Bcl-2在成釉细胞分化、分泌过程中的表达研究

目的:检测凋亡调控抑制蛋白Bcl-2在成釉细胞分化、分泌过程中的表达,观察细胞超微结构的变化,探讨Bcl-2和细胞凋亡在该过程的可能作用。方法:制备出生后2、5、7、9、14 d不同发育阶段的BALB/C小鼠下颌第一磨牙牙胚标本,采用原位末端标记法(TUNEL法)和PV免疫组织化学技术观察成釉细胞分化、分泌和釉质发育完成各阶段细胞凋亡以及Bcl-2的表达情况;透射电镜观察细胞超微结构的变化。结果:出生后第2～5天,小鼠下颌第一磨牙成釉细胞处于分化期,超微结构可见胞浆内有高尔基复合体和线粒体,并有细胞增生的核分裂;免疫组化结果显示Bcl-2阳性表达,TUNEL检测结果发现部分细胞胞核阳性表达,提示细胞凋亡的存在;出生后第7天,成釉细胞已开始分泌,可见新形成的釉质,胞核远基底排列,胞核附近可见大量线粒体,胞质内可见大量高尔基复合体和粗面内质网,胞浆呈Bcl-2强阳性表达,TUNEL检测结果发现少量细胞胞核阳性表达;出生后14 d,釉质发育完成,成釉细胞变短,间隙变大,细胞器数量减少,核膜逐渐不清,核糖体脱颗粒水肿,呈现凋亡征象,胞浆未见Bcl-2阳性表达。结论:细胞凋亡在釉质发育的各期皆有表达,Bcl-2作为凋亡抑制基因可能参与了成釉细胞的分化、分泌的调控。     

小鼠牙胚发育过程中釉原蛋白和釉蛋白的表达特征

目的 观察釉原蛋白和釉蛋白在小鼠牙胚发育中的表达特征,进一步揭示釉原蛋白和釉蛋白的生物学特性.方法 分别制备出生后第1、3、7和14天小鼠下颌第一磨牙牙胚切片,采用免疫组织化学和反转录聚合酶链反应法检测釉原蛋白和釉蛋白的组织学定位和基因转录表达水平.结果 釉原蛋白表达于分泌期成釉细胞的胞质和釉质基质全层,在新生小鼠牙胚成牙本质细胞中有一过性表达;釉蛋白表达于分泌期釉质基质中,在成釉质细胞突边缘和釉质基质深层呈强阳性表达,釉原蛋白和釉蛋白在矿化成熟的釉质中均未见表达.釉原蛋白和釉蛋白在出生后第7天mRNA表达量的相对值分别为0.813±0.085和0.799±0.064,显著高于其他时间的表达水平(P＜0.05).结论 釉原蛋白和釉蛋白主要由分泌期成釉细胞合成并分泌到釉质基质中,其表达具有高度的时空特异性,提示它们在釉质形成和生物矿化中有重要的作用.     

釉成熟蛋白和釉原蛋白基因在小鼠牙胚发育过程中的时空表达研究

目的：观察、比较小鼠釉成熟蛋白（amelotin）和釉原蛋白（amelogenin）基因在牙齿发育过程中的表达。方法：利用Digoxigenin标记的cRNA探针，采用原位杂交技术对amelotin和amelogenin基因表达进行组织学定位；然后从小鼠下颌中提取总RNA，采用RT—PCR技术观察小鼠amelotin和amelogenin基因在小鼠下颌组织中的表达。结果：在出生后1d的小鼠下颌第一磨牙，随着前期牙本质形成，amelogenin在成釉细胞层开始表达，并逐渐增强，但未检测到amelotin基因表达；在出生后5d的小鼠，成釉细胞处于分泌期，amelotin和amelogenin基因在成釉细胞中显著表达；在出生后10d小鼠下颌第一磨牙，amelotin和amelogenin基因在牙尖周围成熟期成釉细胞中的表达信号明显降低。利用RT—PCR技术，在新生小鼠下颌中未检测到amelotin基因表达，但amelogenin已开始表达；从出生1—7d小鼠下颌中均检测到amelotin和amelogenin基因表达；在出生后7d小鼠，amelogenin表达显著下降。结论：amelotin和amelogenin均参与了釉质发育过程；amelotin基因表达迟于amelogenin基因，提示两种釉质基质蛋白在釉质发育过程中可能发挥不同生物学作用。     

c-Jun调控成釉细胞Amelotin基因表达的研究

目的:通过过表达及knockdown c-Jun基因,观察小鼠成釉细胞中釉成熟蛋白(amelotin)的表达变化,以初步确定c-Jun对amelotin的调控作用。方法:①应用免疫组化和细胞免疫荧光方法观察体内外成釉细胞中的c-Jun及amelotin的表达情况;②RT-PCR获得c-Jun基因,克隆至pcDNA3.1/myc-hisA,瞬时转染成釉细胞,real time RT-PCR观察其对amelotin表达的影响;③化学合成c-Jun siRNA,瞬时转染成釉细胞,re-al time RT-PCR观察其对amelotin表达的影响。结果:①体内外成釉细胞中c-Jun和amelotin均有表达,c-Jun主要在成釉细胞胞核中表达,amelotin在成釉细胞和釉质全层均有表达;②成功构建c-Jun真核重组表达载体pcDNA3.1/myc-hisA-c-Jun,转染成釉细胞后amelotin mRNA表达水平显著上升;③成功沉默c-Jun,amelotin在转染c-Jun siRNA组表达水平显著下降。结论:在成釉细胞中,c-Jun在转录水平调控amelotin基因的表达。     

小鼠釉质发育过程中成釉器中间层细胞增殖、凋亡以及组织非特异性碱性磷酸酶的表达

目的:观察小鼠釉质发育过程中成釉器中间层细胞的增殖、凋亡和其组织非特异性碱性磷酸酶(tissue nonspecific alkaline phosphatase,TNSALP)的表达,探讨中间层在釉质形成过程中的可能作用.方法:取出生后1、3、5、7、9、11、15d的BALB/c小鼠56只,解剖分离含下颌第一磨牙区的下颌骨,脱钙,制片,HE染色进行牙釉质发育情况的组织学观察,分别采用原位末端标记法(TUNEL法)、SP免疫组织化学技术、PV二步法观察中间层细胞的凋亡及检测Bcl-2、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),并对TNSALP进行组织学定位.通过Image-pro plus 6.0图像分析软件.对图像进行半定量分析.采用SPSS13.0软件包进行t检验、单因素方差分析及SNK-q检验.结果:出生后1d.中间层细胞增殖细胞核抗原由比成釉细胞表达高的强阳性表达逐渐降低;至7d时,其表达为阴性,而且中间层细胞随釉质的发育逐渐凋亡.中间层细胞先于成釉细胞表达TNSALP,出生后5d时即呈现强阳性染色,而后其表达又逐步减弱;成釉细胞7d时开始出现阳性,且呈逐渐增强趋势.结论:中间层细胞的增殖和凋亡及其TNSALP的表达与成釉细胞具有相关性,提示中间层细胞有可能参与成釉细胞的分化及釉质形成.     

小鼠牙齿发育中Pax-9的表达及意义

目的 通过观察PAX-9在牙发育中的分布及变化规律，探索牙齿发生的机制。方法 标本为妊娠10．5～18．5天昆明小鼠分别取胎鼠，分别作HE染色和免疫组化染色，图像分析仪分析。结果 Pax-9表达于胞浆中。蕾状期，牙蕾上皮及间充质细胞不表达Pax-9；帽状期牙乳头细胞表达较强；钟状期早期成牙本质细胞或前成牙本质细胞表达强阳性，内釉上皮细胞、星网状层细胞及牙乳头细胞有微弱表达；钟状期晚期成牙本质细胞、成釉细胞、及釉牙本质界处集中表达，新形成的釉质和前期牙本质染色较强。结论 PAX-9促进牙胚从蕾状期向帽状期转变，调节钟状期成牙本质细胞及成釉细胞的分化及基质分泌，并参与生物矿化过程。     

钟状期成釉细胞的体外生长研究

目的:通过对乳牙牙胚成釉细胞的体外培养生长情况的研究,了解成釉细胞的生长和分化过程.方法:解剖分离因疾病原因引产遗弃的19周胎儿下颌骨,半侧固定后拍摄数字X线影像,连续切片HE染色;另半侧未固定的组织分离乳牙牙胚成釉器,胶原酶消化法制成细胞悬液,采用差速贴壁和无血清培养基选择性培养成釉细胞.培养的乳牙胚成釉细胞通过细胞角蛋白14免疫细胞染色鉴定,并以RT-PCR方法检测釉质基质蛋白的表达.结果:X线影像显示牙胚冠部有硬组织形成,组织学乳牙牙胚处于钟状期,可见分化的成釉细胞分泌大量细胞外基质,釉牙本质界处釉质已矿化.无血清培养基中牙源性上皮的生长分化良好,细胞角蛋白14染色阳性,RT-PCR显示有釉蛋白、釉原蛋白和成釉蛋白的表达.结论:采用无血清培养基可以选择性的培养乳牙胚钟状期成釉细胞,能够较好反映成釉细胞的生长分化状态.钟状期分化的成釉细胞进入细胞外基质分泌活跃期,并在釉牙本质界处最早出现釉质的矿化.     

釉原蛋白在人牙胚发育不同时期表达的免疫组化研究

目的：探讨釉原蛋白在人牙胚发育不同时期的表达及其意义。方法：采用免疫组织化学方法观察釉原蛋白在不同发育阶段人牙胚中的分布。结果：在前成釉细胞、基底膜及前成牙本质细胞相邻部位都呈阳性反应。牙乳头细胞及牙本质呈阴性反应。釉原蛋白分布于釉质全层，在釉质浅层及与成釉细胞界面处呈线状强阳性染色，在后期矿化成熟时，成釉细胞及釉质染色阴性。结论：釉原蛋白与成釉细胞、成牙本质细胞的分化及釉质的矿化有关。     

过量氟对大鼠下切牙发育过程中釉蛋白表达的影响

目的 研究过量氟对大鼠下切牙发育过程中釉蛋白表达的影响。方法 选择20只Wistar大鼠,随机分成实验组和对照组。饲养8周后处死,利用HE染色和免疫组织化学染色的方法观察过量氟对大鼠成釉细胞形态及釉蛋白表达的影响。结果 实验组大鼠下切牙成釉细胞由原有的高柱状结构变矮,细胞排列成多层,釉基质形成混乱,成釉细胞和成牙本质细胞中釉蛋白表达明显低于对照组,其差异有统计学意义（P<0.01）。结论 过量氟可抑制釉蛋白表达,影响釉质发育。     

转录因子Ets-2调控小鼠成釉细胞MMP-20基因表达的研究

目的:通过研究Ets-2转录因子对调控小鼠成釉细胞金属基质蛋白酶-20(matrix metalloproteinase-20,MMP-20)基因表达的调控作用,进一步明确Ets-2在釉质发育中的作用。方法:应用免疫组化方法观察出生后5d小鼠切牙成釉细胞中Ets-2的表达;在小鼠成釉细胞中分别转染200ng pcDNA3.1/myc-HisA-Ets-2和Ets-2siRNA后,利用实时定量RT-PCR法检测MMP20基因表达的不同变化;用双荧光素酶报告基因检测系统分析Ets-2对MMP-20启动子突变后转录活性的影响。结果:免疫组化显示Ets-2在成釉细胞中呈阳性表达。实时定量RT-PCR研究发现Ets-2过表达后,MMP-20表达水平显著增加;当Ets-2基因沉默后,MMP-20表达水平则显著下降。双荧光素酶报告基因检测系统检测显示,转染pcDNA3.1/myc-HisA-Ets-2的实验组与对照组相比,MMP-20启动子的转录活性升高;对启动子Ets潜在结合部位进行定点突变后,MMP-20启动子的转录活性显著下降,Ets-2丧失上调MMP-20启动子转录活性的作用。结论:小鼠成釉细胞核中Ets-2可通过MMP-20启动子上Ets潜在结合位点,上调MMP-20基因表达水平。     

The immunohistochemical localization of Bax and Bcl-2 and their relation to apoptosis during amelogenesis in developing rat molars

Bax and Bcl-2 are members of a family of intracellular, membrane-associated proteins that regulate programmed cell death. It has been suggested that, when Bax predominates, programmed cell death is accelerated and the apoptosis inhibitory activity of Bcl-2 is suppressed. The present study was undertaken to immunohistochemically (IHC) localize Bax and Bcl-2 in the cells of the enamel organ during amelogenesis in rat molars. Also, apoptotic cells were detected by TUNEL staining. The IHC intense localization of Bcl-2 and light staining for Bax in the pre-ameloblasts suggest that apoptosis is inhibited in the proliferating pre-ameloblasts. This is consistent with an absence of TUNEL staining for apoptosis in these cells. However, in the late secretory and transition ameloblasts, and adjacent stratum intermedium, evidence of apoptosis of the ameloblasts was observed. Bax and Bcl-2 were co-localized in the proximal ends of late secretory, transition and early maturation-stage ameloblasts, but immunoreactivity for Bax markedly increased in the proximal ends of late secretory and transition ameloblasts, while the Bcl-2 staining appeared to be lighter. This suggests that Bax antagonized Bcl-2 function, limiting the ability of Bcl-2 to prolong cell survival. In the early maturation stage, Bax staining faded while the immunoreactivity for Bcl-2 increased. Evidence of distinct apoptosis was reduced in the early maturation stage ameloblasts. When related to the occurrence of apoptosis during amelogenesis, the relative intensity of expression of Bax and Bcl-2 changed in a pattern consistent with that observed in other cell lines. This indicates that these proteins play essential roles in the process of amelogenesis, as predicted by their proposed mechanisms of action in the control of apoptosis.     

High amounts of fluoride induce apoptosis/cell death in matured ameloblast-like LS8 cells by downregulating Bcl-2

Objective

Excessive fluoride intake during enamel formation may result in enamel fluorosis and apoptosis is regarded to be involved in the process by an unclear mechanism. We hypothesize that excessive fluoride might cause apoptosis in the ameloblasts and fluoride-induced apoptosis varies with the maturation stages of ameloblasts.

Methods

We set up an in vitro differentiation model of ameloblasts by using retinoic acid (RA) and dexamethasone (DEX) to induce the maturation of mouse ameloblast-like LS8 cells.

Results

The mRNA and protein levels of two enamel matrix proteins and two enamel proteinases were downregulated and upregulated, respectively, in the RA/DEX induced cells, indicating RA/DEX induced cells possessed the characteristics of matured ameloblasts. The strengthened endocytosis function and decreased intracellular pH value inside RA/DEX treated ameloblasts confirmed the maturation inducing effect of RA/DEX on ameloblasts. Excessive fluoride inhibited cell proliferation of ameloblasts within 72 h. High amounts of fluoride also induced more apoptosis/dead cells and reduced the expression of Bcl-2, but to a different degree in the non-induced cells and RA/DEX induced cells.

Conclusions

We inferred that high doses of fluoride may easily target the transitional/early maturation ameloblasts and cause apoptosis or cell death. Bcl-2 might be involved in this process.     

Organic anion transport during rat enamel formation

ObjectiveThe C-terminal end of nascent amelogenin is dissociated immediately after secretion and rapidly re-absorbed by ameloblasts, presumably by endocytosis. The purpose of this study was to test whether organic anion transporters (OATs) are also involved in the re-absorption process of enamel matrix proteins via non-endocytotic pathways.Materials and methodsLocalization of OAT1, OAT2, and OAT3 in rat tooth germs was examined by immunohistochemistry using specific antibodies. Actual translocation of organic anions through the ameloblast layer was further tested by systemic tracer experiments in rats in which Lucifer Yellow (LY), a fluorescent organic anion, was used as a tracer.ResultsIn rat tooth germs, OAT2 was associated exclusively with the distal cell membranes of secretory ameloblasts where Tomes' processes were developed and disappeared when matrix formation was terminated. On the other hand, OAT1 was absent in secretory ameloblasts and was colocalized with the ruffled border of ruffle-ended ameloblasts in the maturation stage. OAT3 was undetectable in ameloblasts and located instead only in the stratum intermedium cells. Systemic administration of LY resulted in intense labeling of immature enamel and also a transient labeling of the cytosol of secretory ameloblasts immunopositive for OAT2. In the maturation stage, cytosolic labeling of LY was negligible in all cells of the enamel organs, including ameloblasts.ConclusionsThese data suggest the existence of OATs in rat tooth germs and their possible involvement in matrix re-absorption at least in the secretory stage of amelogenesis.     

Fate of fluoride-induced subameloblastic cysts in developing hamster molar tooth germs

White opacities and pits are developmental defects in enamel caused by high intake of fluoride (F) during amelogenesis. We tested the hypothesis that these enamel pits develop at locations where F induces the formation of sub-ameloblastic cysts. We followed the fate of these cysts during molar development over time. Mandibles from hamster pups injected with 20 mg NaF/kg at postnatal day 4 were excised from 1 h after injection till shortly after tooth eruption, 8 days later. Tissues were histologically processed and cysts located and measured. Cysts were formed at early secretory stage and transitional stage of amelogenesis and detected as early 1 h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16 h post-injection. The size of the cysts was about the same, i.e., 0.46 ± 0.29 × 10⁶ μm³ at 2 h and 0.50 ± 0.35 × 10⁷ μm³ at 16 h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16 h post-injection. With time new ameloblasts repopulated and covered the enamel surface in the cystic area. Three days after injection all cysts had disappeared and the integrity of the ameloblastic layer restored. After eruption, white opaque areas with intact enamel surface were found occlusally at similar anatomical locations as late secretory stage cysts were seen pre-eruptively. We conclude that at this moderate F dose, the opaque sub-surface defects with intact surface enamel (white spots) are the consequence of the fluoride-induced cystic lesions formed earlier under the late secretory–transitional stage ameloblasts.     

氟对体外培养大鼠成釉细胞活性和细胞凋亡的影响

目的 观察氟对体外培养的大鼠成釉细胞活性的影响,为探讨氟斑牙的形成机制提供依据。方法 取对数生长期的大鼠成釉细胞,在培养液中加入浓度为0、0.4、0.8、1.6、3.2、6.4 mmol/L的 NaF溶液,培养24、48、72 h后,采用CCK-8检测各组细胞的活性。荧光显微镜下观察成釉细胞核形态的变化,流式细胞术分析氟对细胞凋亡的影响。采用SPSS13.0软件包对数据进行统计学分析。结果 ①NaF浓度为0.4、0.8 mmol/L时,对成釉细胞有促增殖作用;NaF浓度为1.6、3.2、6.4 mmol/L时,对成釉细胞的活性有抑制作用,随着NaF浓度的增加,对细胞的抑制作用也逐渐增强,并且这种双向调节作用呈时间依赖性。②NaF浓度为0.4、0.8 mmol/L时,鲜见核破碎;NaF浓度为1.6、3.2 mmol/L时,存在核破碎,并且随着浓度的提高,核破碎的数量随之增加,即1.6 mmol/L浓度的NaF可引起成釉细胞凋亡,随着浓度的增加,细胞凋亡的数量随之增加。结论 ①氟对体外培养成釉细胞的增殖具有双向调节作用,即低浓度促进,高浓度抑制。②浓度超过1.6 mmol/L时,NaF诱导成釉细胞凋亡。     

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7^fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.     

High-fluoride promoted phagocytosis-induced apoptosis in a matured ameloblast-like cell line

Endocytosis and phagocytosis are important physiologic activities occurring during ameloblast differentiation. We have previously found that excess fluoride inhibited ameloblasts endocytotic functions. Here, we hypothesized that increasing amounts of fluoride may affect ameloblast phagocytotic function during their differentiation. Using cell culture, we first induced maturation of the mouse ameloblast-like LS8 cells by treatment with exogenous retinoic acid (RA) and dexamethasone (DEX). We measured their phagocytotic activity by fluorescent microscopy using a live cell visualization station. We found that ameloblast-like LS8 cells matured with RA/DEX treatment and the increasing amounts of fluoride demonstrated the up-regulated expression of the phagocytotic marker proteins, LAMP1 and CD68. A connection between phagocytosis and apoptosis was confirmed by the increased number of phagocytotic vacuole-like structures and the heterochromatin margination phenomenon observed in the RA/DEX with NaF treatment group. The increase in albumin uptake by ameloblasts was confirmed using whole organ culture of incisor tooth germs. Here, in fluoride treated tooth germs, mature canonical ameloblasts showed greater amounts of albumin uptake, which was accompanied by decreased expression of the anti-apoptosis marker, Bcl-2 along with up-regulated expression of CD68. From these observations, we inferred that high doses of fluoride may cause apoptosis by increasing the phagocytosis of protein particles in mature-stage ameloblasts and loss of Bcl-2 signals might be involved in this process.     

黄芪多糖对大鼠正加速度引起咬肌成肌细胞损伤的保护作用

目的：探讨高正加速度（＋Gz）暴露下黄芪多糖对 SD 大鼠咬肌成肌细胞的影响。方法：原代培养的 SD 大鼠成肌细胞至第3代（P3）,取 P3代细胞随机分为5组,对照组（常规培养）,＋9 Gz 组,黄芪多糖浓度分别为0、19．53、78．13、312．5 mg／L 的处理组。培养72 h,给予＋9 Gz 加速度暴露。用 TUNEL 法检测成肌细胞凋亡率,RT-PCR 法检测 Bcl-2及 Bax 的表达。结果：与对照组比较,正加速度处理组细胞凋亡率增加,Bax 表达增加,Bcl-2表达降低;黄芪多糖各浓度组与加速度处理组比较,细胞凋亡率降低,Bax 表达降低,Bcl-2表达增加,但中浓度组、高浓度组抑制细胞凋亡的作用弱于低浓度组。结论：黄芪多糖能减轻高正加速度对成肌细胞的损伤,降低细胞凋亡率。其机制与调节成肌细胞 Bax,Bcl-2蛋白表达有关。     

Importance of bicarbonate transport in pH control during amelogenesis — need for functional studies

Dental enamel, the hardest mammalian tissue, is produced by ameloblasts. Ameloblasts show many similarities to other transporting epithelia although their secretory product, the enamel matrix, is quite different. Ameloblasts direct the formation of hydroxyapatite crystals, which liberate large quantities of protons that then need to be buffered to allow mineralization to proceed. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. Many investigations have used immunohistochemical and knockout studies to determine the effects of these genes on enamel formation, but up till recently very little functional data were available for mineral ion transport. To address this, we developed a novel 2D in vitro model using HAT‐7 ameloblast cells. HAT‐7 cells can be polarized and develop functional tight junctions. Furthermore, they are able to accumulate bicarbonate ions from the basolateral to the apical fluid spaces. We propose that in the future, the HAT‐7 2D system along with similar cellular models will be useful to functionally model ion transport processes during amelogenesis. Additionally, we also suggest that similar approaches will allow a better understanding of the regulation of the cycling process in maturation‐stage ameloblasts, and the pH sensory mechanisms, which are required to develop sound, healthy enamel.