自产氧抗菌光动力纳米体系对炎症调节和成骨分化的影响

Effect of oxygen self⁃sufficient nanoplatform on the anti⁃inflammation and osteogenesis of periodontitis

目的:探讨自产氧的抗菌光动力纳米体系--F@Ce6⁃M对炎症调节和成骨分化的影响。方法:构建F@Ce6⁃M并对其进行表征、生物安全性的评价。使用630 nm光照射牙龈卟啉单胞菌(P.gingivalis)菌液处理小鼠成纤维细胞L929,建立炎症模型,分别加入F@Ce6和F@Ce6⁃M并设立相应对照组。实时荧光定量PCR检测促炎因子和抗炎因子的基因表达水平,评价F@Ce6⁃M介导的抗菌光动力治疗(aPDT)对炎症调节的影响;通过免疫荧光检测NF⁃κB/p65亚基易位情况,分析F@Ce6⁃M的免疫调节机制;通过实时荧光定量PCR检测成骨相关基因表达,并采用碱性磷酸酶(ALP)进行染色,评价F@Ce6⁃M在炎症微环境中对前成骨细胞成骨分化的调控作用。结果:实时荧光定量PCR结果显示,与阴性对照组相比,F@Ce6⁃M介导aPDT能明显降低白细胞介素(IL)⁃6、IL⁃1β、肿瘤坏死因子(TNF)⁃α基因表达,并且效果明显优于F@Ce6⁃M未光照组(P<0.05);而各组间转化生长因子(TGF)⁃β1、精氨酸(Arg)⁃1和IL⁃10的基因表达差异无统计学意义(P>0.05)。在巨噬细胞中NF⁃κB/p65易位的免疫荧光实验中,F@Ce6⁃M光照组可抑制NF⁃κB/p65亚基转位(P<0.05)。对于成骨标志物基因表达水平,与阴性对照组相比,光照组ALP、Runt相关转录因子2(RUNX2)、Ⅰ型胶原蛋白(COL⁃1)、骨钙蛋白(OCN)基因表达量显著上升,F@Ce6⁃M光照组的基因表达量升高最为显著(P<0.05)。在ALP染色实验中,光照组的ALP活性水平显著上升,F@Ce6⁃M光照组ALP活性水平升高最为显著(P<0.05)。结论:自产氧的抗菌光动力纳米体系能够减少促炎因子的释放,并对成骨细胞的成骨潜能有显著的恢复作用,从而增强aPDT治疗牙周炎的效果。

Objective:To investigate the effect of self⁃oxygenated antibacterial photodynamic nanoplatform⁃F@Ce6⁃M on inflamma⁃tion regulation and osteogenic differentiation.Methods:The mouse fibroblast L929 was treated with Porphyromonas gingivalis(P.gin⁃givalis)bacterial solution by light irradiation,and the inflammation model was established.F@Ce6 and F@Ce6⁃M were respectively added,and the corresponding control group was set up.The gene expression levels of pro⁃inflammatory and anti⁃inflammatory factors were detected by qRT⁃PCR to evaluate the effect of F@Ce6⁃M mediated antimicrobial photodynamic therapy(aPDT)on inflammation regulation.NF⁃κB/p65 subunit translocation was detected by immunofluorescence to analyze the immunomodulatory mechanism of F@Ce6⁃M.The expression of osteoblast⁃related genes was detected by qRT⁃PCR and stained with ALP to evaluate the regulatory effect of F@Ce6⁃M on osteogenic differentiation of preosteoblasts.Results:qRT⁃PCR results showed that F@Ce6⁃M mediated aPDT signifi⁃cantly decreased the expression of interleukin(IL⁃6),IL⁃1β,tumor necrosis factor(TNF)⁃αgene compared with negative control group,and the effect was significantly better than that of F@Ce6⁃M no light group(P<0.05);There were no significant differences in the gene expressions of transforming growth factor(TGF)⁃β1,arginine(Arg)⁃1 and IL⁃10 among all groups(P>0.05).F@Ce6⁃M light group inhibited NF⁃κB/p65 subunit translocation(P<0.05).The osteogenic marker expressions of ALP,runt⁃related transcription factor 2(RUNX2),collagen typeⅠ(COL⁃1)and osteocalcin(OCN)in light group were significantly increased compared with nega⁃tive control group,and the gene expression levels in F@Ce6⁃M group were the most significantly increased(P<0.05).The activity lev⁃el of ALP in the light group was significantly increased,the most significantly increased in the F@Ce6⁃M light group(P<0.05).Con⁃clusions:F@Ce6⁃M could reduce the release of pro⁃inflammatory factors,and significantly restore the osteogenic potential of osteo⁃blasts,thereby enhancing the effect of aPDT on periodontitis.