【作者】 王敏；

【导师】 王萍；

【作者基本信息】 重庆医科大学 ， 口腔临床医学， 2022， 硕士

【摘要】 牙周炎与糖尿病是临床上常见的疾病,都可对人类的健康产生极大的危害,且两者之间可以相互影响。一方面,糖尿病患者体内的高糖环境可以使牙周组织中的微生物群发生过度的炎症反应,从而使破骨活动增强,且骨修复活动减弱,导致骨代谢的平衡失调,加速牙周组织的破坏;另一方面,牙周炎不利于糖尿病患者血糖的控制,重度牙周炎可使糖尿病患者血糖进一步增高。临床上对于伴随糖尿病的牙周炎患者的治疗方式,通常采用的是比较传统的牙周治疗方法,主要包括牙周刮治术(龈上洁治,龈下刮治术)、根面平整术等,并在此基础上联合使用抗生素及降糖药物。传统的治疗方法往往只能减缓牙槽骨的吸收,而无法重建已经破坏的牙周组织。近些年来,随着组织工程学技术飞速发展,牙周再生治疗成为了当今热门的研究项目。如今较成熟的牙周组织再生技术,是通过种子细胞、生物活性因子以及支架材料三者的联合应用从而诱导形成新的牙骨质、牙槽骨及牙周膜纤维。牙周膜干细胞(Periodontal Ligament Stem Cells,PDLSCs)是存在于牙周组织中的一种成体干细胞,具有间充质干细胞的特性,因其取材较容易且避免伦理争议,有望对临床上牙周组织再生技术的应用发挥重大作用。但糖尿病患者的高糖状态往往会影响PDLSCs的生物学功能,阻碍牙周组织修复再生,降低组织工程的成功率。因此对糖尿患者来说,提升高糖状态下PDLSCs的成骨分化能力对牙周组织修复再生显得尤为重要。Exendin-4是GLP-1(胰高血糖素样肽-1)类似物,与GLP-1具有53%的同源性,它可以通过刺激胰腺β细胞分泌胰岛素,降低血糖从而控制血糖水平,目前在临床上广泛用于治疗糖尿病患者。研究发现,Ex-4除具血糖调控作用外,还是骨骼生长和重塑的重要调节因子。动物实验表明,Ex-4和GLP-1受体促进骨髓干细胞和MC3T3-E1成骨细胞前期的成骨分化。我们小组前期实验也表明,10nM Ex-4能够缓解高糖环境下PDLSCs的成骨抑制作用。MAPK通路及WNT通路对细胞的多种生物学过程,包括生长,发育,代谢平衡等都发挥着重要作用。研究发现,MAPK通路对成骨细胞的分化以及骨骼发育的调节和控制尤为重要,Wnt/β-catenin通路在成骨细胞中也发挥着重要作用,可通过上调成骨调节因子,调节骨骼发育及增加骨量。目的:本实验用30mM葡萄糖浓度模拟了体外的高糖环境,探究高糖环境下Ex-4对PDLSCs的成骨分化能力的影响,并进一步探究MAPK通路及WNT通路在PDLSCs成骨分化中发挥的作用,为糖尿病伴牙周炎患者的牙周组织修复再生提供理论依据。方法:第一部分:通过酶组织块消化法获得原代PDLSCs,并对其进行成骨诱导、成脂诱导及单细胞克隆实验检测其干细胞特性。第二部分:选取30mM葡萄糖浓度模拟高糖环境,将PDLSCs分为以下4组:NG(对照组,5.5mM葡萄糖浓度),NG+10nM Ex-4,HG(高糖组,30mM葡萄糖浓度),HG+10nM Ex-4。成骨培养7天后进行碱性磷酸酶(ALP)染色,成骨培养21天后进行茜素红染色,成骨培养7天,14天后通过qRT-PCR实验检测成骨相关基因ALP、Runx2、Osx 的 mRNA 表达。第三部分:1.用 10nM Ex-4 作用于 PDLSCs 0,0.5,1,1.5,2,3h 后,Western-blot检测p38,p-p38,ERK1/2,p-ERK1/2,JNK,p-JNK蛋白的表达,探究Ex-4作用于MAPK通路的时间。2.使用选择性 MAPK 抑制剂,即5μM PD98059、10μM SB203580和 25μM SP600125 处理人 PDLSCs,Western-blot 检测p38,ERK1/2,JNK磷酸化水平;成骨诱导7天后qRT-PCR检测成骨相关基因Runx2、ALP、Osx mRNA表达水平。第四部分:将PDLSCs分为以下四组,NG,NG+10nM Ex-4,HG,HG+10nM Ex-4,成骨培养 7 天后 Western-blot 检测β-catenin,GSK3β,P-GSK3β,LEF,Runx2 的蛋白表达。结果:第一部分:1.成功分离提取出原代PDLSCs,镜下显示细胞呈长梭形生长。2.克隆实验显示生长2周的PDLSCs形成克隆簇,密度较大。3.成脂诱导14天后,油红0染色显示有脂滴形成;成骨诱导21天后,茜素红染色显示有钙结节形成。第二部分:1.成骨诱导7天后ALP染色显示,与对照组(NG)相比,NG+Ex-4组ALP染色深且面积大,HG组染色浅,HG+Ex-4组也较NG组染色浅。与HG组相比,HG+Ex-4组ALP染色较深。2.成骨培养21天后茜素红染色显示,与NG相比,NG+Ex-4组的钙化结节增加,HG组的钙化结节显著减少,HG+Ex-4组的钙化结节也较NG组减少;与HG组比较,HG+Ex-4组的矿化结节增加。3.qRT-PCR结果显示,成骨培养第7天时,与对照组(NG)相比,NG+Ex-4 组的 Osx mRNA 表达增加,HG 组的 Runx2、Osx mRNA 表达减少。与HG组相比,HG+Ex-4组的ALP表达增加。在第14天时,与NG相比,NG+Ex-4的ALP、Runx2和Osx的表达都增加,HG组的ALP、Runx2的表达显著下降。与HG组相比,HG+Ex-4组的ALP、Runx2、Osx的表达都显著增加(P<0.05)。第三部分:1.Western-blot结果显示Ex-4在短时间内激活了 MAPK通路,其中,p38和ERK1/2磷酸化分别在约1h,0.5h达到峰值,JNK磷酸化在约1.5h达到峰值。2.加入MAPK抑制剂后,Western-blot结果显示与NG组相比,NG+Ex-4组PDLSCs的p38,ERK1/2,JNK的磷酸化增加,但HG组的ERK1/2磷酸化下降。与HG组相比,HG+Ex-4组的p38,ERK1/2,JNK的磷酸化增加。MAPK抑制剂有效抑制了 MAPK通路的磷酸化。3.加入MAPK抑制剂,成骨培养7天后,qRT-PCR结果显示,与NG+Ex-4 组相比,NG+Ex-4+SB203580 组的 ALP、Runx2 和 Osx 的mRNA水平分别降低约92%,72%和82%,与HG+Ex-4比,HG+Ex-4+SB203580 组的 ALP、Runx2 和 Osx 的 mRNA 水平降低约80%、6g%和 71%。同样,加入 SP600125 抑制剂后,与 NG+Ex-4 组相比,ALP,Runx2和OSX的mRNA水平分别降低约98%,53%和28%,与 HG+Ex-4 相比,则分别降低约 95%,38%和20%。PD98059同样降低了所有成骨基因mRNA表达,与NG+Ex-4组相比,ALP、Runx2和OSX的表达分别降低约8g%、70%和73%,与HG+Ex-4组比分别降低约 5g%、65%和38%(p<0.05)。第四部分:Western-blot结果显示与NG组相比,NG+Ex-4组p-GSK3β,β-catenin,LEF和Runx2的蛋白表达量增加,HG组的β-catenin,LEF和Runx2的蛋白表达量降低。与HG组相比,HG+Ex-4组的p-GSK3β,β-catenin,LEF和Runx2的蛋白表达量也都增加。结论:1.10nM Ex-4能够缓解高糖环境对PDLSCs的成骨抑制作用,并促进正常糖环境下PDLSCs的成骨分化。2.Ex-4能够通过激活MAPK通路促进高糖环境下PDLSCs的成骨。3.Ex-4可能通过调控WNT通路来促进高糖环境下PDLSCs的成骨更多还原

【Abstract】 Periodontitis and diabetes are common diseases endangering human health,and there is a two-way relationship between them.On the one hand,diabetes can lead to excessive inflammatory reaction of periodontal microflora,which can enhance osteoclast activity and weaken bone repair,leading to imbalance of bone metabolism,thus accelerating periodontal destruction.On the other hand,periodontitis has an adverse effect on diabetic blood sugar control,and severe periodontitis can further increase the blood sugar of diabetic patients.At present,the treatment of periodontitis patients with diabetes mainly adopts conventional periodontal treatment methods such as periodontal scaling and root planing,and on this basis,antibiotics and hypoglycemic drugs are combined.In recent years,with the increasingly rapid progress of tissue engineering and regenerative medicine technologies,periodontal regeneration has been become a popular research area.Among them,the combined application of seed cells,bioactive factors and scaffold materials to guide periodontal tissue regeneration is a mature method to form new cementum and reshape periodontal fibers.As the most promising seed cells of periodontal tissue engineering at present,periodontal ligament stem cells(PDLSCs)are expected to play an important role in the clinical application of periodontal tissue repair and regeneration because of their easy availability of materials and multi-directional differentiation potential.However,the high glucose state of diabetic patients often affects the differentiation of PDLSCs,hinders the repair of periodontal tissue and reduces the regeneration rate of tissue engineering.Therefore,how to improve the osteogenic ability of PDLSCs under high glucose state is particularly important for the regeneration of periodontal tissue of diabetic patients.Exendin-4(Ex-4)is an analog of GLP-1(glucagon-like peptide-1),which has 53%homology with GLP-1.Ex-4 can control the blood sugar level by stimulating pancreatic beta cells to secrete insulin and lowering blood sugar.At present,it is widely used in the clinical treatment of type 2 diabetes.Studies have found that Ex-4 is an important regulator of bone growth and remodeling in addition to its blood sugar regulation.Animal experiments showed that Ex-4 and GLP-1 receptors promoted the early osteogenic differentiation of bone marrow stem cells and MC3T3-E1 osteoblasts.Our group’s previous experiments also showed that 10 nmolEx-4 could alleviate the osteogenic inhibition of PDLSCs under high glucose environment.MAPK pathway and WNT pathway play an important role in many biological processes such as cell growth,development and metabolic balance.It is found that MAPK pathway is particularly important for the differentiation of osteoblasts and the regulation and control of bone development.Ex-4 can promote the osteogenic differentiation of MC3T3-E1 cells by activating MAPK pathway.Wnt/β-catenin pathway can promote the differentiation of osteoblasts by up-regulating osteogenic regulatory factors,and also plays an important role in regulating bone development and increasing bone mass.Objective:In this experiment,30mM glucose concentration was used to simulate the high glucose environment in vitro,to explore the influence of Ex-4 on the osteogenic differentiation of PDLSCs under high glucose environment,and to further explore the role of MAPK pathway and WNT pathway in the osteogenic differentiation of PDLSCs,so as to provide theoretical support and experimental basis for periodontal tissue repair and regeneration of patients with diabetes mellitus and periodontitis.Method:Part I:Primary PDLSCs were obtained by enzyme tissue block digestion,and their stem cell characteristics were detected by osteogenesis induction,adipogenesis induction and single cell cloning experiments.Part II:Select 30mM glucose concentration to simulate high glucose environment,and divide PDLSCs into the following four groups:NG(control group,5.5mM glucose concentration),NG+10nM Ex-4,HG(high glucose group,30mM glucose concentration),HG+10nM Ex-4.After 7,14 and 21 days of osteogenic culture,respectively,alkaline phosphatase(ALP)staining,alizarin red staining and real-time quantitative polymerase chain reaction(qRT-PCR)were used to detect the expression of osteogenic related genes ALP,Runx2 and Osx.Part III:1.After PDLSCs 0,0.5,1,1.5,2,3 hours were treated with 10nM Ex-4,the expressions of p38,p-p38,ERK1/2,p-ERK1/2,JNK and p-JNK proteins were detected by Western-blot,and the time when Ex-4 acted on MAPK pathway was explored.2.The selective MAPK inhibitors,namely PD98059,SB203580 and SP600125,were used to treat human PDLSCs.The phosphorylation levels of p38,ERK1/2 and JNK were detected by Western-blot,and the mRNA expression levels of osteoblast-related genes Runx2,ALP and OSX were detected by qRT-PCR after 7 days of osteogenesis induction.Part Ⅳ:PDLSCs were divided into four groups:NG,NG+10nM Ex-4,HG,HG+10nM Ex-4.After 7 days of osteogenesis,the protein expressions of β-catenin,GSK3β,P-GSK3β,LEF and Runx2 were detected by Western-blo.Results:Part Ⅰ:1.The primary PDLSCs were successfully isolated and extracted,and the cells showed long spindle-shaped growth under microscope.2.Cloning experiment showed that PDLSCs grown for 2 weeks formed a clone cluster with high density.3.After 14 days of adipogenic induction,oil red staining showed the formation of lipid droplets,and after 21 days of osteogenic induction,alizarin red staining showed the formation of calcium nodules.It shows that PDLSCs has the potential of adipogenic and osteogenic differentiation.Part II:1.After 7 days of osteogenic culture,ALP staining showed that compared with the control group(NG),the staining of ALP in NG+Ex-4 group was deeper and larger,that in HG group was lighter,and that in HG+Ex-4 group was also lighter than that in NG group.Compared with HG group,HG+Ex-4 group had darker ALP staining.2.Alizarin red staining after 21 days of osteogenic culture showed that compared with NG,the calcified nodules in NG+Ex-4 group increased,while those in HG+EX-4 group decreased significantly,and those in HG+Ex-4 group also decreased compared with NG group.Compared with HG group,the mineralized nodules in HG+Ex-4 group increased.3.The results of qRT-PCR showed that on the 7th day,compared with the control group(NG),the expression of Osx in NG+Ex-4 group increased,while the expression of Runx2 and Osx in HG group decreased.Compared with HG group,the expression of ALP in HG+Ex-4 group increased.On 14th day,compared with NG,the expression of ALP,Runx2 and Osx of NG+Ex-4 increased.The expression of ALP and Runx2 in HG group decreased significantly.Compared with HG group,the expression of ALP,Runx2 and Osx in HG+Ex-4 group increased significantly(P<0.05).Part Ⅲ:1.The results of Western-blot showed that Ex-4 activated MAPK pathway in a short time.The phosphorylation of p38 and ERK1/2 reached the peak at about 1h and 1h,0.5h,respectively,and the phosphorylation of JNK reached the peak at about 1.5h.2.After adding MAPK inhibitor,the results of Western-blot showed that compared with NG group,the phosphorylation of P38,ERK1/2 and JNK of PDLSCs in NG+Ex-4 group increased,but the phosphorylation of ERK1/2 in HG group decreased.Compared with HG group,the phosphorylation of p38,ERK1/2 and JNK in HG+Ex-4 group increased,and MAPK inhibitor effectively inhibited the phosphorylation of MAPK pathway.3.After the addition of MAPK inhibitor and 7 days of osteogenic culture,qRT-PCR results showed that the mRNA levels of ALP,Runx2 and Osx were reduced by about 92%,72%and 82%in the NG+Ex-4+SB203580 group compared with the NG+Ex-4 group,and by about 80%,69%and 71%in the HG+Ex-4+SB203580 group compared with the HG+Ex-4,and mRNA levels of Runx2 and Osx were reduced by about 80%,69%and 71%,respectively.Similarly,the addition of SP600125 inhibitor reduced the mRNA levels of ALP,Runx2 and OSX by approximately 98%,53%and 28%,respectively,compared to the NG+Ex-4 group,and by approximately 95%,38%and 20%,respectively,compared to HG+Ex-4.PD98059 similarly reduced the mRNA expression of all osteogenic genes,compared to the NG+Ex-4 group Expression of ALP,Runx2 and OSX was reduced by approximately 89%,70%and 73%,respectively,compared to the NG+Ex-4 group,and by approximately 59%,65%and 38%,respectively,compared to the HG+Ex-4 group(p<0.05).Part Ⅳ:Western-blot results showed that compared with NG group,the protein expression of p-GSK3β,β-catenin,LEF and Runx2 in NG+Ex-4 group increased,while the protein expression ofβ-catenin,LEF and Runx2 in HG group decreased.Compared with HG group,the protein expression of p-GSK3β,β-catenin,LEF and Runx2 in HG+Ex-4 group also increased.Conclusion:1.10nM Ex-4 alleviated the osteogenic inhibition of PDLSCs by high glucose environment and promoted the osteogenic differentiation of PDLSCs under normal glucose environment.2.Ex-4 can promote the osteogenesis of PDLSCs in high glucose environment by activating MAPK pathway.3.Ex-4 may promote the osteogenesis of PDLSCs in high glucose environment by regulating WNT pathway.更多还原