bFGF调控成釉细胞中牙釉质基质蛋白的基因表达

本研究旨在说明b FGF可以通过DLX1/DLX2调控成釉细胞中牙釉质基质蛋白的基因表达。首先利用免疫组织化学显示DLX1/DLX2在分泌期成釉细胞中的表达;分离培养成釉细胞,并通过RT-PCR检测Dlx1/Dlx2及牙釉质基质蛋白在成釉细胞中的表达;在成釉细胞中分别过表达Dlx1和Dlx2后,Real time PCR检测牙釉质基质蛋白表达的改变;最后以50 ng/m L的b FGF的刺激成釉细胞,研究b FGF对Dlx1/Dlx2表达的影响。结果显示DLX1在分泌期小鼠成釉细胞中表达信号呈强阳性,而DLX2较弱;成功分离培养成釉细胞后,RT-PCR证实了Dlx1/Dlx2与牙釉质基质蛋白基因的表达在时空上存在同步性。在成釉细胞中分别过表达Dlx1和Dlx2对Ambn、Amelx、Amtn和MMP20的表达均有不同程度的影响;进一步研究发现b FGF能够促进Dlx1和Dlx2的表达。本研究结果提示b FGF可以通过DLX1/DLX2调控成釉细胞中牙釉质基质蛋白的基因表达。     

短期高浓度氟对小鼠磨牙成釉细胞形态及BMP-4表达的影响

目的:研究短期高浓度氟对小鼠磨牙成釉细胞形态及骨形成蛋白-4(BMP-4)表达的影响,探讨氟对牙本质发育的相关机制。方法:选择40只4日龄的ICR小鼠为研究对象,随机分2组各20只,每组对照鼠与实验鼠各半,对实验动物进行单次腹腔注射,剂量分别为10mg/kg和20mg/kg的Na F,对照组动物则进行单次腹腔注射等剂量的Na Cl,24小时后处死动物。采用免疫组化染色观察高浓度氟对小鼠磨牙不同分化阶段成釉细胞形态及BMP-4表达的影响。结果:成釉细胞在分泌过渡期及早、晚期对短期暴露高浓度F-敏感,与对照组小鼠相比,实验组小鼠成熟期成釉细胞无明显变化,过渡期与分泌晚期成釉细胞中BMP-4的表达显著降低,差异有统计学意义(P0.05)。结论:短期高浓度氟对分泌过渡期及晚期成釉细胞中BMP-4的表达有抑制作用。     

Cramp基因敲除对小鼠骨髓造血干细胞的影响

目的研究Cramp基因敲除在衰老过程中对小鼠造血干细胞的作用。方法应用流式细胞仪分析3月龄及12月龄Cramp基因敲除小鼠及同窝野生型小鼠的骨髓造血干细胞的比例及不同发育阶段B淋巴细胞的比例。结果与野生型小鼠相比,12月龄Cramp基因敲除小鼠的骨髓长期造血干细胞增多,多潜能造血祖细胞减少;前体B淋巴细胞和未成熟B淋巴细胞减少,成熟B淋巴细胞增多。结论在衰老过程中,Cramp基因敲除对骨髓造血干细胞及B淋巴细胞发育有重要影响。     

成年大脑神经细胞特异性ADAM10基因敲除小鼠模型的建立与鉴定

去整合素和金属蛋白酶10(ADAM10)是一种能够水解30余种跨膜蛋白质的“脱落酶”(sheddase), 参与诸多生理过程和致病机制, 如胚胎发育、细胞粘附、信号转导、免疫反应、癌症和阿尔茨海默病。迄今, 已报道的ADAM10完全基因敲除小鼠和大脑神经前体细胞特异性ADAM10基因敲除小鼠分别于胚胎期或围产期死亡, 致使无法研究成年小鼠大脑神经细胞ADAM10基因的功能。文章利用本研究小组建立的CaMKIIα-Cre转基因小鼠与ADAM10^loxP/loxP转基因小鼠杂交, 获得了CaMKIIα-Cre/ADAM10^loxP/loxP小鼠, 并对其进行鉴定。利用PCR方法检测成年ADAM10 cKO小鼠大脑基因组DNA表明, ADAM10基因缺失主要发生在前脑皮层和海马中。荧光定量PCR检测结果显示, ADAM10 mRNA的表达水平在前脑皮层和海马中分别降低55.7%和60.8% ; 使用Western blotting方法研究发现, ADAM10成熟蛋白质的含量在前脑皮层和海马中分别减少63%和84.8% 。采用免疫组织化学方法检测表明, 成年ADAM10 cKO小鼠与野生型小鼠相比, 其大脑皮层和海马神经细胞的ADAM10免疫染色明显减弱, 而其它细胞如胶质细胞的免疫染色基本一致。总之, 文章成功制备了首个存活至成年的大脑神经细胞特异性ADAM10基因敲除(cKO)小鼠, 克服了小鼠因ADAM10缺失在胚胎期或围产期死亡的弊端, 为研究成年小鼠大脑神经细胞ADAM10基因的功能奠定了坚实的基础。     

驱动蛋白-1在小鼠脂肪组织糖脂代谢中的作用研究

目的:本文旨在探讨动物体内水平驱动蛋白-1在脂肪组织糖、脂代谢中的作用。方法:通过Cre/Loxp重组系统构建脂肪组织特异性敲除驱动蛋白-1的小鼠模型,在生理水平观察驱动蛋白-1表达缺陷对小鼠糖代谢、脂代谢和脂肪因子分泌的影响。结果:与六月龄对照组小鼠相比,同月龄驱动蛋白-1敲除小鼠的体重、脂肪组织重量和空腹血糖水平没有显著差异,但是其血清胰岛素水平显著升高;使用葡萄糖耐量试验(GTT)和胰岛素耐量实验(ITT)对小鼠的糖代谢水平进行评估,结果显示驱动蛋白-1敲除小鼠表现为葡萄糖不耐受、胰岛素不耐受;进一步血清检测显示驱动蛋白-1敲除小鼠表现为高甘油三酯血症和血清脂联素水平降低。结论:驱动蛋白-1在脂肪组织中参与调节糖、脂代谢过程,其表达或功能障碍是2型糖尿病等代谢性疾病的一个重要的发病因素。     

高脂饮食对小鼠附睾内质网应激的影响及褪黑素的干预作用

目的探讨内质网应激在高脂饮食引起的ApoE基因敲除小鼠附睾损伤中的作用及褪黑素（MT）的干预机制。方法将12只ApoE基因敲除的C57BL／6J雄性小鼠随机分为高脂饮食组及MT处理组。高脂饮食组为ApoE基因敲除小鼠,给予高脂饮食;MT处理组给予高脂饲养外,并MT灌胃。以6只野生型C57BL／6J雄性小鼠作为对照组,给予普通饮食。饲养12w后,取附睾组织制片,HE染色观察附睾的病理学形态,免疫组化检测GRP78和CHOP的表达。结果HE染色显示,高脂饮食组小鼠,附睾上皮细胞形态结构不清,细胞萎缩。对照组和褪黑素处理组小鼠附睾上皮细胞形态结构完整,细胞排列整齐。免疫组化显示高脂饮食组小鼠附睾中GRP78、CHOP表达增强（P〈0．01）。MT处理组和高脂饮食组相比,附睾中GRP78、CHOP表达下调（P〈0．01）。结论内质网应激参与高脂饮食导致的附睾损伤;MT可能通过抑制附睾内质网应激,减轻高脂饮食对小鼠附睾的损伤。     

体内GRK5缺陷加剧Tg2576小鼠海马内的病理改变

目的研究体内G蛋白偶联受体激酶5（GRK5）缺陷是否会加剧转瑞典突变淀粉样肽前体蛋白基因（TgAPPsw,Tg2576）小鼠海马内的病理改变。方法将具有C57／BL6遗传背景的GRK5缺陷／敲除（GRK5KO）杂合子与具有相同遗传背景的Tg2576小鼠杂交,以产生野生型（WT）、GRK5KO杂合子型、转淀粉样肽前体蛋白（APP）基因型以及转基因＆敲除（Double）型4种基因型小鼠。用免疫荧光（IF）染色方法来观察这些动物海马内肿胀轴突丛（SACs）和A8沉积量变化。结果IF染色结果定量分析显示,Tg2576小鼠被灭活一个拷贝的GRK5基因后导致海马内SACs和A8沉积量均显著增加。结论体内GRK5缺陷加剧了AD动物海马内的病理改变。     

Midkine-a通过限制心肌细胞总数的方式阻碍斑马鱼胚胎心脏生长

目的探讨一种可溶性的外分泌因子midkine-a在斑马鱼胚胎心脏发育过程中的功能。方法在整体胚胎上做midkine-a RNA的原位杂交实验;利用原有的转基因斑马鱼系Tg(pmidkine-a:EGFP),动态观察胚胎从出生到心脏发育成形这一段时间心脏荧光表达情况;将原有的转基因斑马鱼体系Tg(phsp:midkine-a:EGFP)胚胎进行热休克而过表达midkine-a,观察胚胎心脏表型;利用Tg(pcmlc2:dsRed)鱼系胚胎的心肌细胞核带有红色荧光,能进行单个心脏心肌细胞计数这一特点,将杂合的Tg(phsp:midkine-a:EGFP)鱼系与纯合的Tg(pcmlc2:dsRed)鱼系交配,以得到Tg(phsp:midkine-a:EGFP/pcmlc2:dsRed)的杂合胚胎,对其进行热休克而过表达midkinea,计算每个胚胎心脏内心肌细胞的总数;用吗啉寡聚核苷酸(morphonino,MO)阻碍新生胚胎内的midkine-a mRNA表达,观察胚胎心脏表型。结果原位杂交试验证实midkine-a在胚胎48 hpf(hour post fertilization,受精后,用来标记斑马鱼胚胎年龄)大时表达于心脏;转基因Tg(pmidkine-a:EGFP)胚胎在72 hpf时其EGFP表达于心脏;Tg(phsp:midkine-a:EGFP)胚胎在过表达midkine-a后心脏变小;吗啉寡聚核苷酸敲除midkine-a对胚胎心脏发育无影响;最后在Tg(phsp:midkine-a:EGFP/pcmlc2:dsRed)鱼系胚胎内过表达midkine-a导致其单个心脏内心肌细胞数目变少,与其小心脏外形吻合。结论 midkine-a在斑马鱼胚胎发育过程中表达于胚胎心脏;过表达midkine-a导致胚胎心脏内心肌细胞总数减少及心脏变小;敲除midkine-a则对胚胎心脏发育无影响。     

肿瘤坏死因子-α对小鼠骨髓间充质干细胞成骨分化影响研究

目的:探讨肿瘤坏死因子-α(Tumor Necrosis Factor alpha, TNF-α)对小鼠骨髓间充质干细胞(Bone Marrow-derived Mesenchymal Stem Cells, BM-MSCs)成骨分化的影响。方法:以在促成骨分化培养基(Osteogenic media, OS)中培养的小鼠BM-MSCs作为对照,用含有TNF-α的OS处理小鼠BM-MSCs。第7天进行碱性磷酸酶染色,或培养第21天进行茜素红染色和定量分析。第7天时用一份细胞提取得到的mRNA通过实时定量RT-PCR测定Runx2和Osterix的mRNA表达,用另一份细胞得到的细胞裂解液通过SDS-PAGE来测定Runx2和Osterix的蛋白质表达。结果:TNF-α抑制碱性磷酸酶活性;TNF-α抑制矿化骨节的形成; TNF-α抑制Runx2和Osterix的mRNA和蛋白质表达。结论:TNF-α对小鼠BM-MSCs成骨分化的抑制,可能部分是通过抑制成骨分化中两个关键的转录因子Runx2和Osterix的mRNA和蛋白质表达来实现的。     

FKBP52对小鼠前列腺发育的影响

目的通过FKBP52基因敲除小鼠模型探索FKBP52在小鼠前列腺发育过程中的作用。方法分别对胚胎第17．5天、新生的和出生后3周的野生型和FKBP52基因敲除小鼠的前列腺进行切片HE染色,观察不同发育时期里野生型和FKBP52基因敲除小鼠前列腺发育的异同。结果（1）小鼠前列腺发育的起始不依赖于FKBP52基因的参与;（2）随着胚胎的发育,FKBP52在雄鼠前列腺发育中的作用逐渐显现出来,即FKBP52的缺失会导致前列腺叶发育受阻,最终不能形成成熟的前列腺。结论FKBP52在小鼠前列腺的发育过程中具有重要作用,它不参与前列腺的发育起始过程,但其缺失会导致前列腺发育受阻,即不能形成成熟的前列腺。     

The odontogenic ameloblast‐associated protein (ODAM) cooperates with RUNX2 and modulates enamel mineralization via regulation of MMP‐20

We have previously reported that the odontogenic ameloblast‐associated protein (ODAM) plays important roles in enamel mineralization through the regulation of matrix metalloproteinase‐20 (MMP‐20). However, the precise function of ODAM in MMP‐20 regulation remains largely unknown. The aim of the present study was to uncover the molecular mechanisms responsible for MMP‐20 regulation. The subcellular localization of ODAM varies in a stage‐specific fashion during ameloblast differentiation. During the secretory stage of amelogenesis ODAM was localized to both the nucleus and cytoplasm of ameloblasts. However, during the maturation stage of amelogenesis, ODAM was observed in the cytoplasm and at the interface between ameloblasts and the enamel layer, but not in the nucleus. Secreted ODAM was detected in the conditioned medium of ameloblast‐lineage cell line (ALC) from days 14 to 21, which coincided with the maturation stage of amelogenesis. Interestingly, the expression of Runx2 and nuclear ODAM correlated with MMP‐20 expression in ALC. We therefore examined whether ODAM cooperates with Runx2 to regulate MMP‐20 and modulate enamel mineralization. Increased expression of ODAM and Runx2 augmented MMP‐20 expression, and Runx2 expression enhanced expression of ODAM, although overexpression of ODAM did not influence Runx2 expression. Conversely, loss of Runx2 in ALC decreased ODAM expression, resulting in down‐regulation of MMP‐20 expression. Increased MMP‐20 expression accelerated amelogenin processing during enamel mineralization. Our data suggest that Runx2 regulates the expression of ODAM and that nuclear ODAM serves an important regulatory function in the mineralization of enamel through the regulation of MMP‐20 apart from a different, currently unidentified, function of extracellular ODAM. J. Cell. Biochem. 111: 755–767, 2010. © 2010 Wiley‐Liss, Inc.     

TGF-? Regulates Enamel Mineralization and Maturation through KLK4 Expression

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.     

E-Cadherin Can Replace N-Cadherin during Secretory-Stage Enamel Development

Background

N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development.

Methodology/Principal Findings

The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. μCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ.

Conclusions/Significance

The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects

Ameloblastin (AMBN) is the second most abundant extracellular matrix protein produced by the epithelial cells called ameloblasts and is found mainly in forming dental enamel. Inactivation of its expression by gene knockout results in absence of the enamel layer and its replacement by a thin layer of dysplastic mineralized matrix. The objective of this study was to further characterize the enamel organ and mineralized matrix produced in the AMBN knockout mouse. However, in the course of our study, we unexpectedly found that this mouse is in fact a mutant that does not express the full-length protein but that produces a truncated form of AMBN. Mandibles from wild type and mutant mice were processed for morphological analyses and immunolabeling. Microdissected enamel organs and associated matrix were also prepared for molecular and biochemical analyses. In incisors from mutants, ameloblasts lost their polarized organization and the enamel organ detached from the tooth surface and became disorganized. A thin layer of dysplastic mineralized material was deposited onto dentin, and mineralized masses were present within the enamel organ. These mineralized materials generated lower backscattered electron contrast than normal enamel, and immunocytochemistry with colloidal gold revealed the presence of amelogenin, bone sialoprotein and osteopontin. In addition, the height of the alveolar bone was reduced, and the junctional epithelium lost its integrity. Immunochemical and RT–PCR results revealed that the altered enamel organ in the mutant mice produced a shorter AMBN protein that is translated from truncated RNA missing exons 5 and 6. These results indicate that absence of full-length protein and/or expression of an incomplete protein have direct/indirect effects beyond structuring of mineral during enamel formation, and highlight potential functional regions on the AMBN molecule.     

Rat <Emphasis Type="Italic">wct</Emphasis> mutation prevents differentiation of maturation-stage ameloblasts resulting in hypo-mineralization in incisor teeth

A recent study provided genetic and morphological evidence that rat autosomal-recessive mutation, whitish chalk-like teeth (wct), induced tooth enamel defects resembling those of human amelogenesis imperfecta (AI). The wct locus maps to a specific interval of rat chromosome 14 corresponding to human chromosome 4q21 where the ameloblastin and enamelin genes exist, although these genes are not included in the wct locus. The effect of the wct gene mutation on the enamel matrix synthesis and calcification remains to be elucidated. This study clarifies how the wct gene mutation influences the synthesis of enamel matrix and its calcification by immunocytochemistry for amelogenin, ameloblastin and enamelin, and by electron probe micro-analysis (EPMA). The immunoreactivity for enamel proteins such as amelogenin, ameloblastin, and enamelin in the ameloblasts in the homozygous teeth was the same as that in the heterozygous teeth from secretory to transitional stages, although the homozygous ameloblasts became detached from the enamel matrix in the transitional stage. The flattened ameloblasts in the maturation stage of the homozygous samples contained enamel proteins in their cytoplasm. Thus, the wct mutation was found to prevent the morphological transition of ameloblasts from secretory to maturation stages without disturbing the synthesis of enamel matrix proteins, resulting in the hypo-mineralization of incisor enamel and cyst formation between the enamel organ and matrix. This mutation also prevents the transfer of iron into the enamel.     

APP processing and synaptic plasticity in presenilin-1 conditional knockout mice

We have developed a presenilin-1 (PS1) conditional knockout mouse (cKO), in which PS1 inactivation is restricted to the postnatal forebrain. The PS1 cKO mouse is viable and exhibits no gross abnormalities. The carboxy-terminal fragments of the amyloid precursor protein differentially accumulate in the cerebral cortex of cKO mice, while generation of beta-amyloid peptides is reduced. Expression of Notch downstream effector genes, Hes1, Hes5, and Dll1, is unaffected in the cKO cortex. Although basal synaptic transmission, long-term potentiation, and long-term depression at hippocampal area CA1 synapses are normal, the PS1 cKO mice exhibit subtle but significant deficits in long-term spatial memory. These results demonstrate that inactivation of PS1 function in the adult cerebral cortex leads to reduced Abeta generation and subtle cognitive deficits without affecting expression of Notch downstream genes.     

The absence of muscle segment homeobox 2 leads to the pyroptosis of ameloblasts by inducing squamous epithelial hyperplasia in the enamel organ

Muscle segment homeobox 2 (MSX2) has been confirmed to be involved in the regulation of early tooth development. However, the role of MSX2 has not been fully elucidated in enamel development. To research the functions of MSX2 in enamel formation, we used a Msx2^−/− (KO) mouse model with no full Msx2 gene. In the present study, the dental appearance and enamel microstructure were detected by scanning electron microscopy and micro-computed tomography. The results showed that the absence of Msx2 resulted in enamel defects, leading to severe tooth wear in KO mice. To further investigate the mechanism behind the phenotype, we performed detailed histological analyses of the enamel organ in KO mice. We discovered that ameloblasts without Msx2 could secrete a small amount of enamel matrix protein in the early stage. However, the enamel epithelium occurred squamous epithelial hyperplasia and partial keratinization in the enamel organ during subsequent developmental stages. Ameloblasts depolarized and underwent pyroptosis. Overall, during the development of enamel, MSX2 affects the formation of enamel by regulating the function of epithelial cells in the enamel organ.