(scaling and root planning,SRP),治疗后随机分为3组:SRP组,仅用生理盐水处理;MB组,仅用吩噻嗪染料MB处理;MB-PDT组,光敏剂联合激光治疗,每周治疗1次。所有大鼠在接受治疗3周后处死,采用免疫荧光实验和Micro CT分别检测各组大鼠牙周组织中巨噬细胞的凋亡情况和牙槽骨吸收水平。结果·体外实验结果显示10μmol/L MB和40 J/cm^2的光剂量组合可引起50%以上巨噬细胞死亡,且此模式下的MB-PDT诱导巨噬细胞发生凋亡的现象最明显,同时MB-PDT诱导巨噬细胞中促凋亡基因Bax/抑凋亡基因Bcl-2的比值上调。体内实验结果显示,经MB-PDT处理后,炎症牙周组织中浸润的巨噬细胞发生凋亡,而SRP组和MB组大鼠牙周组织中浸润的巨噬细胞未出现凋亡。Micro CT结果显示MB-PDT组大鼠的牙槽骨吸收程度较SRP组和MB组轻(P<0.05),而SRP组和MB组大鼠间无明显差异。结论·MB介导的光动力疗法可诱导牙周炎中过度增殖的巨噬细胞发生凋亡,并抑制牙槽骨吸收,较单纯的SRP治疗而言是一种良好的牙周炎辅助治疗方法。

Objective·To investigate whether methylene blue-mediated photodynamic therapy(MB-PDT)can induce the apoptosis of macrophages in periodontitis,and simultaneously reduce the bone absorption.Methods·For in vitro treatments,cells were divided into three experimental groups:control group,no treatment;MB group,methylene blue treatment;MB-PDT group,MB and laser irradiation treatment.Then apoptosis and apoptosis related genes were detected in each group.For in vivo treatments,periodontal disease in SD rats was orthodontic ligature and periodontal pathogen induced at the first maxillary molar.After 6 weeks,the ligature was removed and all animals received scaling and root planning(SRP)and were divided according to the following treatments:SRP group,saline solution;MB group,phenothiazinium dye;and MB-PDT group,MB and laser irradiation,once a week.All animals in each treatment were killed after 3 weeks.Immunofluorescence and Micro CT analyses were used to detect the apoptotic macrophages and alveolar bone resorption in periodontal tissues of rats in each group.Results·In vitro experiments showed that the combination of 10μmol/L MB and 40 J/cm^2 light dose could kill more than 50%macrophages,and the apoptosis of macrophages induced by MB-PDT in this mode was most obvious.Meanwhile,MB-PDT increased the expression ratio of proapoptotic gene Bax/antiapoptotic gene Bcl-2 in macrophages.In vivo experiments showed that the macrophages infiltrated in tissues with periodontitis were apoptotic after MB-PDT treatment,while no obvious apoptosis of macrophages infiltrated in the periodontal tissues of SRP and MB rats was found.Micro CT analysis showed that the alveolar bone resorption in MB-PDT rats was less than that in SRP and MB rats(P<0.05),and there was no significant difference between SRP rats and MB rats.Conclusion·MB-PDT can induce the apoptosis of hyperproliferating macrophages in periodontitis and reduce the bone absorption.Compared with SRP,MB-PDT is an effective adjunctive treatment of periodontitis.