脂多糖刺激人牙髓干细胞分泌的外泌体联合基质细胞衍生因子-1对牙髓再生的影响  

Effect of lipopolysaccharide-stimulated exosomes from human dental pulp stem cells combined with stromal cell-derived factor-1 on dental pulp regeneration

作　　者：蓝彬园 林熹 陈文瑨 谢婧 陈文霞[1] Lan Binyuan;Lin Xi;Chen Wenjin;Xie Jing;Chen Wenxia(Department of Operative Dentistry and Endodontics,College&Hospital of Stomatology,Guangxi Medical University&Guangxi Health Commission Key Laboratory of Prevention and Treatment for Oral Infectious Diseases&Guangxi Key Laboratory of Oral and Maxillofacial Rehabilitation and Reconstruction,Nanning 530021,China)

机构地区：[1]广西医科大学口腔医学院·附属口腔医院牙体牙髓科,广西壮族自治区卫生健康委员会口腔感染性疾病防治重点实验室,广西口腔颌面修复与重建研究重点实验室,南宁530021

出　　处：《中华口腔医学杂志》2022年第1期60-67,共8页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81760195)。

摘　　要：目的探讨轻度炎症状态下人牙髓干细胞(human dental pulp stem cell,hDPSC)产生的外泌体与基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)联合应用对牙髓组织再生的影响。方法分离培养hDPSC,脂多糖(lipopolysaccharide,LPS)刺激hDPSC,超速离心法提取hDPSC经LPS刺激后产生的外泌体(exosomes from lipopolysaccharide-stimulated hDPSC,L-EXO)和正常状态下分泌的外泌体(exosome from normal hDPSC,N-EXO),通过透射电镜和蛋白质印迹法鉴定提取物。将40只6~8周龄的SD大鼠通过随机数字表法分为S组(单独应用SDF-1)、L+S组(SDF-1与L-EXO联合应用)、N+S组(SDF-1与N-EXO联合应用)和空白对照组(根管内不植入任何物质),每组10只。以双侧下颌第一磨牙为实验牙,建立大鼠无髓根管模型,根据分组分别在根管内植入不同的内容物。植入后30 d过量麻醉处死所有大鼠,取大鼠双侧下颌骨组织,应用HE、Masson及免疫组织化学染色法进行组织学评价。结果HE染色结果显示,除空白对照组外,其他3组根管内均可见新生牙髓样组织,其中L+S组根管内新生组织的量及组织中的细胞数量最多,S组最少。Masson染色结果显示,L+S组矿化组织沿根管壁纵向排列,胶原纤维有序排列,N+S组呈无规律紊乱分布。定量分析各组新生血管面积,结果显示L+S组血管密度[(2.03±0.65)%]显著高于S组[(0.65±0.05)%]及N+S组[(1.06±0.38)%](F=5.879,P<0.05)。免疫组织化学结果显示,S组及L+S组的趋化因子受体4表达量显著低于N+S组(F=8.633,P<0.01)。结论hDPSC分泌的外泌体联合SDF-1可提高根管内新生组织的量和组织中的血管密度,L-EXO的作用较N-EXO强,并且新生组织中胶原纤维及矿化组织的排列更规律有序。Objective To investigate the effect of exosomes from mild-inflammation-stimulated human dental pulp stem cells(hDPSC)combined with stromal cell-derived factor-1(SDF-1)on dental pulp regeneration in rats.Methods Primary hDPSCs were isolated,cultured and then stimulated by using lipopolysaccharide(LPS).The exosomes from the hDPSCs with(L-EXO)or without(N-EXO)LPS were extracted by overspeed differential centrifugation and were identified by transmission electron microscopy and Western blotting.Forty SD rats,aged 6-8 weeks,were equally divided into S group(SDF-1 alone),L+S group(L-EXO combined with SDF-1),N+S group(N-EXO combined with SDF-1)and blank control group(no substance implanted into the root canal)by random number table method.Bilateral mandibular first molars were used as the experimental teeth to establish pulpless root canal models and different contents were implanted into the root canals according to the groups.All rats were over-anesthetized and sacrificed at the 30th day after content implantation.Bilateral mandibular tissues were taken for histological evaluation by means of HE,Masson and immunohistochemical stainings.Results The HE staining showed new pulp-like tissue in the root canals of all three experimental groups.The amount of new tissues and the number of cells in the tissues were greatest in L+S group and least in S group.Masson staining showed that the mineralized tissue in L+S group was arranged longitudinally along the root canal wall and the collagen fibers were arranged in an orderly fashion,while those in N+S group showed an irregular and disordered distribution.Quantitative analysis of the area of neovascularization in each group showed that the density of vessels in the L+S group[(2.03±0.65)%]was significantly higher than that in the S group[(0.65±0.05)%]and the N+S group[(1.06±0.38)%]respectively(F=5.879,P<0.05).Immunohistochemical staining showed that the expression of CXC chemokine receptor 4(CXCR4)was significantly lower in S and L+S groups than in N+S group,with a statistical

关 键 词：干细胞 外泌体 牙髓干细胞 脂多糖 基质细胞衍生因子-1 牙髓再生 

分 类 号：R78[医药卫生—口腔医学]