• International Journal of Oral Biology
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• 제43권4호
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• pp.177-183
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• 2018

The objective of this study was to examine the expression pattern of Kelch-like ECH-associated protein 1 (Keap1) in the maxillary $2^{nd}$ molar germs of rats. We used the maxillary $2^{nd}$ molar germs in rats' pup at postnatal day 3 (bell stage), 6 (crown formation stage) and 9 (root formation stage). The investigation on mRNA and protein levels were done using reverse transcription - polymerase chain reaction and western blot. Localization of Keap 1 in the maxillary $2^{nd}$ molar germs were revealed through immunofluorescence staining. Keap1 from the maxillary 2nd molar germs were mostly manifested on postnatal day 3 and dramatically decreased on postnatal day 6 and 9 at mRNA and protein levels, while amelogenin and ameloblastin increased during the development of maxillary 2nd molar germs. During immunofluorescence analysis, the strong immunoreactivity against Keap1 was detected in the apical side of ameloblasts at the presecretory and secretory stages. However, Keap1 expression was hardly observed in the ameloblasts at the maturation stage. These results shows that Keap1 is strongly expressed in the presecretory and secretory ameloblasts of amelogenesis, and suggest that Keap1 may be a crucial molecule for the regulatory mechanisms tasked with the formation of enamel layer.

• Applied Microscopy
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• 제46권1호
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• pp.58-66
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• 2016

Thymosin ${\beta}4$ ($T{\beta}4$) has been recently reported to play a role in dentinogenesis by regulating the expression of dentin matrix proteins. Based on previous studies, it is hypothesized that $T{\beta}4$ is associated with the formation of the enamel matrix and thus plays an important role in ameloblast. However, there is no report on the function of $T{\beta}4$ during tooth development so far. Therefore, in this study, we aimed to investigate the expression of $T{\beta}4$ and its function in ameloblasts during mouse tooth development. $T{\beta}4$ was expressed strongly in the tooth bud at the bud stage and in the dental lamina and oral epithelium at the cap stage. In advanced bell stage at postnatal day 4, large elongated ameloblasts were observed and the expression of the $T{\beta}4$ protein was the highest, with the enamel being was thicker than that in the early bell stage. The length of ameloblasts increased from the presecretory to the secretory stage and decreased from the maturation to the protective stage. These results suggest that $T{\beta}4$ participates not only in the proliferation of oral epithelial cells during the early stage of tooth development but also regulates enamel protein secretion in ameloblasts and enamel mineralization.

• Applied Microscopy
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• 제34권2호
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• pp.145-157
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• 2004

불소 투여가 백서태아 발육에 따른 법랑질형성 과정에 미치는 영향을 알아보고자 임신한 어미흰쥐에 대조군은 증류수만을 음용시켰고, 실험군은 100, 200 및 300 ppm의 불소가 함유된 음용수를 투여하였다. 이후, 생후 11일 된 어린 흰쥐를 희생하였고, 하악절치를 발치하여 법랑질형성 동안에 일어나는 법랑모세포의 형태 및 구조적 변화를 광학현미경과 투과전자현미경으로 관찰하였다. 흰쥐태아 치아기의 조직학적 구성은 전분비대, 분비대 및 성숙대로 관찰되었으며, 특히 성숙대의 법랑질에서 물과 유기물을 선택적으로 제거하는 평탄끝법랑모세포(smooth-ended ameloblast)와 무기이온을 추가로 공급하는 주름끝법랑모세포(ruffle-ended ameloblast)가 관찰되었다. 또한 치아기는 가장 외측으로부터 외법랑상피, 성상세망, 중간층, 법랑모세포층, 법랑기질층, 상아기질층, 치수로 이루어져 있었다. 이러한 조직학적 구성은 백서태아에서도 성체에서 관찰되는 구조들과 동일한 것으로 확인되었다. 한편, 불소투여군에서 각 대(zone)의 법랑모세포들은 구조적 및 형태적인 변화가 확인되었는데, 조면소포체가 공포화되고 막성계의 유실로 붕괴되었으며, 라멜라 형태인 동심원상의 팽윤된 구조가 미토콘드리아의 기질에서 관찰되었다. 특히 고농도 실험군인 300 ppm투여군에서 법랑모세포사이에 세포막이 유실된 세포들이 관찰되었다. 그러므로 임신중인 흰쥐에 투여된 불소는 흰쥐태아의 치아발육과정 중 법랑질형성에 관여하는 법랑모세포에 영향을 미쳐 전환주기에 변화를 주고 법랑모세포의 미세구조적 변화와 형태적인 변화를 초래하였다.

• International Journal of Oral Biology
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• 제41권2호
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• pp.89-96
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• 2016

Tooth development shows dynamic morphological changes from the stages of cap to hard tissue formation and is strictly regulated during development. In the present study, we compared expression and localization of 3 major enamel matrix proteins in rats: amelogenin, enamel and ameloblastin. DD-PCR and RT-PCR revealed differential expression of the major proteins from the cap stage to root stage. Immunofluorescence staining results indicated that amelogenin was not detected in either inner enamel epithelium or reduced enamel epithelium, but highly immunoreactive in preameloblasts and ameloblasts; in addition, it was sporadically expressed in preodontoblasts abutting preameloblasts. Ameloblastin expression was also observed in not only differentiated ameloblasts but also osteoblasts. Immunoreactivity to ameloblastin in ameloblasts was strong in Tomes' processes. Enamelin was exclusively localized along the entire newly formed and maturing enamel. Enamelin was largely localized in near Tomes' processes and enamel rods in maturing enamel. Alendronate treatment resulted in down-regulation of amelogenin and ameloblastin at both transcription and translation levels; whereas, enamelin expression was unchanged in response to the treatment. These results suggested that amelogenin, ameloblastin and enamelin might be implicated in cell differentiation, adhesion of ameloblasts to enamel and enamel crystallization during enamel matrix formation, respectively.

• International Journal of Oral Biology
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• 제36권4호
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• pp.195-201
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• 2011

Matrix metalloproteinases (MMPs) have been implicated in tissue development and re-modeling. Dynamic morphological changes of tooth germs reflect involvement of these enzymes during odontogenesis. The present study was performed to investigate expression and localization of MMP-2 and MMP-9, which have been known to have type IV collagenase activities, in rat tooth germs at different developmental stages. MMP-2 expression was increased gradually in the tooth germs from cap to crown staged germs at both transcription and translation levels. The localization of this molecule was detected in secretory ameloblasts and preameloblasts. The strong immunoreactivities were occasionally seen along the basement membrane between ameloblasts (or preameloblasts) and odontoblasts (preodontoblasts). However, weak reactivity was detected in odontoblasts and reduced enamel epithelium. The level of MMP-9 expression in the tooth germs was higher in cap stage than in crown staged germs at both transcription and translation levels. They were strongly expressed in both ameloblasts and odontoblasts. Even though reduced enamel epithelium after enamel formation and inner enamel epithelium at the cap stage exhibited weak reactivity, strong reactivity was detected in dental follicles and perifollicular tissues surrounding cap staged germs. These results suggested that MMP-2 may involve degradation of the basement membrane during hard tissue formation, whereas MMP-9 might be involved in remodeling of follicular tissues.

• 대한소아치과학회지
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• 제11권1호
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• pp.131-143
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• 1984

150 rats weighting about 150gm were devided into control group of 80 and experimental group of 70. Control group was subdivided into the irradiated vitamin D injection group and X-ray irradiated group. Experimental group was given 2.0mg ergocalciferol by four intramuscular injection prior to X-ray irradiation with single 800 rads and 1,500 rads respectively. Experimental animals from each group was sacrificed after 1, 3, 7, 14, and 28 days and their incisors were investigated by histopathological examination. The results were as follows; 1. In the irradiated groups, it showed dentin hypoplasia and formation of dentinoid substance caused by degeneration of odontoblast at the early stage. Especially, 1,500 rads group which was severely effected showed formation of osteoid dentin at the apical portion and severe injuries of dental papilla at the first week. 2. In the vitamin D2 administration group, it showed thinned dentin layer at the early stage but, taking time, predentin and dentin layer was thickened. At the fourth week, dentin was chiefly composed of interglobular dentin, especially in the lingual portion. 3. Using in combination of overdose vitamin D2 administration and X-ray irradiation, it effected severely odontoblast, undifferentiated mesenchymal cells around tooth germ and pulp tissue. At the early stage, dentin layer was thinned but, taking time, it was thickened and composed of interglobular dentin caused by calcification of predentin layer. 4. In 800 rads irradiation after the overdose vitamin D2 administration, it showed formation of osteoid dentin in the lingual portion at the first week. In the 1,500 rads irradiation after the overdose vitamin D2 administration, it showed formation of osteoid dentin and degeneration of ameloblast in both buccal and lingual portion at the first week, and enamel hypoplasia caused by edema and loss of polarity of ameloblasts at the second week. 5. By the entire experiment, the overdose vitamin D2 administration and X-ray irradiation effected severely odontoblasts, undifferentiated mesenchymal cells of dental papilla, and primitive cells of tooth germ among the dental tissue. Especially using combination of overdose vitamin D2 administration and X-ray irradiation also effected ameloblasts, resulting in enamel hypoplasia.

• 대한소아치과학회지
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• 제31권4호
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• pp.651-658
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• 2004

Runx2는 runt gene family에 속하는 전사조절 인자로써 뼈의 형성과 골아세포의 분화에 중요한 역할을 담당하고 있다. Runx2-haploinsufficency는 쇄골의 저형성 및 두개 봉합의 지연을 특징으로 하는 쇄골두개 이형성증을 일으키며, 치아에 있어서는 법랑질의 저형성, 영구치 맹출지연 등을 보인다. 이에, 치아의 발육 및 맹출에 미치는 Runx2의 영향을 알아보기 위해 in situ hybridization 방법으로 태생 1, 4, 7, 14, 21일 된 쥐의 하악 및 제1대구치를 사용하여 실험을 실시하였다. Runx2-full length는 태생 1일과 4일에 치낭 및 그 주위조직에 보이지만 Runx2-typeII는 보이지 않았다 Runx2-full length는 태생 7일에 치관 교합면 부위의 법랑모세포에 발현하였고, 1주일 후인 태생 14일에는 백악법랑경계 상방의 치관인접면 법랑모세포에서 발현되었다. 이에 반해 Runx2-typeII는 법랑모세포에서 발현하지 않았다. 또한 태생 21일에서는 두 가지 이성질체가 모두 하악골에서 발현을 보였다 이런 결과를 종합해볼 때, Runx2-full length는 치아의 맹출과 연관이 있으며, 법랑모세포의 분화 및 이로 인한 법랑질형성에 영향을 주지만 Runx2-typeII는 하악골의 형성에만 영향을 미치는 것으로 사료된다.

• 치위생과학회지
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• 제8권2호
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• pp.89-96
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• 2008

태아형성 시기에 투여된 불소가 법랑모세포의 법랑질형성과정에 미치는 효과를 알아보고자 생후 11일 된 흰쥐의 하악 절치를 대상으로 대조군과 두 그룹의 실험군으로 나누어 실험하였다. 전자현미경을 이용한 형태학적 분석결과, 흰쥐태아 치아기의 조직학적 구성은 전분비대, 분비대 및 성숙대로 관찰되었으며 특히 성숙대에서는 법랑질에서 물과 유기물질을 선택적으로 제거하는 평탄끝 법랑모세포(smooth-ended ameloblast)와 무기이온을 추가로 공급하는 주름끝 법랑모세포(ruffle-ended ameloblast)가 관찰되었다. 이러한 조직학적 구성은 흰쥐태아에서도 성체에서 관찰되는 구조들과 동일한 것으로 확인되었다. 한편, 법랑모세포의 전환주기를 알아보기 위한 형광물질(calcein)을 이용한 검사결과, 전환주기가 대조군에 비하여 실험군에서 평균 1회가 감소되었는데 불소농도가 증가할수록 평탄끝 법랑모세포의 두께는 감소하는 양상을 나타내었다. 또한 대조군에 비해 실험군에서 시상총길이에 대한 주름끝 법랑모세포의 두께 비율보다 평탄끝 법랑모세포의 두께 비율이 증가함을 볼 수 있었다. 그리고 치아의 총길이에 있어서 100 ppm 불소투여군은 대조군과 유사하였으나 200 ppm 불소투여군에서는 다소 짧아지는 경향을 나타내었다. 그리고 실험군의 평탄끝 법랑모세포의 두께와 주름끝 법랑모세포의 두께가 절단연으로 갈수록 좁아지는 경향을 띄었고 성숙대의 길이도 절단연으로 갈수록 짧아지는 양상을 나타내었다. 따라서 대조군에 비해 실험군에서 불소투여 농도가 높아짐에 따라 전환주기가 감소되고 이것은 치아의 총길이도 감소하게 되어, 결국 치아성장을 저해함을 확인하였다.

• 한국발생생물학회지:발생과생식
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• 제17권3호
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• pp.215-220
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• 2013

Recently, the RNA/DNA-binding protein FUS, Fused in sarcoma, was shown to play a role in growth, differentiation, and morphogenesis in vertebrates. Because little is known about Fus, we investigated its expression pattern in murine tooth development. In situ hybridization of mouse mandibles at specific developmental stages was performed with a DIG-labeled RNA probe. During early tooth development, Fus was detected in the dental epithelium and dental mesenchyme at 11 days postcoitum (dpc) and 12 dpc. From 14 dpc, Fus was strongly expressed in the dental papilla and the cervical loop of the dental epithelium. At postnatal day 4 (PN4), Fus expression was observed in the odontoblasts, ameloblasts, the proliferation zone of the pulp, and the cervical loop. At PN14, the expression pattern of Fus was found to be maintained in the odontoblasts and the proliferation zone of the pulp. Furthermore, Fus expression was especially strong in the Hertwig's epithelial root sheath (HERS). Therefore, this study suggests that Fus may play a role in the HERS during root development.

• International Journal of Oral Biology
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• 제34권3호
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• pp.143-150
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• 2009

Amino acid transporters play important roles in supplying nutrients to cells. In our current study, we investigated the expression of LAT1 and measured the amino acid uptake in ameloblast cultures to further elucidate the roles of this transporter during the differentiation of these cells. RT-PCR, observations of cell morphology, Alizaline red-S staining, and uptake analyses were performed following the experimental induction of differentiation in the cultures. LAT1 mRNA was detectable and found to gradually increase over time whereas LAT2 mRNA was not evident in the ameloblast cultures. Transcripts of 4F2hc, a cofactor of LAT1 and LAT2, were also found to be expressed in ameloblast cultures and increase with time. Amelogenin mRNA was expressed in the early stage ameloblast cultures. L-leucine uptake was observed to increase over 14 days of growth in culture. Our data suggest that LAT1 has a key role in the differentiation of ameloblasts and in providing these cells with neutral amino acids, including several essential amino acids.