瘦素对人牙髓干细胞增殖及分化的影响

目的 探讨瘦素(leptin)对体外培养的牙髓干细胞增殖、分化的影响.方法 体外培养牙髓干细胞,采用MTT法及流式细胞技术检测不同浓度瘦素对牙髓干细胞增殖作用的影响,通过碱性磷酸酶、Yon Kossa染色及矿化相关蛋白(DSP、OCN)的免疫组化染色来检测瘦素对牙髓干细胞分化的影响.结果 瘦素对牙髓干细胞增殖没有显著作用,但是对牙髓干细胞的ALP活性呈浓度依赖性促进.Von Kossa染色可见矿化结节形成,DSP及OCN表达阳性.结论 瘦素可促进牙髓干细胞向成牙本质细胞分化.     

黄精多糖对乳牙牙髓干细胞增殖及成骨向分化的影响

目的:初步探索不同浓度黄精多糖(PSP)对乳牙牙髓干细胞(SHEDs)增殖及成骨分化能力的影响.方法: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.采用不同浓度PSP处理SHEDs后,CCK-8法检测SHEDs的增殖情况,钙钴法染色和碱性磷酸酶(ALP)试剂盒检测其ALP活...     

EPO对人牙髓干细胞增殖及分化的影响

目的体外探究促红细胞生成素(EPO)对人牙髓干细胞(human dental pulp stem cells, hDPSCs)增殖及分化的影响。方法体外常规培养hDPSCs后,不同浓度EPO(0、0.1、1、10、20、50 U/ml)刺激hDPSCs,CCK-8法检测细胞增殖能力变化;采用碱性磷酸酶(ALP)染色及活性测定、茜素红染色、Real-time PCR来研究EPO对hDPSCs分化的影响。结果 10U/ml EPO可以促进hDPSCs的增殖(P<0.05);20U/ml EPO组碱性磷酸酶染色加深,活性提高,钙化结节形成增多;成血管成牙成骨向分化相关基因VEGF、bFGF、DSPP、Runx2、ALP的表达上调(P<0.05)。结论适宜浓度的EPO可以促进hDPSCs的增殖及分化。     

不同浓度黄芩苷对人根尖乳头干细胞成骨分化的影响

目的 探讨不同浓度黄芩苷对人根尖乳头干细胞（stem cells from apical papilla,SCAPs）成骨分化的影响。方法 选取具备稳定传代能力的第3代SCAPs,采用Live/Dead细胞荧光染色,检测细胞在0、10、20、50、100 μmol/L不同黄芩苷浓度干预培养24 h后的存活情况及细胞活力;然后将SCAPs分为普通培养基组（DMEM）和成骨诱导培养基组（ODM）,根据黄芩苷的不同浓度,将实验组分为5个实验亚组,依次为0、10、20、50、100 μmol/L,利用碱性磷酸酶（ALP）染色法和ALP定量检测法,研究不同浓度黄芩苷对SCAPs培养7、14 d早期成骨分化的影响。应用茜素红染色法,定量检测不同浓度黄芩苷对SCAPs培养21 d后成骨矿化的影响。利用MTT法,检测不同浓度黄芩苷对SCAPs生长的影响。采用SPSS 20.0软件包对数据进行统计学分析。结果 活/死细胞荧光染色结果表明,经不同浓度药物干预处理后,细胞活力旺盛,无明显异常;但黄芩苷药物浓度高于50 μmol/L时,细胞死亡数目有上升趋势。ALP定量检测结果显示,ODM组诱导分化第14天时,20 μmol/L浓度组的ALP活性显著高于对照组（P<0.05）,100 μmol/L浓度组的ALP活性显著低于对照组（P<0.05）。DMEM组培养14 d后,20 μmol/L浓度组的ALP活性显著高于对照组（P<0.05）。茜素红定量显示,ODM组中100 μmol/L实验组的A值显著低于对照组（P<0.05）;20 μmol/L的黄芩苷浓度组是促进SCAP生长的最适浓度。结论 高于50 μmol/L浓度的黄芩苷对SCAPs的成骨分化表现出明显抑制作用,而低浓度黄芩苷,尤其是20 μmol/L的黄芩苷对SCAPs成骨分化及细胞生长有促进作用。     

硅酸二钙对人牙髓干细胞增殖及骨向分化的影响

目的:探索硅酸二钙(Dicalcium silicate,C2S)在人牙髓干细胞增殖和成骨向分化能力方面的影响.方法:配制含梯度浓度(1、5、10、50、100μg/mL)的C2S条件培养基,与人牙髓干细胞共培养1、3、5、7天,CCK-8实验及迁移实验分别用于检测C2S对人牙髓干细胞的增殖能力与迁移能力的影响.筛选出...     

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...     

基于MAPK信号通路研究miR-218对牙髓干细胞增殖及成牙分化的作用

目的 基于丝裂原活化蛋白激酶/细胞外信号调节激酶(MAPK/ERK)信号通路研究微小RNA(miR)-218对牙髓干细胞(DPSCs)增殖及成牙分化的影响.方法 将含miR-218抑制物的重组质粒、阴性对照质粒转染至DPSCs,分别设为Anti-miR-218组、空载组,采用p38 MAPK抑制剂SB203580(5 ...     

促红细胞生成素对人牙髓细胞增殖和成骨分化的影响

目的：探讨促红细胞生成素（EPO）对体外培养的人牙髓细胞（hDPCs）增殖和成骨分化的影响。方法：体外培养鉴定人牙髓细胞。使用 EPO 对在成骨诱导培养基中培养的牙髓细胞进行刺激,CCK-8检测 EPO 对牙髓细胞增殖的影响;20 U ／ml EPO 培养 hDPCs 7 d 和14 d,采用碱性磷酸酶活性（ALP）检测和茜素红染色观察 EPO 对牙髓细胞矿化的影响;利用 Real-time PCR 检测加入 EPO 后牙髓细胞成牙本质向分化相关基因的表达情况。结果：EPO 以时间和剂量依赖性方式促进牙髓细胞的增殖;20 U ／ml 的 EPO 后作用,牙髓细胞碱性磷酸酶活性显著提高（P ＜0．05）,钙结节形成明显增多;成牙本质向分化相关基因 DSPP、OCN、OSTERIX、RUNX2的表达明显上调（P ＜0．05）。结论：EPO 能促进人牙髓细胞的增殖和分化。     

GLUD1基因对人牙髓干细胞的体外增殖、分化和矿化的影响

目的:探索谷氨酸脱氢酶1 (Glutamate dehydrogenase 1,GLUD1)基因在人牙髓干细胞(human dental pulp stem cells,hPDCSs)体外增殖、成骨分化和矿化中的作用.方法:体外分离培养hPDCSs,利用慢病毒感染方式沉默GLUD1基因的表达,分别采用RT-PCR和Western免疫印迹检测mRNA及蛋白水平的沉默效率.采用CCK8法检测细胞的体外增殖水平.对hDPSC进行体外成骨诱导分化培养,采用茜素红染色法检测矿化结节的形成,利用RT-PCR和免疫荧光染色分别检测Runx2、OCN基因的表达.采用SPSS 20.0软件包对数据进行统计学分析.结果:hDPSCs经过成骨诱导分化培养后,GLUD1的表达量较对照组明显上升;hDPSCs经shGLUD1病毒转染后,GLUD1表达明显下调;沉默GLUD1表达的hDPSCs体外增殖能力减弱;经成骨诱导分化培养后,与对照组相比,矿化结节形成明显减少;成骨早期标志基因Runx2上调,成骨晚期标志物OCN在mRNA和蛋白水平均显著下调.结论:沉默GLUD1表达抑制hDPSCs的体外增殖和矿化形成,影响晚期成骨分化;GLUD1在hDPSCs的成骨分化中具有重要作用.     

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.     

矿化液对人牙髓干细胞生物学特性的影响

目的 探讨矿化液对人牙髓干细胞(DPSCs)生长特性的影响.方法 用含一定浓度的β-甘油磷酸钠、抗坏血酸和地塞米松的矿化液诱导人牙髓干细胞,通过倒置相差显微镜、透射电镜、免疫细胞化学染色、von Kossa染色方法,观察和检测矿化液诱导后细胞形态、超微结构、表型和矿化基质分泌的变化,以未诱导组为对照.结果 矿化液连续培养7d,人DPSCs细胞形态明显改变,呈牙本质样极化细胞表现,另一侧为增大的胞体;超微结构显示,诱导后的细胞体积增大,核浆比例下降,胞浆细胞器丰富;诱导前细胞不表达牙本质涎磷蛋白(DSPP)和骨钙素(OC),诱导后均表达;连续培养21d,诱导组细胞形成多个矿化结节.结论 矿化液能诱导人DPSCs向成牙本质细胞样细胞分化并产生矿化基质.本研究从细胞水平上揭示了人DPSCs向成牙本质细胞分化机理,为人DPSCs应用于牙髓损伤的修复再生提供了依据.     

miR-124对牙髓间充质干细胞成骨分化的影响及机制探讨

目的: 观察微小RNA（miR）-124对牙髓间充质干细胞（DPSCs）成骨分化的影响,并探讨其可能机制。方法: 取对数期DPSCs,分为空白组、空载组、miR-124 inhibitor组和miR-124 inhibitor联合氮-[氮-(3,5-二氟苯乙酰)-L-丙氨酰]-S-苯基甘氨酸丁酯（DAPT,Notch信号通路抑制剂]组。空白组不处理,空载组转染阴性对照载体inhibitor-NC,miR-124 inhibitor组转染miR-124 inhibitor,miR-124 inhibitor联合DAPT组转染miR-124 inhibitor,并加入DAPT使其终浓度为5 μmol/L。转染48 h后,采用CCK-8法检测增殖能力;经诱导液诱导2周后,采用对-硝基苯磷酸盐（P-NPP）法检测碱性磷酸酶（ALP）活性;茜素红染色法检测钙化结节面积;Western印迹法检测细胞中发状分裂相关增强子1（HEY1）、发状分裂相关增强子2（HEY2）和细胞周期蛋白D1基因（CCND1）蛋白表达量。采用SPSS 19.0软件包对数据进行统计学分析。结果: 与空白组、空载组相比,miR-124 inhibitor组CCK-8 24、48、72 h A₄₅₀值,ALP活性A₄₅₀值,钙化结节面积构成比和HEY1、HEY2、CCND1蛋白表达量显著升高（P<0.05）;与miR-124 inhibitor组相比,miR-124 inhibitor联合DAPT组CCK-8 24、48、72 h A₄₅₀值、ALP活性A₄₅₀值、钙化结节面积构成比和HEY1、HEY2、CCND1蛋白表达量显著降低（P<0.05）。结论: 下调miR-124可促进DPSCs成骨分化,推测其作用机制与激活Notch信号通路有关。     

细胞外基质PDMS硬度对牙髓干细胞增殖和成骨分化的影响

目的:研究细胞外基质聚二甲基硅氧烷(polydimethylsiloxane,PDMS)硬度对牙髓干细胞(DPSCs)增殖和成骨分化的影响及其机制.方法:收集南京医科大学附属常州市第二人民医院因正畸而拔除的前磨牙作为实验材料,分离、培养DPSCs,制作PDMS基质.根据不同硬度,将PDMS基质分为A组(基料/固化剂=1...     

Effects of Notch ligand Delta1 on the proliferation and differentiation of human dental pulp stem cells in vitro

Objective

Notch signalling controls cell fate decisions in adult and embryonic tissues. The Notch ligand Delta1 is known to influence proliferation and differentiation of many kinds of tissue specific stem cells. In the present study, we investigated the role of Delta1 in the regulation of dental pulp stem cells (DPSCs) in vitro.

Methods

DPSCs were isolated from impacted third molars. Expression of human Notch1, 2 and Delta1 in DPSCs were detected by immunochemistry. Delta1 overexpressed DPSCs were constructed by a retroviral method. Delta1 transduced DPSCs proliferation changes were examined by means of colony-forming assay, BrdU incorporation assay and cell cycle analysis. Delta1 transduced DPSCs were cultured in differentiation-inductive medium. The nodule formation and DSPP expression were evaluated.

Results

It was shown that the Notch receptors and Delta1 ligand were expressed throughout the proliferation and differentiation process of cultured dental pulp stem cells. Furthermore, it was found in our study that Delta1 could significantly enhance the proliferation of DPSCs and permit DPSCs differentiating into odontoblast-like cells in differentiation-inductive environments.

Conclusions

Our findings verified that Notch-Delta1 signalling was expressed in human DPSCs in vitro and appeared to play pivotal role in DPSCs proliferation enhancement and differentiation regulation, thereby consistent with the hypothesis that the Notch pathway controls stem cell fate during pulp regeneration.