重组人结缔组织生长因子对牙髓细胞增殖与成牙本质分化的影响

目的：研究重组人结缔组织生长因子（recombinant connective tissue growth factor, rCTGF）对牙髓细胞（human dental pulp cells,hDPCs）增殖及分化的影响。方法：利用不同浓度（0、1、10、100 ng/mL）rCTGF分别处理牙髓细胞,CCK8法检测牙髓细胞增殖情况;茜素红染色和半定量试验检测细胞矿化结节的形成变化,qRT-PCR测定成牙本质分化相关基因DMP-1、DSPP和OC的表达情况,Western 免疫印迹法测定rCTGF刺激牙髓细胞后,ERK1/2信号通路的磷酸化水平。采用SAS 9.3软件包对数据进行统计学分析。结果：高浓度的rCTGF（100 ng/mL）可以促进牙髓细胞增殖;经矿化诱导后,10 ng/mL rCTGF促进牙髓细胞矿化结节形成的效果最好,钙盐沉积量最明显（P<0.05）,成牙本质分化相关基因DMP-1、DSPP的表达显著上调（P<0.05）。Western 免疫印迹结果显示,10 ng/mL rCTGF刺激牙髓细胞后,p-ERK1/2蛋白的表达升高。结论：rCTGF可能通过激活ERK1/2信号通路,促进牙髓细胞的增殖与分化。     

MicroRNA-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响

目的:探讨miR-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响。方法:以小鼠前成骨细胞MC3T3-E1为实验对象,对MC3T3-E1细胞进行成骨诱导,分别在0、3、5、7 d应用实时荧光定量PCR（real-time PCR）检测细胞中Runx2、Osx、ALP、miR-103-3p表达水平,Western免疫印迹（Western blotting）检测Runx2、Osx蛋白表达并进行碱性磷酸酶（ALP）染色。通过脂质体lipofectamine2000瞬时转染miR-103-3p模拟物 （miR-103-3p mimics）及模拟物阴性对照进入MC3T3-E1细胞内,Real-time PCR检测2组细胞miR-103-3p的表达水平,CCK-8试剂盒检测细胞增殖。分别对2组细胞进行成骨诱导,在成骨诱导后0、3、7 d,分别使用Real-time PCR和Western免疫印迹检测2组细胞Runx2、Osx等成骨相关基因mRNA和蛋白的表达变化,并对2组细胞进行ALP染色。实验数据采用SPSS19.0软件包进行统计学分析。结果:MC3T3-E1经成骨诱导0、3、5、7 d后,细胞内Runx2、Osx、ALP转录水平持续显著升高;Runx2、Osx蛋白表达升高。ALP染色逐渐加深。在成骨诱导3、5、7 d的MC3T3-E1细胞中,miR-103-3p水平较诱导前受到持续显著抑制(P<0.05)。瞬时转染miR-103-3p mimics后,MC3T3-E1细胞中的miR-103-3p表达水平较对照组显著上调(P<0.05),细胞增殖受到抑制,Runx2、ALP转录水平显著抑制（P<0.05）,Runx2蛋白表达显著抑制。对转染后的细胞进行成骨诱导3、7 d后,miR-103-3p转染组细胞Runx2、Osx、ALP在转录水平的表达较对照组显著降低,Runx2、Osx在蛋白水平的表达较对照组显著降低,且miR-103-3p转染组细胞ALP活性较对照组显著降低。结论:miR-103-3p可能对小鼠前成骨细胞MC3T3-E1的成骨分化早期起抑制作用。     

米诺环素通过Wnt/β-Catenin通路对大鼠牙髓干细胞成骨分化的影响

目的 探讨米诺环素对大鼠牙髓干细胞(RDPSCs)成骨分化的影响,以及对Wnt/β-连环蛋白(Wnt/β-Catenin)通路的作用.方法 将RDPSCs随机分为对照组和不同浓度(0.1、1.0、10.0、100.0 mg/L)米诺环素组,对细胞进行成骨诱导.CCK-8法检测细胞增殖率,试剂盒检测骨形成指标碱性磷酸酶(ALP)和骨钙蛋白(OCN)含量;茜素红S染色检测细胞钙盐沉积量;qRT-PCR检测ALP和OCN mRNA表达情况;蛋白质免疫印迹技术(WB)检测β-Catenin、糖原合成酶激酶-3β(GSK-3β)和Runt相关转录因子2(Runx2)蛋白表达水平.结果 与对照组相比,0.1、1.0 mg/L米诺环素组RDPSCs细胞增殖率、ALP活性、OCN含量、钙盐沉积量及β-Catenin、Runx2蛋白水平均显著升高,GSK-3β蛋白水平显著降低(P<0.05);10.0、100.0 mg/L米诺环素组RDPSCs细胞增殖率显著升高(P>0.05),ALP活性、OCN含量及表达水平、钙盐沉积量及β-Catenin、Runx2蛋白水平均显著降低,GSK-3β蛋白水平显著升高(P<0.05).结论 低浓度米诺环素能够诱导大鼠牙髓干细胞成骨分化,而高浓度米诺环素抑制细胞成骨分化,该过程可能通过调控Wnt/β-Catenin信号通路实现.     

米诺环素通过Wnt/β-Catenin通路对大鼠牙髓干细胞成骨分化的影响

目的 探讨米诺环素对大鼠牙髓干细胞(RDPSCs)成骨分化的影响,以及对Wnt/β-连环蛋白(Wnt/β-Catenin)通路的作用.方法 将RDPSCs随机分为对照组和不同浓度(0.1、1.0、10.0、100.0 mg/L)米诺环素组,对细胞进行成骨诱导.CCK-8法检测细胞增殖率,试剂盒检测骨形成指标碱性磷酸酶(ALP)和骨钙蛋白(OCN)含量;茜素红S染色检测细胞钙盐沉积量;qRT-PCR检测ALP和OCN mRNA表达情况;蛋白质免疫印迹技术(WB)检测β-Catenin、糖原合成酶激酶-3β(GSK-3β)和Runt相关转录因子2(Runx2)蛋白表达水平.结果 与对照组相比,0.1、1.0 mg/L米诺环素组RDPSCs细胞增殖率、ALP活性、OCN含量、钙盐沉积量及β-Catenin、Runx2蛋白水平均显著升高,GSK-3β蛋白水平显著降低(P<0.05);10.0、100.0 mg/L米诺环素组RDPSCs细胞增殖率显著升高(P>0.05),ALP活性、OCN含量及表达水平、钙盐沉积量及β-Catenin、Runx2蛋白水平均显著降低,GSK-3β蛋白水平显著升高(P<0.05).结论 低浓度米诺环素能够诱导大鼠牙髓干细胞成骨分化,而高浓度米诺环素抑制细胞成骨分化,该过程可能通过调控Wnt/β-Catenin信号通路实现.     

组蛋白去甲基化酶FBXL11抑制牙髓干细胞成骨和成牙本质分化

目的 研究组蛋白去甲基化酶FBXL11对牙髓干细胞定向分化能力的影响.方法 成骨分化诱导培养基诱导牙髓干细胞体外成骨/成牙本质分化.逆转录病毒转染构建过表达FBXL11的牙髓干细胞稳定转染细胞,进行FBXL11获得性功能研究.碱性磷酸酶活性实验及碱性磷酸酶染色检测成骨/成牙本质分化早期分化指标-碱性磷酸酶活性.茜素红染色及钙离子定量分析检测牙髓干细胞体外成骨/成牙本质分化能力.实时定量RT-PCR检测FBXL11及成骨/成牙本质分化相关基因-骨涎蛋白、骨桥蛋白和骨钙素的表达.结果 成骨诱导牙髓干细胞抑制FBXL11的表达.过表达FBXL11明显抑制牙髓干细胞的碱性磷酸酶活性、牙髓干细胞体外矿化能力以及骨涎蛋白和骨桥蛋白的表达.结论 组蛋白去甲基化酶FBXL11具有抑制牙髓干细胞成骨和成牙本质分化的潜能.     

TNF-α影响鼠根尖乳头干细胞成骨/成牙本质能力的实验研究

目的研究肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)对鼠根尖乳头干细胞(stem cells from apical papilla,SCAP)增殖及成骨/成牙本质能力的影响。方法分离大鼠根尖乳头组织,采用酶消化法结合组织块法获得SCAP并通过免疫荧光法进行细胞鉴定;将细胞分为实验组(TNF-α浓度5、10、15、20、30、40、50μg/L)和对照组(TNF-α浓度0μg/L),CCK-8法检测细胞增殖能力;采用碱性磷酸酶活性、茜素红染色及实时定量PCR检测TNF-α对SCAP成骨/成牙本质能力的影响。结果体外培养SCAP符合间充质干细胞来源的特征且具有多向分化能力;细胞增殖能力结果显示:各浓度组均能促进SCAP增殖(P<0.05);ALP活性结果显示:各浓度TNF-α均能明显降低ALP活性(P<0.05);茜素红染色结果显示:随着TNF-α的浓度的增加,染色逐渐变浅,红色结节逐渐变小,形成数量逐渐减少;qRT-PCR结果显示:3 d时,实验组OC、DMP-1表达量明显降低(P<0.05),牙本质涎磷蛋白(DSPP)表达量降低(P>0.05),骨涎蛋白(BSP)表达量稍有增加(P<0.05)。7 d时,OC、DSPP表达量明显降低(P>0.05),DMP-1表达量明显降低(P<0.05),BSP表达量与对照组相比仍稍有增加(P>0.05);14 d时,BSP、OC、DMP-1表达量均明显降低(P<0.05),DSPP表达量稍有增加(P>0.05)。结论炎性因子TNF-α对SCAP的增殖有促进作用同时不同程度抑制SCAP成骨/成牙本质向分化能力。     

Foxc2稳定转染对C3H10T1/2细胞成骨性能的影响

目的:探讨Foxc2基因过表达对体外培养的小鼠胚胎成纤维细胞系C3H10T1/2细胞生物学行为及分化能力的影响.方法:通过慢病毒转染构建Foxc2过表达C3H1OT1/2细胞系,采用实时定量PCR和Western免疫印迹法检测转染后Foxc2蛋白的表达.应用CCK-8试剂盒检测细胞增殖,流式细胞仪检测细胞周期及凋亡,实时定量PCR和Western免疫印迹法检测过表达Foxc2对成骨、成脂相关基因(Runx2、OPN、OCN、PPARγ)表达的影响,分别通过碱性磷酸酶(ALP)染色和油红染色检测细胞成骨和成脂分化能力.采用SPSS 17.0软件包对数据进行t检验.结果:成功构建Foxc2稳定过表达的C3H10T1/2细胞系,发现Foxc2过表达阻滞细胞于G1期,抑制细胞增殖.在成骨诱导过程中,过表达Foxc2可以显著上调Runx2、OPN、OCN等成骨相关基因的表达.ALP染色显示,Foxc2稳定过表达细胞较对照组细胞染色深.在成脂诱导过程中,过表达Foxc2显著下调PPARγ基因的表达;油红染色显示,成脂分化进程被部分抑制.结论:Foxc2过表达抑制细胞增殖,促进细胞分化.其机制是上调Runx2、OPN、OCN成骨相关基因的表达,下调PPARγ的表达,促进C3H10T1/2细胞的成骨分化,抑制其成脂分化.     

低浓度氟化钠对人牙髓细胞的成骨/成牙本质分化的影响

目的 探讨低浓度氟化钠对人牙髓细胞（human dental pulp cells,hDPCs）成骨/成牙本质分化的影响。方法 本研究已通过单位伦理委员会审查批准。原代培养hDPCs，采用MTT法检测不同浓度氟化钠对hDPCs增殖的影响；选取合适浓度的氟化钠加入成骨/成牙本质分化诱导培养液中，对hDPCs进行体外诱导，通过茜素红染色检测hDPCs成骨/成牙本质分化能力的变化，RT-qPCR检测分化相关基因的mRNA表达；同时通过RT-qPCR和Western blot检测h DPCs成骨/成牙本质分化过程中内质网应激相关基因的表达。结果 低浓度氟化钠（0.1 mmol/L）在体外可刺激h DPCs增殖，高浓度氟化钠（5～10 mmol/L）可抑制hDPCs增殖（P<0.05）。选取0.1 mmol/L氟化钠体外混合成骨/成牙本质分化诱导培养后hDPCs的茜素红染色增加，成骨/成牙本质分化相关基因牙本质涎磷蛋白（dentin sialophosphoprotein,DSPP）、骨涎蛋白（bone sialoprotein,BSP）和骨钙蛋白（osteocalcin,OCN）mRNA...     

Comparative characterization of STRO-1neg/CD146pos and STRO-1pos/CD146pos apical papilla stem cells enriched with flow cytometry

ObjectiveStem Cells residing in the Apical Papilla (SCAP) of human permanent teeth represent a promising cell source for dental tissue regeneration. Therefore, the functional and molecular properties of specific subpopulations existing within heterogeneous cultures should be further investigated to give insight whether their selection could be beneficial for targeted therapeutic applications.DesignIn this study we extensively characterized SCAP cultures established from 10 healthy subjects, as well as their STRO-1^pos/CD146^pos and STRO-1^neg/CD146^pos subpopulations isolated with fluorescence-activated cell sorting. SCAP were analyzed for embryonic (Nanog, Oct3/4, SSEA-3, TRA-1-60), mesenchymal (STRO-1, CD146/MUC18, CD105/endoglin, CD24, CD90/Thy-1, CD81-TAPA, CD34, CD49f/a6-integrin), neural (CD271/NGFR, nestin) and hematopoietic (CD117/c-kit, CD45) stem cell (SC) markers using flow cytometry. Multipotentiality was evaluated with culture specific staining (Alizarin-Red-S, Oil- Red-O) and RT-PCR analysis for osteo/odontogenic (DSPP, BSP, ALP, osteocalcin, osteonectin, BMP-2, Runx2), adipogenic (lipoprotein-lipase-LPL) and neurogenic (Neurofilament/NFL-L, nestin, β-tubulin-III, NCAM) markers.ResultsOur results showed that the STRO-1^pos/CD146^pos subpopulation demonstrated higher CFU efficiency and much higher expression of several embryonic and mesenchymal SC markers compared to the non-sorted SCAP. They also showed enhanced odontogenic differentiation potential, as evidenced by higher mineralization capacity and expression of osteo/odontogenic markers. By contrast, absence of STRO-1 in the STRO-1^neg/CD146^pos subpopulation yielded the opposite results and was associated with significant downgrading of the above-mentioned properties.ConclusionsThese results suggest that STRO-1^pos/CD146^pos SCAP cells represent a very promising adult MSCs source with enhanced multipotent SC properties that could be easily isolated with simple flow cytometric methods to be used for tissue engineering applications.     

Tideglusib对LPS刺激的人根尖牙乳头干细胞牙/骨向分化的影响

目的 探讨Tideglusib对LPS刺激的人根尖牙乳头干细胞(stem cells from the apical papilla, SCAPs)的牙/骨向分化的影响。方法 分离培养SCAPs,流式细胞术对SCAPs进行表面分子鉴定,检测Tideglusib对SCAPs细胞增殖是否有影响。通过碱性磷酸酶(alkaline phosphatase, ALP)活性和ALP染色筛选Tideglusib促进SCAPs表达ALP活性的最佳浓度。用大肠杆菌脂多糖(lipopolysaccharide, LPS)模拟炎性微环境刺激SCAPs。采用Western blot及实时定量聚合酶链反应(quantitative real-time polymerase chain reaction, RT-qPCR)等方法检测牙/骨向分化的相关蛋白(OSX、OCN、COL-Ⅰ、DSP、RUNX2)和基因(OSX、OCN、COL-Ⅰ、DSPP、RUNX2)表达变化。结果 CCK-8实验显示：Tideglusib浓度低于50 nmol/L时对细胞增殖无抑制作用;1 nmol/L Tideglusib处理LPS刺...     

Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study

Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental pulp stem cells or DPSCs) and deciduous teeth (stem cells from human exfoliated deciduous teeth). We recently discovered another type of MSCs in the apical papilla of human immature permanent teeth termed stem cells from the apical papilla (SCAP). Here, we further characterized the apical papilla tissue and stem cell properties of SCAP using histologic, immunohistochemical, and immunocytofluorescent analyses. We found that the apical papilla is distinctive to the pulp in terms of containing less cellular and vascular components than those in the pulp. Cells in the apical papilla proliferated 2- to 3-fold greater than those in the pulp in organ cultures. Both SCAP and DPSCs were as potent in osteo/dentinogenic differentiation as MSCs from bone marrows, whereas they were weaker in adipogenic potential. The immunophenotype of SCAP is similar to that of DPSCs on the osteo/dentinogenic and growth factor receptor gene profiles. Double-staining experiments showed that STRO-1 coexpressed with dentinogenic markers such as bone sialophosphoprotein, osteocalcin, and growth factors FGFR1 and TGFbetaRI in cultured SCAP. Additionally, SCAP express a wide variety of neurogenic markers such as nestin and neurofilament M upon stimulation with a neurogenic medium. We conclude that SCAP are similar to DPSCs but a distinct source of potent dental stem/progenitor cells. Their implications in root development and apexogenesis are discussed.     

Evaluation of the Apical Complex and the Coronal Pulp as a Stem Cell Source for Dentin-pulp Regeneration

IntroductionThis study compared the stemness and differentiation potential of stem cells derived from the apical complex (apical complex cells [ACCs]) and coronal pulp (dental pulp stem cells [DPSCs]) of human immature permanent teeth with the aim of determining a more suitable source of stem cells for regeneration of the dentin-pulp complex.MethodsACC and DPSC cultures were established from 13 human immature permanent teeth using the outgrowth method. The proliferation capacity and colony-forming ability of ACCs and DPSCs were evaluated. ACCs and DPSCs were analyzed for mesenchymal stem cell markers using flow cytometry. The adipogenic and osteogenic differentiation potential of ACCs and DPSCs were evaluated using the quantitative real-time polymerase chain reaction and histochemical staining. ACCs and DPSCs were transplanted subcutaneously in immunocompromised mice using macroporous biphasic calcium phosphate as a carrier. The histomorphologic characteristics of the newly formed tissues were verified using hematoxylin-eosin staining and immunohistochemical staining. Quantitative alkaline phosphatase analysis and quantitative real-time polymerase chain reaction using BSP, DSPP, POSTN, and Col XII were performed.ResultsACCs and DPSCs showed similar cell proliferation potential and colony-forming ability. The percentage of mesenchymal stem cell markers was similar between ACCs and DPSCs. In the in vitro study, ACCs and DPSCs showed adipogenic and osteogenic differentiation potential. In the in vivo study, ACCs and DPSCs formed amorphous hard tissue using macroporous biphasic calcium phosphate particles. The quantity and histomorphologic characteristics of the amorphous hard tissue were similar in the ACC and DPSC groups. Formation of periodontal ligament–like tissue, positive to Col XII, was observed in ACC transplants, which was absent in DPSC transplants.ConclusionsACCs and DPSCs showed similar stemness, proliferation rate, and hard tissue–forming capacity. The notable difference was the periodontal ligament–like fiber-forming capacity of ACCs, which indicates the presence of various lineages of stem cells in the apical complex compared with the coronal pulp. Regarding regeneration of the dentin-pulp complex, the coronal pulp can be a suitable source of stem cells considering its homogenous lineages of cells and favorable osteo/odontogenic differentiation potential.     

敲低F-ATPase促进变形链球菌感染人牙髓成纤维细胞成骨/成牙本质分化的研究

目的:研究敲低耐酸因子F-ATPase促进变形链球菌感染人牙髓成纤维细胞(human dental pulp fibro-blasts,HDPFs)成骨/成牙本质分化的作用.方法:培养HDPFs并采用免疫荧光染色、流式细胞术进行鉴定,而后分为对照组、感染组、感染+si-NC组、感染+si-F-ATPase组、感染+si...     

人恒牙牙髓干细胞体外定向诱导分化为成骨细胞的研究

目的:研究人恒牙牙髓组织来源的牙髓干细胞在体外分化为成骨细胞的能力,探讨其作为骨组织工程种子细胞的可行性.方法:从正畸治疗减数拔除的恒前磨牙中分离牙髓组织,应用酶消化法获得牙髓细胞.单抗Stro-1标记、免疫磁珠阳性分选系统分选获得牙髓干细胞,第3代牙髓干细胞用成骨向诱导培养基向成骨细胞诱导分化.用碱性磷酸酶染色和Vo...     

Insulin-like growth factor 1 promotes the proliferation and committed differentiation of human dental pulp stem cells through MAPK pathways

ObjectivesInsulin-like growth factor 1 (IGF-1) is a broad-spectrum growth-promoting factor that plays a key role in natural tooth development. Human dental pulp stem cells (hDPSCs) are multipotent and can influence the reparative regeneration of dental pulp and dentin. This study was designed to evaluate the effects of IGF-1 on the proliferation and differentiation of human dental pulp stem cells.MethodsHDPSCs were isolated and purified from human dental pulps. The proliferation and osteo/odontogenic differentiation of hDPSCs treated with 100 ng/ml exogenous IGF-1 were subsequently investigated.ResultsMTT assays revealed that IGF-1 enhanced the proliferation of hDPSCs. ALP activity in IGF-1-treated group was obviously enhanced compared to the control group from days 3 to 9. Alizarin red staining revealed that the IGF-1-treated cells contained a greater number of mineralization nodules and had higher calcium concentrations. Moreover, western blot and qRT-PCR analyses demonstrated that the expression levels of several osteogenic genes (e.g., RUNX2, OSX, and OCN) and an odontoblast-specific marker (DSPP) were significantly up-regulated in IGF-1-treated hDPSCs as compared with untreated cells (P < 0.01). Interestingly, the expression of phospho-ERK and phospho-p38 were also up-regulated, indicating that the MAPK signaling pathway is activated during the differentiation of hDPSCs.ConclusionsIGF-1 can promote the proliferation and osteo/odontogenic differentiation of hDPSCs by activating MAPK pathways.