大鼠切牙apical bud上皮细胞的分离与培养

目的：建立一种分离、培养大鼠切牙apical bud上皮细胞的有效方法。方法：选择出生后5d SD仔鼠,分离下颌切牙牙胚。将切牙颈部末端apical bud切下,酶消化后原代培养。经差别消化及选择性培养液纯化,并对培养的细胞进行角蛋白14和釉原蛋白、波形丝蛋白免疫组织化学染色鉴定。结果：培养的原代细胞混杂部分问充质细胞,经差别消化和选择性培养液纯化后获得了单一的上皮样细胞,此类细胞角蛋白14和釉原蛋白染色阳性,波形丝蛋白染色阴性。结论：经本方法的分离、培养成功地获得了纯化的大鼠切牙颈环上皮细胞。     

Noteh1在体外培养的大鼠根尖蕾细胞中的表达

目的：分离、培养和纯化大鼠切牙根尖蕾细胞，观察Notch1在根尖蕾细胞中的表达。方法：分离出生后7dSD大鼠下颌切牙胚，切取根尖唇侧部分，酶消化法原代培养，差别胰酶消化法纯化上皮细胞。角蛋白14和波形丝蛋白染色鉴定细胞来源。Notch1免疫细胞化学染色。结果：原代培养的细胞经2～3次差别胰酶消化后可获得纯化的根尖蕾上皮细胞。免疫细胞化学染色角蛋白14阳性，波形丝蛋白阴性，并且在根尖蕾细胞中存在Notch1阳性细胞。结论：体外培养的根尖蕾细胞中存在Notch1表达阳性的细胞，Notch信号途径可能参与了根尖蕾中干细胞增殖和分化的调控。     

双向差速法纯化大鼠牙囊细胞

目的：利用牙囊细胞和成釉细胞贴壁速度及酶消化分离速度不同的特点,建立一种简便、快速纯化大鼠牙囊细胞的方法。方法取新生5~6 d SD大鼠下颌第一磨牙牙胚,在体视显微镜下剥离牙囊及成釉器,剪碎后酶消化并混合培养,再利用差速贴壁法和差速传代法纯化牙囊细胞。结果原代细胞为牙囊细胞和成釉器细胞混合生长,差速传代培养到第2~3代可获得纯化的牙囊细胞。倒置显微镜下观察牙囊细胞呈梭形或三角形,免疫组织化学染色显示抗波形丝蛋白阳性,抗角蛋白阴性。结论双向差速法是一种高效、简便的纯化牙囊细胞的方法。     

大鼠切牙颈环上皮细胞的培养

目的：培养大鼠切牙颈环上皮细胞。方法：选择出生后（postnatal,PN）8d的SD大鼠,分离下颌切牙牙胚,切取切牙颈环结构,酶消化法细胞培养。通过差别消化法初步纯化颈环上皮细胞,抗角蛋白免疫化学染色初步鉴定颈环上皮细胞。结果：原代培养细胞为上皮和间充质混杂的细胞,经初步纯化获得铺路石样排列的,角蛋白阳性的颈环上皮细胞团。结论：实验成功培养了大鼠切牙颈环上皮细胞。     

差速传代纯化大鼠牙囊细胞

目的:建立一种简捷的分离培养和纯化大鼠牙囊细胞的方法。方法:分离出生后6dSD大鼠上下颌第一和第二磨牙完整牙胚,剥离牙囊和成釉器,剪碎后酶消化并混合培养,再利用多次差速传代纯化牙囊细胞。结果:原代细胞为牙囊细胞和成釉器细胞混合生长,差速传代培养到第4代可获得纯化的牙囊细胞。倒置显微镜下观察牙囊细胞呈长梭形或三角形,免疫组化染色抗波形丝蛋白阳性,抗角蛋白阴性。结论:利用多次差速传代可从混合培养的原代细胞中获得纯化的牙囊细胞。     

大鼠Hertwig＇s上皮根鞘细胞的分离培养

目的：建立大鼠Hertwig＇s上皮根鞘细胞分离、培养和纯化的方法。方法：分离出生后7dSD大鼠上下颌第一、二磨牙牙胚,切取牙冠颈部组织,酶消化法原代培养,利用差速传代纯化上皮细胞。用角蛋白14和波形丝蛋白染色确定细胞来源。结果：原代培养细胞为上皮和间充质混合细胞,经2～3次差速传代后可获得纯化的上皮根鞘细胞。免疫组化染色角蛋白14阳性,波形丝蛋白阴性。结论：利用酶消化法及差速传代法培养并获得纯化的上皮根鞘细胞。     

Notch 1在体外培养的大鼠根尖蕾细胞中的表达

目的:分离、培养和纯化大鼠切牙根尖蕾细胞,观察Notch 1在根尖蕾细胞中的表达.方法:分离出生后7d SD大鼠下颌切牙胚,切取根尖唇侧部分,酶消化法原代培养,差别胰酶消化法纯化上皮细胞.角蛋白14和波形丝蛋白染色鉴定细胞来源.Notch 1免疫细胞化学染色.结果:原代培养的细胞经2～3次差别胰酶消化后可获得纯化的根尖蕾上皮细胞.免疫细胞化学染色角蛋白14阳性,波形丝蛋白阴性,并且在根尖蕾细胞中存在Notch 1阳性细胞.结论:体外培养的根尖蕾细胞中存在Notch 1表达阳性的细胞,Notch信号途径可能参与了根尖蕾中干细胞增殖和分化的调控.     

人牙源性上皮细胞的体外培养研究

目的：建立人牙源性上皮细胞的体外培养方法，为牙齿组织工程研究提供可靠的种子细胞来源。方法：采用组织块法原代培养人牙源性上皮细胞，用更换培养液类型、多次差别消化法和反复贴壁法进行纯化，在倒置显微镜下观察细胞形态及生长状况，用免疫荧光法检测细胞表达角蛋白和成釉蛋白的情况。结果：原代培养的人牙源性上皮细胞生长良好，但有少量成纤维细胞混杂，经纯化后明显好转，上皮细胞呈片状生长，表达角蛋白及成釉蛋白。传至第5代后，细胞逐渐变为长梭形，失去上皮细胞形态。结论：体外培养的人牙源性上皮细胞在较长时间内可保持其特性，有望作为牙齿组织工程研究的种子细胞。     

大鼠根尖蕾细胞的分离培养

目的研究大鼠切牙根尖蕾细胞的分离、培养和纯化的方法。方法分离出生后3dSD大鼠下颌切牙胚,切取根尖唇侧部分组织,酶消化法原代培养,利用差速传代纯化上皮细胞。用角蛋白14和波形丝蛋白染色确定细胞来源。结果原代培养的细胞为上皮和间充质混合细胞,经2～3次差速传代后可获得纯化的根间蕾上皮细胞。免疫组化染色角蛋白14阳性,波形丝蛋白阴性。结论利用酶消化法和差速传代法培养获得了纯化的根尖蕾细胞。     

大鼠Hertwig''''s上皮根鞘细胞的分离培养

目的:建立大鼠Hertwig's上皮根鞘细胞分离、培养和纯化的方法.方法:分离出生后7d SD大鼠上下颌第一、二磨牙牙胚,切取牙冠颈部组织,酶消化法原代培养,利用差速传代纯化上皮细胞.用角蛋白14和波形丝蛋白染色确定细胞来源.结果:原代培养细胞为上皮和间充质混合细胞,经2～3次差速传代后可获得纯化的上皮根鞘细胞.免疫组化染色角蛋白14阳性,波形丝蛋白阴性.结论:利用酶消化法及差速传代法培养并获得纯化的上皮根鞘细胞.     

牙胚细胞的分离培养

目的：分离培养牙胚细胞。方法：选择出生后（PN）8天的SD大鼠,从第一磨牙牙胚上撕脱牙囊和成釉器组织,切取牙胚颈部组织,剁离牙乳头组织,分别进行细胞培养,通过差速消化法分离符种牙胚细胞。结果：牙囊、牙乳头、成釉器和上皮根鞘细胞被分离纯化出来。结论：利用差速消化法可同时分离培养4种主要的牙胍细胞。     

Ameloblastin and amelogenin expression in posnatal developing mouse molars

Ameloblastin and amelogenin are structural proteins present in the enamel matrix of developing teeth. Here we report the results of in situ hybridization analyses with DNA probes of ameloblastin and amelogenin expression in the mandibular first molars of ICR/Jcl mice from postnatal day 1 to day 15. Ameloblastin mRNA expression was observed in ameloblasts at day 2 while amelogenin mRNA was detected in secretory ameloblasts at day 3. Significant expression of both molecules was observed at days 4, 5 and 6, after which the levels decreased. Amelogenin expression ended on day 10, while ameloblastin mRNA was only weakly detected on day 12. Neither amelogenin nor ameloblastin expression was observed in day 15 mouse molars. Amelogenin and ameloblastin mRNAs were restricted to ameloblasts. We conclude that amelogenin and ameloblastin expression is enamel-specific, and suggest that these genes might be involved in the mineralization of enamel. It is possible that ameloblastin could participate in the attachment of ameloblasts to the enamel surface. In this case, the downregulation of expression may indicate the beginning of the maturation stage in which the ameloblasts tend to detach from the enamel layer.     

Identification of proteins from human permanent erupted enamel

Proteins from the extracellular matrix of enamel are highly specific and necessary for proper enamel formation. Most proteins are removed from the matrix by enamel proteases before complete mineralization is achieved; however, some residual protein fragments persist in the mineralized matrix of erupted enamel. So far, only amelogenin peptides obtained by traditional bottom‐up proteomics have been recovered and identified in human permanent erupted enamel. In this study, we hypothesize that other enamel‐specific proteins are also found in human permanent enamel, by analysing human erupted third molars. Pulverized enamel was used to extract proteins, and the protein extract was subjected directly to liquid‐chromatography coupled to tandem mass spectrometry (LC‐MS/MS) without a previous trypsin‐digestion step. Amelogenin and non‐amelogenin proteins (ameloblastin and enamelin) were succesfully identified. The sequences of the naturally occurring peptides of these proteins are reported, finding in particular that most of the peptides from the amelogenin X‐isoform come from the tyrosine‐rich amelogenin peptide (TRAP) and that some were identified in all specimens. In conclusion, our LC‐MS/MS method without trypsin digestion increased the coverage of identification of the enamel proteome from a few amelogenin peptides to a higher number of peptides from three enamel‐specific proteins.     

Effects of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on the formation of dental hard tissue of mouse molar tooth germs in organ culture system

In the developing tooth, 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) causes hypoplasia and hypomineralization of enamel and dentine. The present study was undertaken to clarify the effects of HEBP on the formation of dental tissues of tooth germs in an organ culture system. Mandibular first molars from 17.5-day-old mouse embryos were cultured with or without 250 microM HEBP in culture medium. Cultured tooth germs were analyzed by histological examination and by immunohistochemical localization using anti-amelogenin antibody. In cultured tooth germs treated with HEBP before the commencement of calcification in dentine, calcification of dentine matrix was inhibited completely and enamel formation was not observed. Ameloblasts were directly adjacent to dentine matrix. However, immunohistochemical data indicated that these ameloblasts secreted amelogenin. In the experiments of adding HEBP to cultured tooth germs on culture day 13, calcified dentine and enamel had formed before the administration of HEBP, but the dentine matrix newly formed after the administration of HEBP had not calcified. It was confirmed by immunohistochemical observations that enamel matrix-like material had penetrated into uncalcified dentine matrix and accumulated in dental papilla of tooth germs. However, no enamel matrix-like material was observed in calcified dentine and predentine underneath the calcified dentine by immunohistochemical staining. From these results, it might be concluded that ameloblasts secreted enamel matrix in the presence of HEBP and diffused through uncalcified dentine matrix into dental papilla. These findings suggests the calcification of dentine might be essential for the physical barrier to accumulate the enamel matrix and form a distinct layer of enamel as enamel.     

Relative levels of mRNA encoding enamel proteins in enamel organ epithelia and odontoblasts

Amelogenin, enamelin, sheathlin (ameloblastin/ amelin), enamelysin (MMP-20), and KLK4 (EMSP-1) are the major structural proteins and proteinases in developing tooth enamel. Recently, odontoblasts were reported to express amelogenin, the most abundant enamel protein. In this study, we hypothesized that odontoblasts express all enamel proteins and proteases, and we measured their relative mRNA levels in enamel organ epithelia and odontoblasts associated with porcine secretory- and maturation-stage enamel by RT-PCR, using a LightCycler instrument. The results showed that amelogenin mRNA in secretory-stage EOE is 320-fold higher than in odontoblasts beneath secretory-stage enamel, and over 20,000-fold higher than in odontoblasts under maturation-stage enamel. Similar results were obtained for enamelin and sheathlin. Enamelysin mRNA levels were equivalent in these two tissues, while KLK4 mRNA was higher in odontoblasts than in secretory-stage EOE. These results support the conclusion that odontoblasts are involved in the formation of the enamel layer adjacent to enamel-dentin junction.     

IGFs increase enamel formation by inducing expression of enamel mineralizing specific genes

Insulin-like growth factors (IGF-I and IGF-II) have been shown to play an important role in growth and differentiation in a number of tissues including mineralizing bone. Little is known about their role in tooth mineralization. Previous work in our laboratory has shown the presence of IGFs ligands, their receptors, and their binding proteins during mouse tooth morphogenesis. The expression of IGF I coincides with the expression of amelogenin, ameloblastin and enamelin at the late bell and secretory stage. The objective of this study is to determine the mechanisms by which IGFs modulate enamel and dentin formation. Mouse first mandibular molars were dissected from E16 and E17 mouse embryos and placed in organ culture in the presence of IGF-I or IGF-II. The molars were harvested after 12 days for histological examination or 1 day for mRNA expression analysis by real-time RT-PCR. Our results show an increase in enamel deposition, and an induction of enamelin, amelogenin and collagen type I mRNA expression, while expression of DSPP was down-regulated. These results suggest that IGFs increase enamel formation by the induction of gene expression of enamel related genes. Studies are underway to determine a possible mechanism for these factors.