PDLs22 재조합 단백질의 합성과 평가

이경연,최용석,이유진,배현숙,김흥중,조광희,장현선,박주철,Lee, Kyoung Yeon,Choi, Yong-Seok,Lee, You-Jin,Bae, Hyun-Sook,Kim, Heung-Jeong,Cho, Kwang-Hee,Jang, Hyun-Seon,Park, Joo-Cheol 대한치주과학회 2007 Journal of Periodontal & Implant Science Vol.37 No.1

Periodontal ligament (PDL) is the connective tissue located between the tooth root and alveolar bone. In a previous study, PDLs22 was isolated as a PDL-specific gene by using subtractive hybrid-ization between cultured PDL fibroblasts and gingival fibroblasts. It was also suggested that PDLs22 plays important roles in the development, differentiation and maintenance of periodontal tissues. However, little is known about functional study of PDLs22 using recombinant protein in PDL fibroblast differentiation and periodontium formation. In this study, in order to produce the PDLs22 recombinat protein, PDLs22 expression vector were constructed and expressed its protein in various host cell and temperature conditions. The results were as follows: 1. PDLs22 protein was not strongly expressed In the induction system using pRSET-PDLs22 construct. 2. When the BL21(DE3) pLysS was used as a expression host, PDLS22 protein was strongly ex-pressed in the induction system using pHCEIIBNd-PDLs22 construct. 3. The PDLs22 protein was recognized at a molecular weight of 28 kDa in western blots. 4. Almost of the expressed PDLs22 protein was not soluble and observed like as inclusion body. 5. The protein solubility was not improved after modification of induction time and temperature during PDLs22 protein production. In this study, the system for the PDLs22 protein production was connstructed. However, the re-results suggest that further studies will be needed to produce the considerable amount of PDLs22 re-combinat protein, which can use for the periodontal regeneration.

치주인대, 이틀뼈 및 시멘트질의 발생 과정에서 치주인대

지숙(Suk Ji),김병옥(Byung-Ock Kim),김홍중(Heung-Joong Kim),김성미9Sung-Mi Kim),박주철(Joo-Cheol Park) 대한해부학회 2003 Anatomy & Cell Biology Vol.36 No.2

치주인대 (periodontal ligament)는 치아를 지지할 뿐만 아니라 인접 이틀뼈 및 시멘트질의 수복과 재생에 관여하는 것으 로 알려져 있으나, 치주인대 세포의 발생과 분화에 선택적으로 관여하는 유전자에 대한 연구는 미미한 실정이다. 치주인 대 세포의 분화와 뼈와 시멘트질의 형성과정에서 치주인대-특이 유전자인 PDLs22의 명확한 기능을 알아보기 위하여 배 양 치주인대 세포의 분화 과정에서 PDLs22, osteonectin과 osteocalcin mRNA 발현을 RT-PCR법으로 비교하였고, PDLs22에 대한 항체를 제작한 후 흰쥐 치아와 치주조직 발생과정에서 PDLs22 단백질의 분포를 면역조직화학적으로 확인하였다. 치 주인대 세포들은 배양 14일째에 석회화 결절을 형성하였고, 세포의 배양과정에서 osteocalcin mRNA는 배양 초기부터 발 현되어 배양 기간 동안에 강하게 발현되었으며, osteonectin PDLs22 mRNA는 배양 초기부터 발현되었으나 석회화 결절이 형성되면서 발현이 감소되었다. 면역조직화학적 염색소견에서 PDLs22 단백질은 치아관 형성시기의 바깥치아상피와 별그 물에서 발현되기 시작하여 HERS (Hertwig’s epithelial root sheath)의 바깥치아상피로 이어진 다음 치주인대, 이틀뼈 및 시멘 트질을 형성하기 위한 전구세포에서 강하게 발현되었다. 이상의 결과를 종합하면 치주인대-특이 유전자 PDLs22는 치주조 직 발생에 있어 상피-간엽간의 중요한 매개물질로 작용하며, 뼈와 시멘트질의 초기 형성과정에 중요한 역할을 하는 것으 로 생각된다. Identifying specific factors and/or mechanism regulating development of periodontal tissue will provide important information as to which molecules and cells are required for regulation of periodontal tissue lost as a consequence of disease. The origin and location of cementoblast and osteoblast precursor cells in adult periodontal tissues is not definitely known but it has been suggested that tooth related periodontal ligament may be the source of cementoblasts and the bone-related periodontal ligament for osteoblasts. However, little is known of the molecular mechanism controlling PDL function. PDL-specific protein; PDLs22 had been previously identified as a novel protein isolated from cultured human PDL fibroblasts using subtraction hybridization between human gingival fibroblasts and PDL fibroblasts. The aim of this study was to examine the functional characterization of PDLs22 in differentiation of periodontal ligament, alveolar bone and cementum. Human osteocalcin (OC), osteonectin (ON) and PDLs22 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) in primary cell cultures of periodontal ligament fibroblast during mineral nodule formation in vitro. And the localization of PDLs22 in rat tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows: 1. PDL cells were capable of producing mineral-like nodules in vitro. 2. PDLs22 mRNA was expressed in the initial stages whereas it was not expressed in the calcification stage, during mineral nodule formation of PDL cells in vitro. 3. PDLs22 protein was expressed in external dental epithelium and stellate reticulum during crown formation stage, and was continued in external dental epithelium of Hertwig’s epithelial sheath. Also PDLs22 protein was strongly expressed in the bone and cementum-related side of the PDL and weakly expressed in the middle of PDL. In the developing bone, PDLs22 protein is only expressed in preosteoblast not osteocyte and osteoblast. The results suggest PDLs22 is important mediator of epithelial-mesenchymal reaction in development of PDL, alveolar bone and cementum and is related to initial differentiation of cementum and alveolar bone.

치주조직의 발생과 분화과정에서 치주인대-특이 유전자 PDLs22의 발현

김흥중,정문진,김병옥,국중기,김종관,박주철 대한구강악안면병리학회 2005 대한구강악안면병리학회지 Vol.29 No.1

Periodontalligament (PDL) fibroblasts have an ectomesenchymal origin and are known to participate not only in formation of PDL but also in the repair and regeneration of the a이acent alveolar bone and cementum. However, little is known about the molecular mechanism which is related to the development and differentiation of PDL cells. Recendy, we reported the PDLs (a periodontalligament-specific) 22 as a PDL fibroblast-specific mRNA which is not expressed in gingival fibroblasts. In this study, to examine the expression and functional characterization of PDμ22 mRNA and prαein in development and differentiation of periodontal 따sue , we carried out northem analysis, insitu hybridization, immunofluorescence and immunohistochemistry. The expression of PDLs22 mRNA was increased with PDL cell differentiation from the confluent to multilayer stage but decreased slighdy with mineralized nodule formation in vitro. πle PDLs22 protein was localized on the nuclear membrane and expressed throughout the differentiation of PDL fibroblasts in vitro. The PDLs22 mRNA and protein were expressed in the differentiating cementoblasts, PDL fibroblasts and osteoblasts along the r∞t surface and alveolar bone of the developing rat teeth. These results indicate that the PDLs22 plays an irnportant role in the differentiation of cementoblasts and osteoblasts and thus homeostasis of cementum, PDL and alveolar bone.

흰쥐의 치아 맹출과 치간 이개 과정에서 수종의 치주인대 단백질 발현의 변화에관한 면역 조직화학적 연구

임성훈(Sung-Hoon Lim),박형수(Hyung-Soo Park),윤영주(Young-Jooh Yoon),김광원(Kwang-Won Kim),김흥중(Heung-Joong Kim),정문진(Moon-Jin Jeong),박주철(Joo-Cheol Park) 대한치과교정학회 2004 대한치과교정학회지 Vol.34 No.1

치아의 맹출 과정과 치간이개로 유도된 치아 및 치조골의 흡수 과정에서 치주인대 세포와 치주인대 단백질의 기능을 알아보기 위하여, 발육 중인 흰쥐를 치근 형성 전, 치근 형성 시작과 치근 형성 및 맹출 시기로 구분하여 표본을 제작하고, 또한 성장 중인 흰쥐를 2주간 치간 이개시켜 조직표본을 제작하였다. 치주인대 섬유모세포에서 특이적으로 발현되며 치주인대의 분화와 성숙에 관여하는 PDLs22 단백질과 치아와 치조골의 파괴와 흡수를 조절하는 것으로 알려진 RANKL과 OPG의 발현을 면역 조직화학적으로 연구하였다. PDLs22 단백질은 치근 형성이 시작되면서부터 치낭세포와 골모세포에서 발현되어, 치아가 맹출하는 과정에서도 그 발현이 계속 유지되었으나, 치간이개에 의하여 치주인대가 개조되는 부위에서는 발현이 감소하였다. RANKL은 치근 형성 과정에서는 미약한 발현을 나타내었으나, 치아가 맹출하면서 발현이 증대되었으며, 치간이개에 의한 치근과 치조골 흡수과정에서는 치주인대세포, 골모세포, 치수세포 및 파치세포에서 발현이 증대 되었다. OPG는 치근이 형성되는 시기에는 강한 발현을 보였으나, 치아가 맹출하면서 발현이 현저히 감소하였ㄱ, 치아와 치조골의 흡수가 진행됨에 따라서 발현이 다소 감소하였다. In this study, we attempt to investigate the mechanisms by which PDL cells regulate osteoclast formation and also tc know whether PDL retained their characteristic phenotype during tooth eruption and interdental separation. Rats were prepared at developmental days 21 (pre-root formation), 27 (root development), 34 (advanced root formation/ eruption) and at later times(adult rats). To induce severe resorption state of alveolar bone and tooth root, interdental separation with brass wire was performed between the lower first and second molars for 2 weeks in adult rats. Rat mandibles were dernineralized and embedded in paraffin, and horizontal and frontal section were prepared for immunohistochenucal analysis using PDL-specific protein 22 (PDLs22), receptor activator of NFKB ligand (RANKL) and ostei protegerin (OPG) antibodies. 1. Root formation and eruption stage of tooth development. 1) PDLs22 immunolocalization was observed in tooth folliclelPDL cells and osteoblasts throught out the root formation and eruption stages of tooth development 2) RANKL expression became stronger at eruption stage than root formation stage of tooth development. 3) Strong expression of OPG was detected in follice/PDL cells of root formation stage but it was decreased with tooth eruption. 2. Interdental separation between lower first and second molar. 1) Con parared to normal animal, multinucleated osteoclasts and odontoclasts were markedly induced in the alveolar bone and tooth root with PDL remodeling in hematoxylin-eosin section. 2) PDLs22 expression was decreased with interdental separation. 3) RANKL expression was increased with interdental separation in PDL fibroblasts, osteoblasts, odontoclasts and it lacunae, resorbing dentin, cementum and bone matrix. 4) OPG expression was slightly decreased in the PDL cells adjacent to the alveolar bone and root surface with interdental separation. These results suggested that during tooth eruption and tooth movement, RANKL and OPG in the periodontal tissues are important determinants regulating balanced alveolar bone and tooth root resorption. And it is also suggested that PDL cells retained their characteristic phenotype during tooth eruption and interdental separation except for the short period of PDL remodeling.

Enamel Matrix Derivatives가 사람 치주인대 세포의 특이유전자인 PDLs17, PDLs22의 발현에 끼치는 효과

한근아,장현선,국중기,박주철,김흥중,김종관,김병옥,Han, Geun-A,Jang, Hyun-seon,Kok, Jung-Ki,Park, Ju-Chol,Kim, Heoung-Jung,Kim, Jung-Gwan,Kim, Byung-Ock 대한치주과학회 2004 Journal of Periodontal & Implant Science Vol.34 No.2

The enamel matrix derivative (EMD) has been recently used in the periodontal regenerative techniques. The present study was established to investigate the influence of EMD on human periodontal ligament cells using expression of mRNA of periodontal ligament specific gene (PDLs)17, PDLs22, type I collagen when EMD applied to periodontal ligament cells. Periodontal ligament cells were obtained from a healthy periodontium and cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum and ${\beta}-glycerophosphate$ with ascorbic acid. Test groups were two; One adds EMD in culture media and another added EMD and Dexamethasone (DEX) in culture media. Positive control group added DEX in culture media, and negative control group adds niether of EMD nor DEX. $Emdogain^{(R)}$ (Biora, Sweden, 30 mg/ml) was diluted by 75 ${\mu}g/ml$ concentration to culture media. For reverse transcription-polymerase chain reaction (RT-PCR), total RNA isolated on days 0, 7, 14 and 21. mRNA of PDLs17 was expressed on days 14 and 21 in EMD or DEX group, and expressed on days 7, 14 and 21 in EMD plus DEX group, the other side, expressed on days 21 in negative control group. mRNA of PDLs22 expressed on days 7, 14 and 21 in EMD group, and expressed on days 14 and 21 in DEX group, and expressed on days 7, 14 and 21 in EMD plus DEX group. Negative control group expressed on days 14 and 21. Type I collagen was expressed on all days and all groups. These results indicate that EMD promotes differentiation of periodontal ligament cells, and this is considered to offer basis that can apply EMD to periodontal tissue regeneration technique.