甲氨蝶呤载药囊泡在小鼠实验性牙周炎治疗中的作用研究  

Study of the methotrexate loaded extracellular vesicles in the treatment of experimental periodontitis in mice

作　　者：杨建花 何小宁 刘治 王文哲 李蓓 

机构地区：[1]第四军医大学口腔医院口腔组织病理学教研室、口颌系统重建与再生全国重点实验室、国家口腔疾病临床医学研究中心、陕西省口腔疾病国际联合研究中心,西安710032 [2]第四军医大学口腔医院口腔正畸科、口颌系统重建与再生全国重点实验室、国家口腔疾病临床医学研究中心、陕西省口腔疾病临床医学研究中心,西安710032

出　　处：《中华口腔医学杂志》2024年第7期681-689,共9页Chinese Journal of Stomatology

基　　金：国家重点研发计划(2021YFA1100600)。

摘　　要：目的探究甲氨蝶呤载药囊泡对小鼠实验性牙周炎的治疗作用。方法分离人脐带间充质干细胞(hUC-MSC)的细胞外囊泡(EVs)。构建甲氨蝶呤(MTX)载药囊泡(MTX-EVs)并通过扫描电镜及动态光散射仪(DLS)对构建的载药囊泡进行形态大小分析,通过蛋白质印迹法对其表面特异性蛋白进行鉴定。选取4~5周C57BL/6J雄性小鼠,其中通过盲抓法随机抽取8只不做处理,正常饲养作为对照组(由第四军医大学实验动物中心提供),其余小鼠利用脂多糖(LPS)(2 g/L,5μl)牙周局部注射诱导小鼠牙周炎模型,间隔1天注射1次,诱导2周。将成功诱导牙周炎模型小鼠通过盲抓法随机分为4组,每组8只。分别为LPS组、LPS+MTX组、LPS+EVs组及LPS+MTX-EVs组。牙周局部治疗2周后,酶联免疫吸附测定(ELISA)检测牙龈组织炎症因子白细胞介素(IL)-1β、IL-6及肿瘤坏死因子α(TNF-α)的质量浓度。通过显微CT(micro-CT)、HE染色评估对照组、LPS组、LPS+MTX组、LPS+EVs组及LPS+MTX-EVs组小鼠的牙槽骨吸收情况。流式细胞仪分析牙龈组织中γ干扰素(IFN-γ)阳性细胞的占比。结果扫描电镜结果显示EVs和MTX-EVs呈圆形或椭圆形,DLS粒径分析证明EVs粒径在200 nm左右,MTX-EVs粒径在300 nm左右。ELISA结果显示,对照组、LPS组、LPS+MTX组、LPS+EVs组及LPS+MTX-EVs组IL-1β质量浓度分别为(28.86±2.76)、(51.50±2.04)、(35.26±2.40)、(45.49±2.04)、(35.77±3.49)ng/L;LPS+MTX组、LPS+EVs组及LPS+MTX-EVs组IL-1β质量浓度均显著低于LPS组(均P<0.05);LPS+MTX-EVs组IL-1β质量浓度显著低于LPS+EVs组(P<0.05)。对照组、LPS组、LPS+MTX组、LPS+EVs组及LPS+MTX-EVs组IL-6质量浓度分别为(125.44±4.12)、(221.64±10.59)、(178.16±16.90)、(181.09±18.22)、(170.15±9.04)ng/L;LPS+MTX组、LPS+EVs组及LPS+MTX-EVs组IL-6质量浓度均显著低于LPS组(均P<0.05);LPS+MTX-EVs组IL-6质量浓度显著低于LPS+EVs组和LPS+MTX组(均P<0.05)。对照组、LPS组、LPS+MTX组、LPS+EVs组及LPS+MTObjective To investigate the therapeutic effect of methotrexate loaded vesicles on experimental periodontitis in mice.Methods Extracellular vesicles(EVs)were isolated from human umbilical cord mesenchymal stem cells(hUC-MSC).Methotrexate loaded vesicles(MTX-EVs)were constructed,whose morphology and size were analyzed by using scanning electron microscopy and particle size analyzer.Western blotting was used to identify their surface specific proteins.C57BL/6J male mice of 4-5 weeks(provided by Experimental Animal Center of The Fourth Military Medical University)were selected,among which 8 were randomly selected by blind grasp method without treatment and fed normally as normal group,and others were induced to periodontitis models by local injection of lipopolysaccharide(LPS)into the periodontium.The LPS was injected once every day with a concentration of 2 g/L and a volume of 5μl,lasting for two weeks.The mice with successfully induced periodontitis were randomly divided into 4 groups by blind grasping method,with 8 mice in each group.The LPS group was with no treatment,and the other three groups were treated with periodontal local injection of MTX,EVs or MTX-EVs,respectively.Two weeks later,enzyme-linked immunosorbent assay(ELISA)was used to detect the expressions of inflammatory cytokine interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)in gingival tissue.The amount of alveolar bone resorption of four groups was detected by using micro-CT scanning and HE staining.The expression proportion of the inflammatory factor in gingival tissue was analyzed by using flow cytometry.Results The scanning electron microscopy results showed that EVs and MTX-EVs were circular or elliptical in shape.Dynamic light scattering(DLS)particle size analysis showed that the particle size of EVs was around 200 nm,while that of MTX-EVs was around 300 nm.The ELISA results showed IL-1βlevels in the normal group,LPS group,LPS+MTX group,LPS+EVs group and LPS+MTX-EVs group were(28.86±2.76),(51.50±2.04),(35.26±2.40),(45.49±2.04)

关 键 词：牙周炎 细胞外囊泡 甲氨蝶呤载药囊泡 牙槽骨质丢失 促炎因子 

分 类 号：R781.42[医药卫生—口腔医学]