【作者】 张晓静；

【导师】 许彦枝；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2013， 硕士

【摘要】 目的：牙周炎是侵犯牙龈和牙周组织的慢性炎症，是一种细菌感染的破坏性疾病，其主要特征为牙周袋的形成及袋壁的炎症，牙槽骨吸收和牙齿逐渐松动。目前牙周病的治疗方法很多，在临床上虽取得了一定成效，但均未取得理想的效果。如何使牙周病治疗获得牙周新附着，实现牙周组织的再生修复,一直是人们努力的方向，近年来，牙周组织工程的出现及其技术的不断完备，为牙周组织工程在牙周病的治疗及牙周组织缺损再生修复方面提供了崭新的研究空间。牙周组织工程包括三大要素即具有增殖分化潜能的种子细胞、促进细胞增殖分化的生长因子以及利于细胞粘附生长的细胞外基质（支架）材料。牙周膜细胞（periodontal ligament cells，PDLCs）是牙周组织工程中理想的种子细胞，在适宜的刺激下，可分化为成纤维细胞、成骨细胞和成牙骨质细胞,实现牙周病变组织的再生修复，但PDLCs来源有限，须拔除一定量自体牙后才能获得，且其培养成功率相对较低而不能被广泛应用。目前研究发现牙龈成纤维细胞在一定刺激下也具有一定的成骨表型，且因取材方便、创伤小、繁殖能力强、培养成功率高而成为牙周组织工程种子细胞研究的热点。中草药因对人体刺激小、毒副作用小等特点得到人们的关注，中药双黄补由黄连、黄芩、骨碎补按一定比例熬制而成,其中黄连提取物及黄芩提取物对牙周炎致病菌有明显抑制作用，并且适当浓度的骨碎补也可增强牙龈成纤维细胞的繁殖能力及诱导其成骨表型。本实验以双黄补作为牙周组织工程中的生长因子，通过构建牙龈成纤维细胞（Gingival fibroblasts，GF）-玉米醇溶蛋白支架（Zein Scaffold）-双黄补复合体并应用于比格犬牙周缺损模型中，探讨双黄补在牙周组织工程中对牙周组织再生修复的作用，为双黄补在临床治疗牙周病提供实验依据和理论基础。方法：1牙龈成纤维细胞培养及来源鉴定将切取的Beagle犬颊侧牙龈组织，转移至无菌环境，DMEM冲洗3次，然后移入含少量DMEM的培养皿中，将其剪成1mm×1mm×1mm大小的组织块，采用改良组织块法培养牙龈成纤维细胞。取生长状态良好的第3代细胞，免疫组化ABC法进行波形丝蛋白和角蛋白染色鉴定其来源,PBS代替一抗作阴性对照，光镜下观察。2玉米醇溶蛋白支架制备将玉米醇溶蛋白溶于酒精溶液后，加入氯化钠致孔剂，采用溶剂浇铸/粒子沥滤法制备支架，将支架切成5mm×4mm×1mm大小，紫外线消毒2小时备用。3双黄补培养液的制备将黄连、黄芩、骨碎补按一定比例混合，蒸馏水浸泡30min，分别加8倍体积及6倍体积的水煎煮两次，水提醇沉法配制成1g/ml生药的双黄补煎液，活性炭脱色，调节PH值为7.07.1，滤膜过滤除菌，4℃保存备用。4牙龈成纤维细胞-支架复合体及牙龈成纤维细胞-支架-双黄补复合体制备37℃下，将玉米醇溶蛋白支架分别置于含10%FBS的DMEM培养液及含100μg/ml质量浓度的中药双黄补培养液的96孔板中浸提24小时，取第4代生长良好的细胞，以1.5×105的细胞浓度接种于支架上，扫描电镜下观察细胞在支架粘附情况，以备用于动物实验。5动物实验选用健康雄性清洁级Beagle犬2只，体重约15Kg，月龄6-12月,以每犬上颌5445，下颌87433478作为手术部位，每组5个牙位，在每牙位颊侧制备牙周缺损,分别将Zein支架、GF-Zein复合体及GF-Zein-双黄补复合体植入相应牙位，空白对照组不做任何处理，间断缝合牙龈组织。术后3个月，取88标本行扫描电镜观察，其余标本行组织学观察及测量（组织学测量指标：骨缺损高度、新生牙槽骨高度、新生牙骨质高度、结合上皮长度）以及免疫组织化学染色定量分析胰岛素样生长因子（insulinlike growth factor-1，IGF-l）及骨桥蛋白（osteopontin，OPN）的表达。6统计学方法所有数据均以X--±S表示，采用SPSS13.0统计软件进行统计学分析，采用方差分析比较各组缺损区牙槽骨生成的差异以及比较各组IGF-1和OPN阳性细胞率的差异，P﹤0.05有统计学意义。结果：1牙龈成纤维细胞培养及来源鉴定牙龈成纤维细胞大约在培养的第7天由组织块游出，围绕组织快周围呈放射状排列，细胞呈长梭形，胞体丰满，胞浆均匀，可见多个胞浆突，核圆形或卵圆形。经免疫组织化学ABC法染色后，显示抗波形蛋白染色阳性、抗角蛋白染色阴性，证实为中胚层来源,，符合成纤维细胞的特性，可用于实验。2牙龈成纤维细胞-支架复合体及牙龈成纤维细胞-支架-双黄补复合体扫描电镜观察扫描电镜下可见，双黄补作用下粘附在玉米醇溶蛋白支架上的牙龈成纤维细胞数目较多，呈长梭形或星形，伸出伪足并横跨孔隙交织成网状。无双黄补诱导作用下粘附在玉米醇溶蛋白支架上的细胞数目较少，多呈星形，且主要黏附生长在孔隙陷窝内。3牙体牙周联合标本观察3.1组织学观察术后3个月, GF-Zein-双黄补复合体组与其余三组相比，牙周组织中炎症细胞明显减少，可见再生的牙槽骨及牙骨质。3.2组织学测量术后3个月，GF-Zein-双黄补复合体组新生牙槽骨生长高度（1.16±0.06）mm较其余三组明显增高（P0.05）。而新生牙骨质高度与结合上皮长度各组之间无显著差别（P0.05）。3.3扫描电镜观察术后3个月，GF-Zein-双黄补复合体组的再生牙槽骨较其余三组可见排列规则的骨小梁，趋于成熟的哈弗氏系统。3.4免疫组化行IGF-l及OPN定量分析术后3个月，GF-Zein-双黄补复合体组新生牙槽骨周IGF-l的阳性细胞率(24.32±1.85)%高于其余组的阳性细胞率，有统计学意义（P0.05）。GF-Zein-双黄补复合体组新生牙槽骨周围OPN的阳性细胞率(12.68±1.25)%与空白对照组相比有统计学意义（P0.05），而GF-Zein-双黄补复合体组与GF-Zein复合体组及Zein组新生牙槽骨周围OPN的阳性细胞率相比无统计学意义（P0.05），但是GF-Zein-双黄补复合体组新生牙槽骨周围可见阳性表达的成骨细胞数目明显较多。结论：1双黄补在牙周组织工程中可有效控制牙周组织中炎症反应，为牙周组织修复提供良好环境。2双黄补牙周组织工程中可刺激牙龈成纤维细胞向成骨细胞分化，促进IGF-l及OPN在牙周组织的表达，利于牙周缺损处牙槽骨及牙骨质的修复再生。3双黄补可以作为一种牙周组织工程的生长因子，具有良好的临床应用前景。更多还原

【Abstract】 Objective：Periodontitis is bacterial infectious diseases for the characteristic,andresult in formation of periodontal pocket and absorption of alveolarbone,leading to lost of teeth.Nowadays,the routine clinical treatments failed toachieve the ideal effect.How to achieve the regeneration of periodontalsupporting tissue,including aveolar bone,cementum,and periodontalligament,is the direction of us. As the progress of technology of tissueengineering,it has been introduced into the field of periodontology,whichprovides a new research space for the treatment of periodontitis.Periodontal tissue engineering involve three basic biological factors,which are seed cells,differentiation inducing factors and matrical materials.Periodontal ligament (PDL) cell is regarded as the ideal cell to regenerateperiodontal tissues,because of their potential of differentiation to fibroblasts,steoblasts,and cementoblasts under appropriate stimulus.However,PDLCs aredifficult to carried out in clinic,and the success rate of cell culture is relativelylow.Nowadays,some researchers has found that gingival fibroblast cells showsome characters of osteoblasts by some bone-inducing factors,easier to beobtained clinically than PDLCs,and have strong proliferation potential.Now,people pay more and more attention to radix scutellariae for its smallstimulations and side effects. Shuanghuangbu as traditional chinese medicinemainly includes Rhizoma coptidis,Radix scutellariae and Rhizona drynariae inthe reasonable proportion.Among them,rhizoma coptidis and radix scutellariaecould inhibit periodontal pathogenic bacteria obviously. Rhizoma drynariaecould promote proliferation and differentiation of human gingival fibroblastcells into osteoblasts.In our study,shuanghuangbu as a growth factor in periodontal tissue engineering,establish the complex of GF-Zein-shuanghuangbu and implant inthe model of periodontal defects,in order to investigate the effect ofshuanghuangbu on periodontal regeneration and provide experimental basisand theoretical basis for clinical treatment of periodontitis.Methods:1The cultivation of gingival fibroblasts and source identificationGingival tissues romoved from buccal gums of beagle dog weretransferred to sterile environment,washed three times with DMEM,and thentransferred to petri dish.The tissue block was cut into1mm×1mm×1mm piecesand used for primary cultures with the tissue block cultivation.Selecting the third passage cells,keratin staining and vimentin staining byimmunohistochemical and ABC staining identified the source.2Preparation of zein scaffoldsZein dissolved in alcohol.Nacl as porogen was added into the solution.Zein scaffolds were prepared by solvent casting/particle leaching, irradiatedfor2hours under UV and stored for using.3Preparation of shuanghuangbuAccording a certain proportion,coptidis rhizome,scutellariae radix andrhizoma drynariae were mixed together.The mixture was soaked in distilledwater for30minutes and then boiled respectively in8volumes and6volumesof water.Shuanghuangbu was prepared to concentration of1g/ml with waterextraction and alcohol precipitation method,discolored by active carbons forthree times,adjusted pH to7.0-7.1,stored at4℃.4Preparation of GF-Zein complexs and GF-Zein-shuanghaungbu complexThe Zein scaffold were soaked in DMEM containing10%FBS and inshuanghuangbu at37℃for24h in a96-well plate.The forth passage cells werecollected and made into cell suspensions at1.5×105/ml to implant into the zeinscaffolds,and then observed under scanning electron microscope.5Animal experimentTow male clean beagle dogs weighted about15kg and aged6-12monthswere selected in this study. Prepare model of periodontal defects at buccal alveolar bone of the4th and5th premolars of double upper jaws and the3rd,4th,7th and8th of double lower jaw.Zein, GF-Zein complex and GF–Zein-shuanghuangbu complex were separately implanted into orrespondingmodel,while the control group did not give any disposition.Then sutured thegingival tissues.3months after surgery, animals were sacrificed.Collect aspecimen of88in each group to be viewed under scanning electronmicroscope,and the other specimens were evaluated by histology observationand histological measurement (experimental defect,new alveolar boneformation, new cementum formation,the lenghth of junctional epithelium) andimmunohistochemical staining quantitative analysis (insulin like growthfactor-1, osteopontin).6Statistical methodsAll datas are expressed byX--±s, Using SPSS13.0statistical software forstatistical analysis. The amount of alveolar bone formation and the positiverate of insulin like growth factor-1(IGF-1) and osteopontin(OPN) inperiodontic area were compared with a single factor analysis of variance.There is statistical significance when probability is less than0.05.Results:1Morphological observation and source identificationThe gingival fibroblasts cultured with tissue bloke cultivation takethe shape of long spindle and the cell body were plump. After immunohisto-chemical staining,the the anti-vimentin staining was positive,and anti-keratinstaining was negative，which confirmed the cells originated from mesoderm.2Observations of GF adhered to zein scaffolds under scanning electronmicroscopeThrough scanning electron microscopy, the number of gingivalfibroblasts adhered to Zein scaffolds was more under the action of shuang-huangbu,while the number was less without the action of shuanghuangbu.3Dontal and periodontal specimen observation3.1Histology observationThree months after surgery, compared with the rest of the three groups， inflammatory cells were not observed in GF-Zein-shuanghuangbu complexgroups,while New bone and cementum was obviously observed.3.2Histological measurementsThree months after surgery,height of NB in GF-Zein-shuanghuangbucomplex group(1.16±0.06)mm was obviously larger than that in other groups（P<0.05）,while Height of NC and the length of JE has no significantdifference in the four groups（P<0.05）.3.3SEM observationThree months after surgery, trabeculae arranged in order and maturehaversian system were viewed in the GF-Zein-shuanghuangbu complexgroups under SEM.3.4Quantative analysis of IGF-l and OPN in periodontal tissueThree months after surgery, the cell positive rate of IGF-1surroundingnew alveolar bone of GF-Zein-shuanghuangbu complex groups(24.32±1.85)%was significantly higher than the rest of the three groups（P0.05）.The cellpositive rate of OPN surrounding new alveolar bone are significant differencebetween the GF-Zein-shuanghuangbu complex groups(12.68±1.25)%and thecontrol groups(P0.05).The cell positive rate of OPN in periodontic surround-ding new alveolar bone didn’t see anything statistically significant among thegroups of Zein,GF-Zein complex and GF-Zein-shuanghuangbu complex (P0.05),but in GF-Zein-shuanghuangbu complex groups,the number of OPNpositive cells was more.Conclusions：1Shuanghuangbu can effectively control the inflammatory reaction inperiodontal tissues, thus providing a well environment for periodontal tissuerepairing in periodontal tissue engineering.2Shuanghuangbu can stimulates gingival fibroblast cells transform intothe osteoblast and increase the expression of IGF-l and OPN in periodontaltissue,which benefit the regeneration of alveolar bone and cementum.3Shuanghuangbu can serve as a growth factor in periodontal tissueengineering, and has a good prospect in clinical application.更多还原