组蛋白去甲基化酶KDM5A在人牙髓细胞及成牙本质细胞分化中的表达

目的探讨组蛋白赖氨酸去甲基化酶KDM5A在人牙髓细胞（hDPC）中的表达模式及成牙本质分化诱导对其表达量的影响。方法体外培养hDPC,实时荧光定量聚合酶链反应和Western blot检测第1代至第8代（P1-P8代）hDPC中KDM5A mRNA和蛋白的表达量;免疫荧光检测KDM5A在hDPC中的分布;对P3代细胞进行成牙本质分化诱导,于第7天和第14天分别检测KDM5A mRNA和蛋白的表达水平。结果体外传代培养hDPC中可检测到KDM5A的表达,KDM5A mRNA和蛋白量均呈先增加后减少的趋势;hDPC细胞质及细胞核中均表达KDM5A;成牙本质向分化诱导7和14 d,KDM5A mRNA和蛋白量高于未诱导组细胞,诱导14 d表达量高于诱导7 d（P〈0.05）。结论 hDPC表达KDM5A,矿化诱导可提高KDM5A的表达。     

组蛋白去甲基化酶Jmjd3对牙髓干细胞成牙本质向分化的影响研究

目的研究组蛋白去甲基化酶Jmjd3对牙髓干细胞(dental pulp stem cells,DPSCs)成牙本质向分化的影响。方法原代分离培养DPSCs,用流式细胞术和茜素红染色鉴定DPSCs。体外诱导DPSCs成牙本质向分化不同时间(0、3、5、7、14 d),qRT-PCR检测Jmjd3及成牙本质细胞标志物牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、牙本质基质蛋白1(dentin matrix protein1,DMP1)的表达情况。用不同浓度(0、1、10μmol/L)的Jmjd3抑制剂GSK-J4处理DPSCs 14 d,q RT-PCR检测DSPP、DMP1的表达情况。结果原代培养的DPSCs表达间充质干细胞表面标记物CD44、CD29、CD146,体外诱导培养具有成骨分化潜能。DPSCs成牙本质向分化过程中,Jmjd3、DSPP、DMP1表达水平均上调(P<0.05),且可能具有时间依赖性。10μmol/L GSK-J4处理DPSCs可明显抑制DSPP、DMP1的表达(P<0.05)。结论Jmjd3在DPSCs成牙本质向分化过程中发挥正调节作用,且与其去甲基化酶活性相关。     

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.     

组蛋白去甲基化酶KDM6A促进骨髓间充质干细胞成骨分化研究

目的研究组蛋白去甲基化酶KDM6A对骨髓间充质干细胞成骨定向分化能力的影响。方法构建Flag-KDM6A慢病毒表达质粒。利用慢病毒转染构建稳定过表达KDM6A的骨髓间充质干细胞。碱性磷酸酶活性实验检测成骨分化早期指标-碱性磷酸酶活性。茜素红染色及钙离子定量分析检测干细胞体外矿化能力。实时：毫量RT-PCR检测成骨分化相关基因．骨桥蛋白和骨钙素的表达。结果过表达KDM6A促进骨髓间充质干细胞碱性磷酸酶活性、骨髓间充质干细胞体外矿化能力以及骨桥蛋白和骨钙素的表达。结论组蛋白去甲基化酶KDM6A具有促进骨髓间充质干细胞成骨分化的潜能,可以作为一个候选的靶基因用于骨组织工程技术,促进骨组织再生。．     

TERT过表达对人骨髓间充质干细胞精氨酸组蛋白甲基化酶表达影响的研究

目的探索端粒酶对间充质干细胞精氨酸组蛋白甲基化酶和组蛋白甲基化状态的影响。方法利用逆转录病毒将端粒酶催化亚基转染到人骨髓间充质干细胞建立稳定转染细胞系,研究TERT过表达后对精氨酸组蛋白甲基化酶基因族的主要相关基因表达的影响。结果人骨髓间充质干细胞转染端粒酶催化亚基后,精氨酸组蛋白甲基酶基因超家族中的9个基因PRMT1～9的表达明显下降,组蛋白甲基化水平整体降低。结论 TERT过表达抑制了骨髓间充质干细胞精氨酸组蛋白甲基酶的表达,从而可能抑制精氨酸组蛋白的甲基化,精氨酸组蛋白甲基化水平降低有利于间充质干细胞维持年轻的状态。     

组蛋白去甲基化酶KDM4B促进根尖牙乳头干细胞中成骨和成牙本质分化

目的 研究组蛋白去甲基化酶KDM4B对根尖牙乳头干细胞定向分化能力的影响.方法 人重组骨形成蛋白4(BMP4)刺激根尖牙乳头干细胞后检测KDM4B的表达;利用慢病毒转染过表达或者基因敲除KDM4B进行获得性或丧失性功能研究.通过检测碱性磷酸酶(ALP)活性、茜素红染色、钙离子定量分析研究根尖牙乳头干细胞体外成骨和成牙本质分化能力.结果 BMP4促进根尖牙乳头干细胞KDM4B的表达.基因敲除KDM4B抑制根尖牙乳头干细胞ALP活性及体外矿化能力、促进转录因子PPAR-gamma的表达.过表达KDM4B增强根尖牙乳头干细胞ALP活性和体外矿化能力.结论 组蛋白去甲基化酶KDM4B具有促进根尖牙乳头干细胞成骨和成牙本质分化的潜能.     

组蛋白去乙酰化酶1、3在炎症牙周膜中的表达

目的:比较组蛋白去乙酰化酶1、3在炎症牙周膜和健康牙周膜中的表达差异。方法:分别收集炎症牙周膜和健康牙周膜,采用RT-PCR方法分别检测组蛋白去乙酰化酶1、3的mRNA表达,以管家基因作为内参,进行灰度值比较。结果:与健康牙周膜相比,炎症牙周膜中组蛋白去乙酰化酶1、3mRNA的表达均显著升高,差异有统计学意义(P<0.05)。结论:炎症牙周膜组织中组蛋白去乙酰化酶1、3表达升高,提示其可能参与牙周炎症反应的基因调控。     

Shh在鼠磨牙牙胚发育晚期的基因表达

目的：观察信号分子Sonic hedgehog(Shh)在小鼠下颌第一磨牙牙胚发育晚期的基因表达，探讨其在成釉细胞、成牙本质细胞分化中的作用。方法：制备昆明小鼠磨牙牙胚发育晚期(E16．5—P1．5)标本，用原位杂交法分析Shh mRNA在牙胚中的表达和分布。结果：Shh mRNA在牙胚发育晚期的前成釉细胞和中间层细胞呈阳性表达，在成牙本质细胞层表达较弱。结论：在牙胚发育晚期，Shh可能通过自分泌途径和旁分泌途径，参与了成釉细胞和成牙本质细胞分化。     

Expression of neural cell-adhesion molecule mRNA during mouse molar tooth development

This study employed in situ hybridisation using a probe recognising all isoforms of the molecule. Expression of the molecule in tooth germs started at embryonic day 13, when they were at the bud stage. Both inner cells of the epithelial bud and peripheral cells of the dental mesenchyme were positive. At the cap stage, positive cells were found in the inner part of the enamel organ but only in a limited area near the outer enamel epithelium. In the mesenchyme at the cap stage, expression was weak in the dental papilla and strong in the follicle. From the bell stage onward, epithelial cells in the enamel organ were negative except for the cells of the stratum intermedium, which were transiently positive at early and late bell stages. In the dental papilla, expression had mostly ceased during and after the bell stage, although transient expression was found in cuspal areas at the early bell stage. The dental follicle strongly expressed neural cell-adhesion molecule (NCAM) to the end of the experimental period, at post-natal day 4. In contrast to the first molar at its earliest stage of appearance, in which both the thickened epithelium and surrounding mesenchyme were negative for the expression of the molecule, the second molar appeared as a combination of extending epithelial thickenings and mesenchymal cells strongly positive for its expression. This study newly identifies the dental papilla and the stratum intermedium as NCAM-expressing sites.     

尼古丁对体外培养鼠磨牙牙胚发育的影响

目的:探讨尼古丁对体外鼠磨牙牙胚发育的影响.方法:采用RPMI-1640半固态培养基,在培养基中加入不同浓度的尼古丁,将15d胎龄的胎鼠的下颌第一磨牙牙胚分别置于各浓度组RPMI-1640半固态培养基表面培养4、8d,每48h更换培养基一次.结果:实验组与对照组相比较,主要表现在实验组发育中的牙胚体积缩小,成牙本质样细胞的分化程度不足、数量和速度均低于对照组,前期牙本质样基质形成减少或不能形成,并且呈时间、浓度依赖性.结论:尼古丁对体外培养胎鼠牙胚的形态、细胞的增殖和分化有抑制作用.     

Notch1在小鼠下颌第一磨牙牙胚发育中的表达

目的探讨Notch1在小鼠下颌第一磨牙胚胎发育过程中的组织学分布。方法制作ICR小鼠下颌第一磨牙不同发育阶段的冰冻组织切片,对小鼠下颌第一磨牙自牙胚发育起始期至出生后2天不同发育阶段组织的Notch1分布情况进行免疫组织化学染色。结果 Notch1在小鼠下颌第一磨牙牙胚发育起始期和蕾状期牙板上皮上方或其包绕的口腔上皮中表达,在牙板上皮中没有表达。自帽状期至钟状期,Notch1在牙胚的中间层表达,而在内釉上皮中无表达。至牙釉质和牙本质分泌期,Notch1仍在颈环部位的中间层表达。此外,Notch1还在牙胚发育不同时期的间充质、牙乳头和早期牙髓中表达。结论 Notch1可能在小鼠下颌第一磨牙发育过程中的牙上皮特别是内釉上皮的细胞分化,以及牙囊和牙乳头细胞分化及分化完成后的牙髓干细胞的稳定性方面有重要作用。     

Expression of typical calpains in mouse molar

Objective

Calpain is a calcium ion-dependent cysteine protease, consisting of two primary isoforms (calpain1/calpain2) which mediate crucial cellular functions. The activity of the calpains is tightly regulated by the endogenous inhibitor calpastatin. Calpains have been detected in several studies during the embryonic and foetal stages. The aim of this study is to investigate the temporal transition of typical calpains and their inhibitor calpastatin during odontogenesis.

Design

We used the first molar of foetal ICR mice from embryonic day (E) 14 to postnatal day (PN) 7. Using laser microdissection and semi-quantitative real-time PCR, we investigated calpain1, calpain2 and calpastatin expressions in each enamel epithelium, inner enamel epithelium, stellate reticulum and outer enamel epithelium.

Results

We found calpain1 and calpain2 mRNA increased in the all enamel epithelia between E18 and PN1. In addition calpastatin mRNA expression increased in the ameloblasts from PN1 to PN7. The immunohistochemistry results demonstrated that calpain1/calpain2 was present in the distal side of ameloblasts from PN1 to PN7, and calpastatin was present in the extracellular enamel matrix from E16 to PN1. Furthermore calpain1/calpain2 was present in the dentin, and calpastatin was detected in dentin producing odontoblasts and predentin at PN7.

Conclusions

In this study the temporal transition of calpain1, calpain2 and calpastatin mRNA and the immunolocalization are identified during tooth development. Our results indicate that the calcium-dependent proteases may play an important role in mouse molar development and extracellular calpain and calpastatin may be involved in molar mineralization.     

Morphogenesis and bone integration of the mouse mandibular third molar

The mouse third molar (M3) develops postnatally and is thus a unique model for studying the integration of a non-mineralized tooth with mineralized bone. This study assessed the morphogenesis of the mouse M3, related to the alveolar bone, comparing M3 development with that of the first molar (M1), the most common model in odontogenesis. The mandibular M3 was evaluated from initiation to eruption by morphology and by assessing patterns of proliferation, apoptosis, osteoclast distribution, and gene expression. Three-dimensional reconstruction and explant cultures were also used. Initiation of M3 occurred perinatally, as an extension of the second molar (M2) which grew into a region of soft mesenchymal tissue above the M2, still far away from the alveolar bone. The bone-free M3 bud gradually became encapsulated by bone at the cap stage at postnatal day 3. Osteoclasts were first visible at postnatal day 4 when the M3 came into close contact with the bone. The number of osteoclasts increased from postnatal day 8 to postnatal day 12 to form a space for the growing tooth. The M3 had erupted by postnatal day 26. The M3, although smaller than the M1, passed through the same developmental stages over a similar time span but showed differences in initiation and in the timing of bone encapsulation.     

Expression and processing of small integrin-binding ligand N-linked glycoproteins in mouse odontoblastic cells

OBJECTIVE: Small integrin-binding ligand N-linked glycoproteins (SIBLINGs) are expressed in dentin and believed to control dentinogenesis. Five members of SIBLING family include bone sialoprotein (BSP), osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP). These genes are clustered on chromosome 4q in humans and share similar biological features. DSPP and DMP1 are processed into given structural/functional fragments in rat and porcine. It still remains unclear whether these evidences occur in mouse and other SIBLING members are also processed into given fragments from their parent precursors. The aim of this study was to identify expression and processing of the five proteins in two mouse odontoblastic cell lines. DESIGN: Two mouse odontoblastic cells were used to study expression and processing of the five SIBLING proteins by immunohistochemistry and Western blot analyses. RESULTS: Immunohistochemistry study showed that all of the five SIBLING members were expressed within the cytoplasm and cellular processes in the mouse odontoblastic cell lines. Expression levels of DMP1 and DSPP were higher in differentiated mouse odontoblasts than undifferentiated mouse odontoblasts. Immunolabelling signal of DSP and MEPE was also detected within the nucleus in the two cell lines. Western blot assay indicated that all five members were processed into at least two fragments in these cells. CONCLUSIONS: These results suggest that different processed products and expression levels of the SIBLING proteins may play distinct biological functions in tooth development and mineralisation.     

核心结合因子a1在小鼠牙齿发育过程中的表达

目的：观察Cbfal蛋白在小鼠牙胚发育过程中的表达情况。方法：用自制的Cbfal多克隆抗体，采用免疫组化染色观察其时空表达特性。结果：Cbfal蛋白在帽状期牙胚中表达较广泛，在钟状早、中期牙胚表达于内釉细胞、成釉细胞以及紧靠成牙本质细胞层的牙乳头细胞，而在钟状晚期，Cbfal在牙乳头表达为阴性，成牙本质细胞层弱阳性，成釉细胞为阳性或强阳性。结论：Cbfal可能在小鼠牙胚发育，尤其是在成牙本质细胞和成釉细胞分化过程中具有重要作用。