Transplantation of stem cells from human exfoliated deciduous teeth for bone regeneration in the dog mandibular defect

AIM: To investigate the effect of stem cells from human exfoliated deciduous teeth(SHED) transplanted for bone regeneration in the dog mandibular defect.METHODS: In this prospective comparative study, SHEDs had been isolated 5 years ago from human exfoliated deciduous teeth. The undifferentiated stem cells were seeded into mandibular bone through-andthrough defects of 4 dogs. Similar defects in control group were filled with cell-free collagen scaffold. After 12 wk, biopsies were taken and morphometric analysis was performed. The percentage of new bone formation and foreign body reaction were measured in each case. The data were subject to statistical analysis using the Mann-Whitney U and Kruskalwalis statistical tests. Differences at P < 0.05 was considered as significant level.RESULTS: There were no significant differences between control and SHED-seeded groups in connective tissue(P = 0.248), woven bone(P = 0.248) and compact bone(P = 0.082). There were not any side effects in transplanted SHED group such as teratoma or malignancy and abnormalities in this period.CONCLUSION: SHEDs which had been isolated and characterized 5 years ago and stored with cryopreservation banking were capable of proliferation and osteogenesis after 5 years, and no immune response was observed after three months of seeded SHEDs.

Stem cells from human exfoliated deciduous teeth ameliorate concanavalin A-induced autoimmune hepatitis by protecting hepatocytes from apoptosis

BACKGROUND Autoimmune hepatitis is a serious autoimmune liver disease that threatens human health worldwide,which emphasizes the urgent need to identify novel treatments.Stem cells from human exfoliated deciduous teeth(SHED),which are easy to obtain in a non-invasive manner,show pronounced proliferative and immunomodulatory capacities.AIM To investigate the protective effects of SHED on concanavalin A(ConA)-induced hepatitis in mice,and to elucidate the associated regulatory mechanisms.METHODS We used a ConA-induced acute hepatitis mouse model and an in vitro co-culture system to study the protective effects of SHED on ConA-induced autoimmune hepatitis,as well as the associated underlying mechanisms.RESULTS SHED infusion could prevent aberrant histopathological liver architecture caused by ConA-induced infiltration of CD3+,CD4+,tumor necrosis-alpha+,and interferon-gamma+inflammatory cells.Alanine aminotransferase and aspartate aminotransferase were significantly elevated in hepatitis mice.SHED infusion could therefore block ConA-induced alanine aminotransferase and aspartate aminotransferase elevations.Mechanistically,ConA upregulated tumor necrosisalpha and interferon-gamma expression,which was activated by the nuclear factor-kappa B pathway to induce hepatocyte apoptosis,resulting in acute liver injury.SHED administration protected hepatocytes from ConA-induced apoptosis.CONCLUSION SHED alleviates ConA-induced acute liver injury via inhibition of hepatocyte apoptosis mediated by the nuclear factor-kappa B pathway.Our findings could provide a potential treatment strategy for hepatitis.

Application of dental stem cells in three-dimensional tissue regeneration

Dental stem cells can differentiate into different types of cells.Dental pulp stem cells,stem cells from human exfoliated deciduous teeth,periodontal ligament stem cells,stem cells from apical papilla,and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development.The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering.In recent years,three-dimensional(3D)tissue scaffolds have been used to reconstruct and restore different anatomical defects.With rapid advances in 3D tissue engineering,dental stem cells have been used in the regeneration of 3D engineered tissue.This review presents an overview of different types of dental stem cells used in 3D tissue regeneration,which are currently the most common type of stem cells used to treat human tissue conditions.

基于RNA测序的SHED体外连续扩增后差异表达基因的研究

目的:利用RNA测序(RNA sequencing,RNA-seq)技术研究人脱落乳牙干细胞(stem cells derived from human exfoliated deciduous teeth,SHED)体外长时期扩增至20代(P20)后基因表达的差异,初步探讨其体外扩增衰老相关的信号通路。方法:从健康儿童脱落的乳牙中分离牙髓干细胞,在常规条件下将其扩增培养至20代,利用RNA-seq筛选出差异表达的基因,并对其进行相关生物学信息分析,寻找SHED体外连续扩增衰老相关的信号通路。结果:RNA-seq结果显示,SHED第4代(P4)和P20代差异表达的基因共有1884个,其中上调表达的基因有575个,下调表达基因1309个,这些差异表达的基因分布在生物过程(biological progress,BP)、细胞组成(cellular component,CC)和分子功能(molecular function,MF)等生物学过程中。早、晚期代次的SHED差异基因及相关蛋白之间的作用主要富集在剪接体、核糖体、细胞周期、p53通路相关的信号通路上。结论:本研究揭示了SHED体外连续扩增培养至第20代后基因表达谱的改变,为后续进一步研究其细胞体外衰老机制指明了方向。

miR-26b对人脱落乳牙牙髓干细胞及人脐带间充质干细胞增殖、迁移和成骨分化的影响

背景:microRNA-26b(miR-26b)对干细胞的多种功能具有重要的调节作用,但其对人脱落乳牙牙髓干细胞和人脐带间充质干细胞生物学特性的影响尚不清楚。目的:探讨miR-26b对人脱落乳牙牙髓干细胞和人脐带间充质干细胞增殖、迁移和成骨分化的影响。方法:培养及鉴定人脱落乳牙牙髓干细胞和人脐带间充质干细胞;用miR-26 mimics(实验组)和miRNAs mimics对照(对照组)转染2种细胞,构建过表达模型用于后续实验。CCK-8法检测过表达miR-26b细胞的增殖能力;Transwell和划痕实验分析过表达miR-26b细胞的迁移能力;RT-qPCR法检测过表达miR-26b细胞成骨诱导后成骨标志物的表达。结果与结论:①转染miR-26b模拟物可提高2种细胞中miR-26b表达量,促进人脱落乳牙牙髓干细胞增殖,对人脐带间充质干细胞的扩增无明显影响;②相较于对照组,2种细胞过表达miR-26b后迁移能力增强;③miR-26b在2种细胞成骨分化过程中表达水平降低;④相较于对照组,2种细胞过表达miR-26b后,成骨相关基因骨钙素、骨桥素、碱性磷酸酶、Ⅰ型胶原蛋白mRNA水平均下调。结果表明,过表达miR-26b能促进人脱落乳牙牙髓干细胞增殖、迁移,抑制其成骨分化;也促进人脐带间充质干细胞迁移,抑制其成骨分化,但对其增殖无明显影响。

牙源性干细胞治疗帕金森病的研究进展

帕金森病(PD)是第二常见的神经退行性疾病,主要是由于中脑黑质(SN)多巴胺能神经元的丧失而导致运动功能障碍。牙源性干细胞(DSCs)来自颅神经嵴,易于采集,具有很强的增殖和分化能力,能够促进神经修复和再生。DSCs可以分化成新的多巴胺神经元并分泌大量神经营养因子(NTFs)改善神经功能,同时还可以通过免疫调节抑制神经炎性反应,DSCs可以有效减轻PD大鼠的运动功能障碍,并在再生医学领域中扮演着重要的角色。

高糖对乳牙牙髓干细胞生长及成骨分化能力的影响

目的探讨高糖对乳牙牙髓干细胞(SHED)生长及成骨分化能力的影响。方法从河南大学赛思口腔医院口腔颌面外科收集拔除的6~8岁无牙体牙髓牙周疾病的健康儿童的滞留乳牙,体外培养获得SHED,实验分为5.5 mmol·L^(-1)低糖组及25 mmol·L^(-1)高糖组。用细胞计数CCK-8试剂盒(CCK-8)检测两组细胞的增殖速度。分别用含两种不同糖浓度的成骨矿化液诱导21 d后,行茜素红染色实验,实时荧光定量PCR(QPCR)技术检测骨向分化相关基因碱性磷酸酶(ALP)、Runt相关转录因子2(RUNX2)和骨钙素(OCN)、骨桥蛋白(OPN)表达的差异。结果CCK-8检测结果显示,培养2~6 d时高糖组的OD值小于低糖组(P<0.05);成骨诱导后,高糖组的钙化结节数量少于低糖组,且高糖组的RUNX-2、ALP、OCN和OPN的相对表达量低于低糖组(P<0.05)。结论高糖抑制SHED的生长及成骨分化能力。

不同浓度的大黄素对人乳牙牙髓干细胞增殖和成骨分化的影响

目的:研究大黄素对人乳牙牙髓干细胞的增殖和成骨分化的影响。方法:分离人乳牙牙髓干细胞,镜下观察细胞形态,成骨诱导后经茜素红染色和碱性磷酸酶染色鉴定其成骨分化能力,流式细胞术检测细胞表面标记物鉴定其干细胞源性。不同浓度的大黄素预处理人乳牙牙髓干细胞,通过CCK-8法检测细胞的增殖能力,茜素红染色、碱性磷酸酶染色检测细胞成骨分化能力,通过RT-qPCR检测碱性磷酸酶、RUNX2和骨钙素的mRNA表达,Western blot检测成骨相关蛋白RUNX2的蛋白表达。结果:①CCK-8结果显示,大黄素在浓度为0.1-10μmol/L时可以明显促进人乳牙牙髓干细胞的增殖,而浓度为100μmol/L则显著抑制了细胞的增殖;②碱性磷酸酶染色和茜素红染色显示,随着大黄素浓度的升高,碱性磷酸酶染色和茜素红染色逐渐加深,10μmol/L大黄素处理组碱性磷酸酶染色最深,矿化结节数量也最多;③RT-qPCR结果显示,大黄素促进碱性磷酸酶、RUNX2和骨钙素mRNA的表达量(P<0.05)。④Western blot结果显示,成骨相关蛋白RUNX2的表达随着大黄素的处理浓度增加而增加。结论:大黄素在1-10μmol/L范围内对人乳牙牙髓干细胞的增殖和成骨分化具有促进作用,且浓度为10μmol/L时效果最明显。

Dental pulp cell bank as a possible future source of individual hepatocytes

Mesenchymal stem cells(MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth(SHEDs) are the best candidates for personal cell banking(dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immuneregulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity.

乳牙牙髓干细胞分化前后的外泌体miRNA筛选研究

目的 分析乳牙牙髓干细胞分化前后的外泌体中miRNA差异表达谱。方法 原代提取乳牙牙髓干细胞,在矿化诱导培养基中进行分化。超速离心法提纯外泌体并进行鉴定;利用miRNA基因芯片检测分化前后的外泌体中miRNA的表达谱,验证差异表达的miRNA,预测其靶基因,进行GO分析和KEGG通路的功能分析。结果 在未分化和分化的乳牙牙髓干细胞的外泌体中共发现215个差异表达的miRNA,最相关的KEGG通路为FoxO、Ras和MAPK等信号通路。GO分析包括多细胞生物的发育和结构形态发生的调控。结论 乳牙牙髓干细胞分化前后的外泌体miRNA可能会参加调控成牙本质向分化过程,为牙髓修复提供了理论依据。

骨碎补总黄酮对乳牙牙髓干细胞增殖及成骨向分化的影响

目的初步探索不同浓度骨碎补总黄酮(total flavone of rhizoma drynariae)对乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth)增殖及成骨分化能力的影响。方法2020年3月—2021年8月黑龙江省医院检验科初步筛选出适宜浓度的RDTF,三代分离、培养SHEDs,通过流式细胞术及茜素红染色分别对细胞表面标志物和成骨分化潜能进行鉴定。实验分为4组(0时段为对照组,24、48、72 h组),每组设置3个复孔,采用不同浓度RDTF处理SHEDs后钙钴法染色,Western blot法检测其成骨相关蛋白表达情况。结果结果显示,所获取的细胞为具有成骨分化能力的SHEDs。与对照组相比,24、48、72 h内各浓度组均对SHEDs增殖无明显影响,差异无统计学意义(P>0.05),12.5、25 mg/L RDTF组的Runt相关转录因子2(Runx2)及骨钙素(OCN)的蛋白表达均显著上调,其中25 mg/L RDTF组对Runx2表达的促进效果最明显(249.681±14.653),差异有统计学意义(F=102.7,P<0.001)。结论RDTF对SHEDs的增殖无显著影响,可促进SHEDs成骨分化。

白藜芦醇促进人乳牙牙髓干细胞的增殖和成骨分化

背景:白藜芦醇是一种天然非黄酮类多酚化合物。研究表明,白藜芦醇具有抗氧化、抗炎、抗衰老和抗肿瘤等多种生物活性,并且能促进骨髓间充质干细胞的成骨分化,但其是否能调节乳牙牙髓干细胞的成骨分化尚不清楚。目的:研究白藜芦醇对人乳牙牙髓干细胞增殖和成骨分化能力的影响。方法:组织块法分离培养人乳牙牙髓干细胞,不同浓度的白藜芦醇处理成骨诱导的人乳牙牙髓干细胞,CCK-8检测细胞活性,茜素红染色检测矿化结节数量,RT-qPCR检测成骨相关基因的mRNA表达及Western blot检测Runt相关转录因子2的蛋白水平。结果与结论:①CCK-8检测结果显示,处理第3天时,较高浓度(20,40,80,100μmol/L)白藜芦醇显著抑制细胞增殖,因此,后续实验中选用1,5,10μmol/L白藜芦醇处理乳牙牙髓干细胞,检测其对成骨分化的作用;②与成骨诱导对照组相比,10μmol/L白藜芦醇组乳牙牙髓干细胞的矿化结节数量显著增多;③与成骨诱导对照组相比,10μmol/L白藜芦醇组乳牙牙髓干细胞的碱性磷酸酶、Runt相关转录因子2和骨钙素的mRNA表达量升高;④与成骨诱导对照组相比,白藜芦醇组乳牙牙髓干细胞的Runt相关转录因子2蛋白水平呈浓度依赖性增加;⑤结果表明,适宜浓度的白藜芦醇可提高人乳牙牙髓干细胞的增殖和成骨分化能力。

人脱落乳牙牙髓干细胞治疗非口腔疾病的研究进展

人脱落乳牙牙髓干细胞(SHEDs)作为一类具有较强增殖能力且能够多向分化的牙源性干细胞,易于获取且符合伦理要求,在干细胞治疗中具有很大潜力。目前关于SHEDs在非口腔疾病中的治疗作用缺乏阐述,本文就SHEDs在神经系统、消化系统、心血管系统、泌尿系统、免疫系统、内分泌系统及呼吸系统疾病中的最新治疗进展做一综述,以期为SHEDs在非口腔疾病中的临床应用提供参考。

人乳牙牙髓干细胞在干细胞治疗中的应用

乳牙牙髓干细胞(SHED)是牙源性干细胞的一种,属外胚间充质干细胞。作为一种理想的干细胞来源,SHED在干细胞治疗中有良好的应用前景。本文阐述了SHED的生物学特征及其在干细胞治疗中的优势,探讨了SHED在组织再生和修复中发挥的多向分化潜能、细胞分泌功能和免疫调节功能等方面的功能作用。此外,本文还介绍了SHED在各系统、器官疾病治疗中的临床应用,重点阐述了用SHED进行干细胞移植在牙髓—牙本质再生、颌骨再生、神经系统疾病治疗和免疫系统疾病治疗方面的研究进展。

Wnt信号通路在调控牙髓干细胞多向分化及炎症损伤修复中的作用

人牙髓干细胞(hDPSC)和乳牙牙髓干细胞(SHED)均属于间充质干细胞,具有多向分化潜能。Wnt信号通路包括经典Wnt/β-catenin信号通路和非经典Wnt信号通路,参与牙齿的生长发育过程,能促进牙损伤的修复。在炎症状态下,激活的Wnt信号通路可加快牙损伤修复,参与hDPSC的增殖与成牙本质向分化过程。在非炎症状态下,Wnt信号通路参与调控hDPSC和SHED成骨、成牙本质方向分化,抑制其成脂向分化。

转化生长因子β3联合肝素在人乳牙牙髓干细胞向成牙本质样细胞分化中的作用

目的:探讨转化生长因子β3(TGF-β3)诱导人乳牙牙髓干细胞(SHED)向成牙本质样细胞分化的能力。方法:采用酶消化法将人乳牙牙髓分离培养,获得SHED;对体外培养的第3代SHED分别单独加入25 ng/mL的重组TGF-β3,或与肝素联合进行培养;通过Q-PCR和Western-blotting方法,分别观察SHED表达成牙本质细胞特异性标志物-牙本质涎磷蛋白(DSPP)基因及其基因表达产物-牙本质涎蛋白(DSP)的情况;通过碱性磷酸酶(AKP)试剂盒检测SHED的AKP活性的改变;细胞爬片行免疫化学染色和茜素红染色。结果:SHED在诱导体系中生长状态良好。25 ng/mL TGF-β3联合10 U/mL肝素作用组的AKP活性明显增强,与TGF-β3单独作用组、肝素单独作用组以及对照组相比差异具有统计学意义(P<0.01);TGF-β3联合肝素作用组的茜素红染色呈强阳性,Q-PCR和Western-blotting结果均显示,该组的DSPP表达在mRNA水平和蛋白质水平上均明显升高。结论:在TGF-β3与肝素联合作用的诱导下,可促进SHED分化为成牙本质样细胞。

TNF-α对人脱落乳牙牙髓干细胞促破骨细胞形成能力的影响

目的:探讨肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)对人脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHED)促破骨细胞形成能力的影响。方法:通过酶消化法体外分离、培养处于生理性根吸收时期的自然脱落乳牙牙髓干细胞;建立SHED与破骨前体细胞外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)的间接共培养模型,在破骨诱导培养液中加入0、5、10、50、100 ng/m L不同浓度的TNF-α,通过实时定量RT-PCR和Western免疫印迹检测破骨相关基因及核转录因子κB(nuclear factor-κB,NF-κB)信号通路相关基因的表达。采用SPSS 19.0软件包对数据进行统计学分析。结果:在10 ng/mL的TNF-α刺激下,PBMCs中破骨相关蛋白CTSK和TRAP的表达水平均显著增高。Western免疫印迹和实时定量RT-PCR检测结果显示,SHED胞质内p-IκBα和胞核内p65蛋白、基因的表达水平在10 ng/m L的TNF-α刺激下显著高于无TNF-α刺激组。结论:炎症细胞因子TNF-α通过NF-κB信号通路对SHED促破骨细胞形成能力具有调控作用。

改良富血小板血浆促进乳牙牙髓干细胞成骨分化作用研究

目的:体外研究改良富血小板血浆(modified platelet-rich plasma,mPRP)促进人乳牙牙髓干细胞成骨分化的作用。方法:以α-MEM作为基础培养基,分别加入1%、2%、5%、10%4种不同浓度mPRP或者10%胎牛血清(对照),对第4代SHED连续培养并诱导矿化,碱性磷酸酶试剂盒检测ALP活性的变化,qRT-PCR方法检测细胞内RUNX2和骨钙素mRNA含量的改变。结果:不同浓度的mPRP均可以促进乳牙牙髓干细胞的ALP活性,且浓度为2%时A值最高;qRT-PCR检测显示2%mPRP可以上调乳牙牙髓干细胞内RUNX2及骨钙素mRNA的含量。结论:一定浓度的mPRP对乳牙牙髓干细胞的成骨分化具有一定的促进作用。

脱落乳牙牙髓干细胞的生物学特性

在乳恒牙交替过程中,乳牙不断脱落,即脱落乳牙牙髓干细胞(SHED)获取容易,且SHED较骨髓间质干细胞和牙髓干细胞有更强的细胞增殖能力。SHED含有的成纤维细胞生长因子、转化生长因子-β、结缔组织生长因子、神经生长因子、骨形态发生蛋白等促进SHED增殖。SHED可向成牙本质细胞、神经细胞、软骨细胞、脂肪细胞和肝细胞向分化及诱导宿主细胞分化为成骨细胞,还有免疫调节和促进伤口愈合功能,是理想的骨组织工程和皮肤组织工程种子细胞。有鉴于此,SHED在治疗牙体缺失、骨缺损,神经系统疾病和免疫系统疾病,皮肤外伤和肝脏疾病方面有着广泛的应用前景。

牙本质细胞外基质蛋白对乳牙牙髓干细胞体外增殖、分化能力的影响

目的:提取牙本质细胞外基质蛋白(Dentin extracellular matrix proteins,DEMPs)研究其对人脱落的乳牙牙髓干细胞(Stem cell from human exfoliated deciduous teeth,SHED)体外增殖分化能力的影响。方法:采用组织块联合酶消化法获得SHED并进行体外培养。成骨诱导液诱导细胞,鉴定其多向分化能力;拔取健康牛牙,用4mol/L盐酸胍和0.5mol/L EDTA提取DEMPs,四唑盐比色法(MTT)检测不同浓度DEMPs对SHED增殖能力的影响,同时测定DEMPs对SHED碱性磷酸酶(ALP)活性的影响;实时荧光定量PCR(RT-PCR)检测牙本质磷蛋白与牙本质基质蛋白1的mRNA表达情况。结果:DEMPs诱导细胞培养3d、5d可促进细胞增殖。细胞培养5、7d上调ALP活性,RT-PCR结果显示,DEMPs诱导后可促进DSPP与DMP-1基因的表达。结论:牙本质细胞外基质蛋白可以促进SHED的增殖与牙向分化能力。