促丝裂原激活蛋白激酶在牙髓干细胞向成牙本质细胞分化和牙髓损伤修复中的作用

通过诱导牙髓干细胞（DPSC）向成牙本质细胞方向分化,龋源性牙髓炎的治疗将不再局限于根管治疗这一临床选择,修复治疗也不再成为缺失牙治疗的唯一方案。促丝裂原激活蛋白激酶（MAPK）,尤其是P38MAPK通过直接或间接磷酸化特定的转录因子,将细胞外刺激信号转导至细胞及其核内,从而引起一系列细胞生物学反应,如细胞增殖、分化、转化和程序性死亡。骨形态发生蛋白-2、矿物三氧化物聚合体和Biodentine皆可诱导DPSC向成牙本质细胞分化,而三者正是通过MAPK信号转导通路发挥作用的。在组织工程支架诱导DPSC分化过程中,支架材料通过激活P38MAPK信号转导通路促进了DPSC的分化。此外,MAPK信号转导通路参与牙髓损伤修复中DPSC的迁移、黏附和分化,参与牙髓损伤修复中牙本质的形成。由于MAPK信号转导通路在细胞增殖、分化和生存等过程中都起着十分关键的作用,因此,深入研究其反应分子、作用底物和作用机制有着重要的理论和临床意义。     

体外持续培养对人牙髓细胞分化能力的影响

目的：探讨人牙髓细胞在体外持续培养时，其分化能力的变化趋势。方法：体外持续培养人牙髓细胞，检测不同代次细胞的碱性磷酸酶活性、钙化能力及Ⅰ型胶原表达，并对比分析。结果：随着体外培养细胞代次的增多，在同等刺激分化条件下，人牙髓细胞的碱性磷酸酶活性整体呈下降趋势，钙化结节的数目和面积逐渐减少，Ⅰ型胶原表达合成逐渐减少。结论：人牙髓细胞在体外持续培养时，分化能力逐渐下降，可能与其含有的未分化细胞（人牙髓干细胞）数目逐渐减少有直接关系。     

釉基质蛋白对人牙髓细胞成牙本质分化的影响

目的 探讨釉基质蛋白对人牙髓细胞(human dental pulp cells,HDPC)成牙本质分化的影响,为牙本质的组织工程学研究提供有效的生物活性物质.方法 将HDPC接种于6孔板(2×105个/孔)后分为5组,分别加入含1、10、100 mg/L釉基质蛋白(enamel matrix proteins,EMP)(分别为EMP 1、10、100 mg/L组)、10-8 mol/L地塞米松及100 mg/L抗坏血酸(dexamethasone and ascorbic acid,Dex-AA组)、单纯基础培养液(对照组).于培养1、5、10 d分别用碱性磷酸酶(alkaline phosphatase,ALP)试剂盒检测ALP活性,实时荧光定量反转录聚合酶链反应技术检测成牙本质相关基因牙本质基质蛋白1(dentine matrix protein-1,DMP-1)及牙本质涎磷蛋白(dentine sialophosphoprotein,DSPP)的表达,应用2-△△CT方法得出具体数值并进行单因素方差分析与Post Hoe 检验,用茜素红染色检测并定量分析矿化情况.结果 各组ALP水平呈时间依赖性上调,培养1d后各组ALP活性与对照组相比差异均无统计学意义;5 d后EMP 10、100 mg/L组及Dex-AA组ALP活性显著增高,分别达到7.573±0.267、6.119±0.502、5.846±0.096,与对照组相比差异均有统计学意义(P＜0.05);10 d后10 mg/L EMP组及Dex-AA组ALP活性增高更显著,分别达21.035±0.149、13.223±0.797,与对照组(5.825±0.404)相比差异均有统计学意义(P＜0.01).培养1d后各组DMP-1及DSPP mRNA水平均较对照组显著升高,差异均有统计学意义(P＜0.01);培养10 d后10 mg/L EMP组DMP-1和DSPP表达水平均显著增高,分别达到14.791±0.164、12.238±0.421.培养10 d后各组均有矿化,10 mg/L EMP组钙离子浓度[(191.8±2.0) μmol/L]显著高于对照组[(81.1±8.1)μmol/L],差异有统计学意义(P＜0.01).结论 适当浓度的EMP对HDPC的成牙本质分化有一定的促进作用.     

DLX3 mutation negatively regulates odontogenic differentiation of human dental pulp cells

ObjectivesThe purpose of this study was to investigate the role of a novel mutant DLX3 on the odontogenic differentiation of human dental pulp cells (hDPCs) in tricho-dento-osseous (TDO) syndrome.DesignhDPCs were obtained from the healthy premolars, stably-expressing wild-type DLX3 (WT), novel mutant DLX3 (Mu) and control vector (NC) cells were generated using recombinant lentiviruses. The proliferation rates of WT-hDPCs and Mu-hDPCs were measured by CCK8 assay. Odonto-differentiation of hDPCs was assessed by alkaline phosphatase (ALP) activity assay, and mineralization ability was assessed by Alizarin red staining. Odontogenic markers, including DMP-1, DSPP, Nes, ALP, and DLX5, were analyzed using real-time polymerase chain reaction (qPCR). DMP-1 and DSPP expressions were further confirmed by Western blotting.ResultsCCK8 results showed that the novel mutant DLX3 decreased the proliferation rate of hDPCs compared with wild-type DLX3. qPCR showed that the novel mutant DLX3 weakened odontogenic differentiation by downregulating the expression of odontogenic genes. These results were further confirmed by Western blotting and ALP activity assay. Additionally, Alizarin red staining showed that the novel mutant DLX3 decreased the mineralization of hDPCs compared with wild-type DLX3.ConclusionsNovel de novo mutation of DLX3 significantly decreases the proliferation rate and inhibits the odontogenic differentiation and mineralization of hDPCs, suggesting that this novel mutation of DLX3 can influence the dentinogenesis in TDO syndrome.     

人牙髓细胞条件培养液对人牙囊细胞成骨分化作用的体外研究

目的:探索人牙髓细胞条件培养液(HDPSCs-CM)对人牙囊细胞(HDFSCs)向成骨分化的作用.方法:利用胶原酶消化法获得HDFSCs,经纯化鉴定后培养于HDPSCs-CM中;CCK-8检测HDFSCs增殖活性,观察细胞形态改变;碱性磷酸酶(ALP)染色、茜素红S染色定性分析细胞成骨能力的改变.qRT-PCR检测HDFSCs中骨膜蛋白(POSTN)、Ⅰ型胶原(Col-Ⅰ)、碱性磷酸酶(ALP)、骨涎蛋白(BSP)以及骨桥蛋白(OPN)的mRNA表达情况.结果:经HDPSCs-CM诱导后,HDFSCs形态发生成牙骨质或成骨样改变;诱导组HDFSCs增殖活性受到明显抑制(P<0.05或P<0.01);诱导组ALP染色强于对照组,茜素红S染色矿化结节多于对照组;诱导组POSTN、Col-Ⅰ、ALP、BSP、OPN的表达量明显增高(P<0.05或P<0.01).结论:HDPSCs-CM能促进HDFSCs成骨分化.     

人牙髓细胞与牙周韧带细胞多向分化能力的比较

目的 比较人牙髓细胞(dental pulp cells,DPC)和牙周韧带细胞(periodontal ligamentcells,PDLC)的多向分化能力,揭示其干细胞成分特征,为开展干细胞介导的生物牙根再生奠定实验基础.方法 酶消化法分离培养人DPC和PDLC,流式细胞术检测STRO-1的表达.诱导细胞成牙本质及成骨分化、成脂分化和成软骨分化,von Kossa染色、抗骨钙素(osteocalcin,OCN)和牙本质涎蛋白(dentin sialoprotein,DSP)免疫组化染色、油红O染色、阿新蓝染色、抗Ⅱ型胶原免疫组化染色以及实时荧光定量反转录聚合酶链反应(RT-PCR)等检测DPC和PDLC的多向分化.结果 DPC和PDLC体外呈克隆样生长,STRO-1阳性率分别是(16.5%±4.2%)和(11.6%±1.1%).100%的DPC和83.3%的PDLC样本可多向分化.细胞诱导分化后,OCN、牙本质涎磷蛋白(dentinsialophosphoprotein,DSPP)、过氧化物酶体激活物增生受体2(peroxisomal proliferator activated receptorgamma 2,PPARγ2)、脂蛋白脂酶(lipoprotein lipase,LPL)和Ⅱ型胶原mRNA表达上调,与诱导前相比差异均有统计学意义(P<0.001),DPC和PDLC间OCN和PPARγ2基因上调倍数的差异有统计学意义(P<0.001).结论 人DPC和PDLC的间充质干细胞比例和多向分化能力相似.     

凝溶胶蛋白gelsolin对人牙髓细胞增殖及迁移的影响

目的研究凝溶胶蛋白gelsolin（GSN）对人牙髓细胞（hDPC）增殖及迁移的影响。 方法激光共聚焦检测gelsolin在hDPC中的表达。以小干扰RNA（siRNA）沉默hDPC中的gelsolin,CCK-8法检测细胞增殖;流式细胞仪检测细胞凋亡及细胞周期;Transwell实验观察细胞的迁移情况。采用单因素方差分析数据。 结果激光共聚焦显示gelsolin普遍表达于hDPC胞浆。沉默gelsolin,hDPC的增殖率在24、48、72 h时分别下降47.8%（F= 6.182,P= 0.035）、48.9%（F= 5.520,P= 0.044）、64.1%（F= 15.440,P= 0.004）;流式结果显示干扰gelsolin,hDPC的凋亡率增加约4.5%（F= 21.162,P= 0.002）,而细胞周期未发生明显阻滞,S期细胞比例升高约5%（F= 4.526,P>0.05）,G1期细胞比例下降约8%（F= 4.743,P>0.05）;Transwell结果显示,24 h内hDPC的迁移能力降低约60%（F= 12.781,P<0.001）。 结论gelsolin对hDPC的增殖及迁移具有调控作用。     

KLF4 promotes the odontoblastic differentiation of human dental pulp cells

Introduction

Krüppel-like factor 4 (KLF4) plays an important role in cytodifferentiation and proliferation. Our previous study showed that KLF4 was specifically expressed in polarizing and elongating odontoblasts. However, the role of KLF4 in odontoblast differentiation was still unknown. The purpose of this study was to investigate the role of KLF4 in odontoblastic differentiation of human dental pulp cells (hDPCs).

Methods

hDPCs were treated with odontoblastic induction medium. Odontoblastic differentiation was determined by the detection of alkaline phosphatase (ALPase) activity and the expression of mineralization-related genes including ALP, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1). Also, cell proliferation ability was examined by the 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay. Simultaneously, messenger RNA and protein levels of KLF4 were detected. pKLF4-IRES2-EGFP plasmid encoding full-length KLF4 was constructed to overexpress KLF4, and biologic effects of KLF4 on hDPCs were investigated by the evaluation of ALPase activity and the detection of ALP, DSPP, and DMP-1 expression and analysis of cell proliferation ability.

Results

ALPase activity and the expression of odontoblastic differentiation markers progressively increased in hDPCs cultured with odontoblastic induction medium. Meanwhile, the proliferation ability decreased in this procedure; messenger RNA and protein levels of KLF4 increased significantly on day 5 after the odontoblastic induction of hDPCs and kept increasing until day 14. hDPCs showed up-regulated activity of ALPase and the expression of mineralization-related genes, including ALP, DMP-1, and dentin sialoprotein (DSP), after KLF4 overexpression. Besides, the proliferation ability of hDPCs decreased significantly in the KLF4 overexpression group by EdU incorporation assay.

Conclusions

Our findings suggest that KLF4 is able to promote odontoblastic differentiation of hDPCs and inhibit proliferation of hDPCs.     

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cultured human dental pulp cells by low-power gallium-aluminium-arsenic laser irradiation

AIM: To examine whether low-power laser irradiation (LPLI) promotes cellular proliferation of human dental pulp-derived fibroblast-like cells (dental pulp cells). METHODOLOGY: Dental pulp cells were obtained by primary culture of human dental pulp tissues from extracted third molar teeth. The phosphorylation of the mitogen-activated protein kinase (MAPK) family after LPLI of these cells was investigated by Western blotting. By using a specific MAPK/ERK kinase (MEK) inhibitor (PD098059), the specific effect of LPLI on the MAPK pathway was also investigated by Western blotting as described above. The incorporation of [3H]thymidine into the cells after LPLI was determined, and statistical analysis was performed by Wilcoxon signed-ranks test. RESULTS: Extracellular signal-regulated protein kinase (ERK) 1/2 was phosphorylated between 5 and 30 min after LPLI. Moreover, PD098059 inhibited LPLI-mediated ERK1/2 activation. LPLI did not affect p38 MAPK or c-Jun N-terminal kinase (JNK) phosphorylation. But LPLI did not stimulate [3H]thymidine incorporation into these cells. CONCLUSIONS: These results indicated that LPLI activated MAPK/ERK, a signal for proliferation, differentiation and survival, but did not activate the stress signals p38 MAPK and JNK in human dental pulp cells.     

应用不同方法进行人牙髓细胞原代培养的比较研究

目的比较并建立人牙髓细胞原代培养的理想方法.方法分别采用组织块法、酶消化法和组织块消化法进行人牙髓细胞的体外原代培养,倒置显微镜及台盼蓝染色观察并比较三者的培养成活率、细胞活力、细胞形态和数量、培养耗时等,免疫组化法鉴定细胞来源.结果三种方法均可从人牙髓组织分离培养获得外胚间充质来源的牙髓细胞.组织块法耗时较长,4周能进行首次传代,传代成功率50%,细胞形态较单一;酶消化法能在较短时间内获得形态多样的人牙髓细胞,但培养成活率较低,且细胞数量较少,8天时传代成功率为20%;组织块消化法不仅培养成活率(75%)和8天传代成功率(62.5%)较高,而且在较短时间内可多量获得多种形态类型的人牙髓细胞.结论组织块消化法是一种更加科学可行的人牙髓细胞原代培养的优化方法,可为进行人牙髓干细胞的分离培养及特性研究提供实验方法学基础.     

Stimulating effects of quercetin and phenamil on differentiation of human dental pulp cells

Dentin formation is preferred in the healing response of the pulp to pulp‐capping agents during vital pulp therapy. Enhancement of the dentinogenic differentiation of dental pulp cells is thought to accelerate pulp repair. The aim of this study was to evaluate the dentinogenic activity of small molecules (three flavonoids and phenamil) that have been shown previously to induce osteoblast differentiation. Among the flavonoids (quercetin, genistein and baicalin), quercetin induced the highest alkaline phosphatase (ALP) activity of human dental pulp (HDP) cells. Phenamil, an amiloride derivative, elicited higher ALP activity than quercetin. However, increased expression of dentin sialophosphoprotein (DSPP) mRNA and mineral deposition were seen in cultures treated with quercetin compared with phenamil. This would seem to suggest that quercetin is the most dentinogenic agent among the tested chemicals. The increase in ALP activity in the quercetin‐treated cells was not affected by ICI 182,780, an estrogen receptor inhibitor, and was partially blocked by PD98059, an extracellular signal‐regulated kinase 1/2 (ERK1/2) inhibitor. This suggests that ERK1/2 is activated in the quercetin‐induced differentiation of HDP cells without the mediation of estrogen receptors, which are known to be involved in osteoblast differentiation induced by quercetin.     

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。     

碱性成纤维细胞生长因子对体外培养人牙髓细胞增殖和迁移的影响

目的研究碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）对体外培养人牙髓细胞增殖、迁移活性的影响。方法以常规组织块培养法体外获得人牙髓细胞,实验组分别加入终浓度为1.0、5.0、10.0和50.0ng/ml的bFGF,对照组仅加入等量的空白培养基,采用CCK-8法分别测定450nm下各组光吸收度A值,研究bFGF对牙髓细胞增殖活性的影响;应用Transwell小室培养法,研究bFGF对牙髓细胞迁移活性的影响。结果与对照组相比,bFGF在1.0～50.0ng/ml浓度下可促进牙髓细胞的增殖（P〈0.05）,bFGF最低显效浓度为1.0ng/ml,最佳显效浓度为10.0ng/ml。bFGF在1.0～50.0ng/ml浓度下诱导细胞迁移（P〈0.05）。结论 bFGF可诱导体外培养人牙髓细胞的增殖和迁移,在牙髓牙本质复合体修复中可能发挥重要作用。     

miR-103对牙髓干细胞增殖及其向成牙本质细胞分化的影响

目的:探讨miR-103对牙髓干细胞向成牙本质细胞分化的影响.方法:从即刻拔除的成入第三磨牙中分离培养人牙髓干细胞,将培养的细胞分为对照组、miRNA阴性对照组、miR-103过表达组和miR-103降低表达组,利用CCK-8法检测人牙髓细胞的增殖能力变化,利用q-PCR和Western Bolt技术检测人牙髓细胞中R...     

小檗碱对牙髓干细胞MAPK/ERK信号通路及成牙本质向分化的影响研究

目的 探讨小檗碱对牙髓干细胞(dental pulp stem cells,DPSCs)成牙本质向分化及p38丝裂原活化蛋白激酶(MAPK)/细胞外调节蛋白激酶(ERK)信号通路的影响.方法 采用酶消化法提取DPSCs,将培养鉴定的DPSCs传代培养至第3～5代,用于以下实验.实验分为对照组(正常培养DPSCs)、矿化...     

Inhibition of Delta1 promotes differentiation of odontoblasts and inhibits proliferation of human dental pulp stem cell in vitro

Objective

Dental pulp stem cells (DPSCs) have been receiving more attentions recently as an important biomaterial for tissue engineering. Notch signalling plays a key role in regulating self-renewal and differentiation of a variety of cells. The objective of this study is to investigate the effects of Notch-Delta1 RNA interference (RNAi) on the proliferation and differentiation of human dental pulp stem cells in vitro.

Design

In the present study, we performed gene knockdown of Notch ligand Delta1 in DPSCs using lentivirus-mediated Delta1-RNAi. Changes of proliferation in DPSCs/Delta1-RNAi were examined by cell cycle analysis, Cell viability assay (CCK-8) and Western blot analysis of proliferating cell nuclear antigen (PCNA). Cells were cultured in odontoblast differentiation-inducing medium, and the differentiation of cells was detected with Alkaline phosphatase ALP activity assay, Alizarin red S staining, calcium concentration measurement, and Western blot analysis of Dentine sialophosphoprotein (DSPP).

Results

Lentivirus-mediated Delta1-RNAi stably knocked-down the expression of Delta1 and Notch signalling, and some of DPSCs/Delta1-RNAi displayed changes in morphology or DSPP expression. The growth rate of Delta1-deficient DPSCs was significantly suppressed as compared with wild type DPSCs and control lentivirus vector transfected DPSCs. Furthermore, the differentiating capability of DPSCs/Delta1-RNAi into odontoblasts is much higher than the two control groups.

Conclusions

Notch signalling plays a crucial role in regulating self-renewal and differentiation in DPSCs. The deficient Notch signalling inhibits the self-renewal capacity of DPSCs and tends to induce DPSCs differentiation under odontoblast differentiation-inducing conditions. These findings suggested that DPSCs/Delta1-RNAi might be applicable to stem cell therapies and tooth tissue engineering.