Protein microarray를 이용한 Apin-단백질의 상호작용에 관한 연구

박주철,박선화,김흥중,박종태,윤성호,김지웅,이태연,손호현 대한치과보존학회 2007 Restorative Dentistry & Endodontics Vol.32 No.5

법랑모세포 분화와 법랑질 형성과정에서 OD314, Apin protein의 발현 및 기능

박종태,최용석,김흥중,정문진,오현주,신인철,박주철,손호현 대한치과보존학회 2006 Restorative Dentistry & Endodontics Vol.31 No.6

본 연구에서는 법랑모세포 분화와 법랑질 형성에 연관이 있는 OD314 일명 Apin protein의 기능을 밝힐 목적으로, in-situ hybridization에 의한 OD314 mRNA 발현과 법랑모세포 세포주에서 OD314 enamel matrix protein의 발현, 그리고 OD314 유전자를 과발현/억제시킬 수 있는 construct를 제작한 후 법랑질 형성 중에 OD314의 기능을 알아보고자 RT-PCR를 시행하여 다음과 같은 결과를 얻었다. 1. OD314 mRNA는 발생중인 상아모세포보다 법랑모세포에서 강하게 발현되었다. 2. Tuftelin은 석회화 결정이 형성되는 14일까지 발현이 지속되고, 그 이후부터 점차 감소하였다. Amelogenin과enamelin은 7일부터 그 발현이 점점 감소하였다. 3. U6-OD314 siRNA construct를 이용하여 transfection한 법랑모세포 세포주는 OD314와 tuftelin,MMP2 mRNA 발현이 감소하였으며, CM-OD314를 transfection하여 OD314의 과발현을 유도한 경우에는 OD314와 MMP20 mRNA의 발현이 뚜렷이 증대되었다. 이 결과는 OD314가 법랑모세포의 분화와 법랑질의 형성 그리고 석회화 과정에 중요한 역할을 하는 새로운 인자임을 시사한다. This study was aimed to elucidate the biological function of OD314 (Apin protein), which is related to ameloblast differentiation and amelogenesis. Apin protein, calcifying epithelial odontogenic (pindborg) tumors (CEOTs)-associated amyloid, were isolated from CEOTs, and has similar nucleotide sequences to OD314. We examined expression of the OD314 mRNA using in-situ hybridization during tooth development in mice. Expression of OD314 and several enamel matrix proteins were examined in the cultured ameloblast cell line up to 28 days by reverse transcription-polymerase chain reaction (RT-PCR) amplification. After inactivation and over-expression of the OD314 gene in ameloblast cell lines using U6 vector-driven RNA interference and CMV-OD314 construct, RT-PCR were performed to evaluate the effect of the OD314 during amelogenesis. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were more strongly expressed in ameloblast than odontoblast. 2. When ameloblast cells were cultured in the differentiation and mineralization medium for 28 days, the tuftelin mRNA expression was maintained from the beginning to day 14, and then gradually decreased to day 28. The expressions of amelogenin and enamelin were gradually decreased according to the ameloblast differentiation. 3. Inactivation of OD314 by U6-OD314 siRNA construct down-regulated the expression of OD314, MMP-20, and tuftelin, whereas over-expression of OD314 by CMV-OD314 construct up-regulated the expression of OD314 and MMP-20 without change in tuftelin. These results suggest that OD314 is considered as an ameloblast-enriched gene and may play the important roles in ameloblast differentiation and mineralization.

법랑모세포 분화와 성숙과정에서 OD314의 발현

박주철,안성민,김흥중,정문진,박민주,신인철,손호현 大韓齒科保存學會 2005 Restorative Dentistry & Endodontics Vol.30 No.5

법랑모세포는 법랑질을 형성하고 유지하는 세포로, 법랑질의 유기기질을 분비하고 법랑질 석회화 과정에도 관여한다. 치아 발생과정에서 법랑모세포의 분화는 순차적인 상피-간엽 상호작용에 의하여 조절되나, 분화나 성숙과정의 정확한 기전은 아직까지 잘 알려져 있지 않다. 최근에 상아모세포에서 처음 발견된 OD314가 치아 발생과정에서 상아질을 형성하는 상아모세포 뿐 아니라 법랑모세포에도 발현된다고 하였다. 이에 본 연구에서는 생쥐 하악 전치의 다양한 시기의 법랑모세포를 이용하여, 형태학적 분석과 in-situ hybridization에 의한 OD314 mRNA의 발현 그리고 OD314 항체를 이용한 면역조직화학적 분석을 통하여 OD314 유전자의 법랑 모세포 분화와 성숙과정에서의 역할을 연구하여 다음과 같은 결과를 얻었다. 1. 형태학적으로 법랑모세포는 분화 단계에 따라 분비 전단계 법랑모세포, 분비기 법랑모세포, 성숙기의 평탄끝 법랑모세포와 성숙기의 주름끝 법랑모세포로 구분되었다. 2. OD314 mRNA는 분비기의 법랑모세포에서부터 발현되기 시작하여 법랑모세포가 성숙해갈 수록 그 발현이 증가하였다. 3. OD314 단백질은 분비 전단계의 법랑모세포에서는 발현되지 않고, 분비기의 법랑모세포에서는 세포질에 전체적으로 발현되었다. 성숙기의 평탄끝 법랑모세포와 주름끝 법랑모세포에서는 세포의 근심과 원심끝단에 OD314 단백질이 강하게 발현되었다. 이상의 결과를 종합하여 OD314는 법랑모세포의 분화와 성숙과정에서 세포질 내부에서 특징적인 역할을 하는 것으로 사료된다. Ameloblasts are responsible for the formation and maintenance of enamel which is an epithelially derived protective covering for teeth. Ameloblast differentiation is controlled by sequential epithelial-mesenchymal interactions. However, little is known about the differentiation and maturation mechanisms. OD314 was firstly identifled from odontoblasts by subtraction between odontoblast/pulp cells and osteoblast/dental papilla cells, even though OD314 protein was also expressed in ameloblast during tooth formation. In this study, to better understand the biologcal function of OD314 during amelogenesis, we examined expression of the OD314 mRNA and protein in various stages of ameloblast differentiation using in-situ hybridization and immunohistochemistry. The results were as follows : 1. The ameloblast showed 4 main morphological and functional stages referred to as the presecretory, secretory, smooth-ended, and ruffle-ended. 2. ○D314 mRNA was expressed in secretory ameloblast and increased according to the maturation of the cells. 3. OD314 protein was not expressed in presecretory ameloblast but expressed in secretory ameloblast and maturative ameloblast. OD314 protein was distributed in entire cytoplasm of secretory ameloblast. However, OD314 was localized at the proxiamal and distal portion of the cytoplasm of smooth- ended and ruffle-ended ameloblast. These results suggest that ○D314 may play important roles in the ameloblast differentiation and maturation.

치수제거후 흰쥐 삼차신경절에서 VIP 면역반응세포의 변화: 공초점레이저주사현미경적 연구

김흥중,김승재,박주철,박주철,이상호 대한소아치과학회 2001 大韓小兒齒科學會誌 Vol.28 No.1

말초신경 손상에 의한 VIP의 변화를 연구하기 위해 흰쥐 하악대구치 치수제거 후 삼차신경 절에서 VIP의 분포 및 반응강도를 공초점레이저주사현미경을 이용하여 관찰하였다. 체중 200g내외의 Sprague-Dawley계 흰쥐를 대조군과 하악대구치 치수제거 후 14일군으로 분리하여 희생시켰다. 1차 항체로 rabbit anti-VIP, 2차 항체로 fluorescein isothiocyanate(FITC) conjugated anti-rabbit IgG를 사용하여 면역형광염색을 시행한 후 공초점레이저주사현미경으로 관찰하여 다음과 같은 결론을 얻었다. 1.삼차신경절 하악부위에서 VIP 양성반응세포의 비율은 대조군에서 7.40%를, 실험군에서는 28.42%를 보였다. 대조군에 비해 실험군에서 양성반응세포의 증가를 보였다. 2.삼차신경절 하악부위에서 VIP 면역반응세포체에 대한 상대성 형광강도는 대조군에서 87.78을, 실험군에서는 138.65를 보였다. 대조군과 비교하였을 때 실험군에서 상대성 형광강도의 증가를 보였다. 3.실험군의 광연속절편(1㎛) 관찰에서 VIP 면역반응세포는 9개의 절편 대부분에서 강하게 나타났다. 축삭의 면역반응을 살펴보면, 대조군의 축삭에서는 약한 반응을 보였으며, 실험군의 축삭에서는 강한 면역반응을 보였다. 또한 양성반응 세포체의 크기는 20∼25㎛의 중간 크기의 세포체에서 강한 면역반응을 보였다. 위의 결과로 보아 치수제거 후에 삼차신경절 하악부위에서 VIP면역반응세포의 증가와 함께 상대성 형광강도가 높아졌음을 알 수 있었다. The purpose of this study was to investigate the distribution and fluorescence intensity of vasoactive intestinal polypeptide immunoreactive(VIP-IR) cells in rat trigeminal ganglion following pulp extirpation of rat mandibular molar. The animals were divided into control group(n=6) and experimental group(n=6). The experimental animals were sacrificed at 14 days after pulp extirpation. The trigeminal ganglion was removed and immersed in the 4% paraformaldehyde in 0.1M phosphate buffer. Serial frozen sections about 20㎛ in thickness were cut with a cryostat The immunofluorescence staining was performed. The rabbit anti-VIP(1:8,000) was used as primary antibody and fluorescene isothiocynate(FITC) cojugated anti-rabbit IgG(1:80) as secondary antibody. The slides were observed under confocal laser scanning microscope(CLSM). Unprocessed optical sections were obtained and stored on a optical disk. Color pictures were printed by a video copy processor. The results were as follows ; 1. The positive ratio of VIP-IR cells in mandibular part of trigeminal ganglion were 7.40% in control group and 28.42% in experimental group(14 days affter pulp extirpation). 2. The relative fluorescence intensity of VIP-IR cells in mandibular part of trigeminal ganglion were 87.78 in control group and 138.65 in experimental group. The relative fluorescence intensity of experimental group was 58% higher than that of control group. 3. In optical serial section analysis of VIP-IR cells of experimental group, most of the 9 section showed high fluorescence intensity. At high magnification, axons of the experimental group displayed greater VIP-IR than in the control group, and the positive cells were mainly of medium size. The result indicate that number and fluorescence intensity of VTP-IR cells were increased in the mandibular part of trigeminal ganglion following pulp extirpation of mandibular molar, and it suggests that VIP could play a role in processing of nociception.

분편 인플루엔자백신(split influenza vaccine)의 임상효과 및 면역원성에 관한 연구

우흥정,김동림,정희진,천병철,이주연,안정배,김지희,박찬,신영규,김우주,김민자,박승철 대한화학요법학회 1999 대한화학요법학회지 Vol.17 No.1

목적 : 아단위 인플루엔자 백신 접종 후 백신의 인플루엔자 예방효과, 인플루엔자 방어 항체형성, 인플루엔자 백신의 안전성을 조사하고자 하였다. 방법 : 총 571명을 대상으로 인플루엔자 백신 접종을 하였고, 이들 접종자에서 인플루엔자 양질환의 이환을 조사하여 인플루엔자 백신의 인플루엔자예방효과를 알아보았고, 백신의 접종 전 및 접종 4주 후 혈청에서 혈구응집억제물(Hemagglutination Inhibition : HAI) 항체 검사를 실시하여 백신의 방어항체생성을 조사하였고, 백신의 안전성을 알아보기 위해 백신접종 후 1주일 이내의 부작용을 조사하였다. 결과 :백신 접종군과 백신 비접종군에서 인플루엔자 양 질환의 이환을 조사한 결과 접종군 28.35%, 비접종군 35.88%으로 나왔으며, p 값이 0.001로 통계적으로 접종군에서 유의하게 낮았고, 인플루엔자 양 질환의 예방 효과는 20.97%를 보였다. 백신의 방어항체 형성의 평가를 위해 유럽의 인플루엔자 백신 허가 기준을 조사하였는데 B/Guangdong/5/94균주의 백신접종 후 항체가 40이상의 비율을 제외한 다른 기준은 모두 만족 시켰다. 부작용은 전체 조사자 521명중 149명(29%)으로 주로 접종 부위의 국소 부작용을 호소했고, 전신 부작용은 2% 내외였으며 특별히 심각한 부작용은 발견되지 않았다. 결론 : 분편 인플루엔자 백신은 인플루엔자양질환의 예방과 방어항체생성에 효과 있으며 안전한 것으로 사료된다. Background : The safety and effectiveness of influenza vaccine are well known in developed country. The influenza vaccination has been recommended as one of the tentative immunization schedule for indicated persons since 1997 in Korea. But there are still no available data about them, even though nearly 5 million doses of influenza vaccine were used in 1997-1998 season. So it is immediately needed to investigate the safety. efficacy and immunogenicity of influenza vaccine among Korean. Methods : We studied the clinical efficacy of influenza vaccine by monitoring Occurrence of influenza-like illness in influenza risk group(vaccination ; 300, non-vaccination; 215) from December in 1997 to March in 1998. We used the split quadrivalent influenza vaccine containing 15 microgram of hemagglutinin of A/Beijing/262/95(HlNl), A/Wuhan/359/95(H3N2), B/Mie/1/93 and B/Guangdong/5/94. Hemagglutination inhibition(HA1) antibody titers were determined before immunization and 1 months after vaccination And we evaluated adverse effect of influenza vaccination at 7 days after vaccination. Results : Influenza vaccination was associated with si@icant reductions in influenza-like spptoms(vaccination group; 28.35%, non-vaccination group, 35.88%, p=0.001). The preventive effect of influenza-like i3lne.s among influenza risk goup was 20.97%. And immunogenicity of influenza A and B exceeded all of the European licensure criteria for immunogenicity except postvaccination proportion of titers 240 of B/Guangdong/5/94 strain. And the adverse effects were mainly local injection site problem and no serious adverse effect was noted. Conclusion : Split influenza vaccine is safe, inmunogenic and eff'tive in influenza risk group in Korea.

마멸입자 형태해석에 의한 유압피스톤용 모터의 상태감시

문병주,조연상,박흥식,전태옥 韓國工作機械學會 2000 한국생산제조학회지 Vol.9 No.6

Morphological analysis of wear particles is one of useful methods for machine condition monitoring because it is well reflected in machine driving state. This paper was undertaken to apply to the condition monitoring of hydraulic piston motor. The lubricating wear test was performed under different experimental conditions using the wear test device and wear specimens of the pin on disk type was rubbed in paraffinic base oil by three kinds of lubricating materials, varying applied load, sliding distance. The four shape parameters (50% volumetric diameter, aspect, roundness and reflectivity) arc used for morphological analysis of wear particles. The results showed that the four shape parameters of wear particles depend on a kind of the lubricating materials. It was capable of calculating presumed wear volume for three kinds of mate-rials on driving time to foresee a damage condition of lubricating materials.

정신분열병 환자의 인터류킨-2 생산능

이선미,박주홍,김동인,은헌정,김임,김경용 大韓神經精神醫學會 1996 신경정신의학 Vol.35 No.4

급성기 및 관해상태의 정신분열병 환자와 정상 대조군의 IL-2 생산능을 비교함으로써, 정신분열병의 여러 발병원인 중 자가면역 병인론의 증거인 세포성 면역방응의 저하 및 질병상태와 관련한 세포성 면역기능의 변화를 일부 한국인 정신분열병 환자에서 조사에서 조사하고자 하였다. 방 법 : DSM-IV의 정신분열병 진단기준에 적합한 급성기 환자 19명(남 : 10, 여 : 9)과 관해상태의 환자 21명(남 :12, 여: 9) 및 정상대조군 40명(남 : 20, 여 : 20)을 연구대상으로 하였다. 피험자로부터 채혈한 혈액에서 임파구를 분리하여 phytohemagglutinin으로 T임파구를 자극하고 세포배양하여 증식시킨후 IL-2immunoassay kit을 이용하여 T임파구의 IL-2 생산능을 측정하였다. 결 과 : IL-2 생산능은 성별에 관계없이 정신분열병 환자군에서 정상 대조군에 비하여 유의하게 감소하였다. (t=5. 97, p<0.001). 질병상태에 따라 급성기 정신분열병 환자군 및 관해상태 정신분열병 환자군으로 구분하여 정상대조군의 IL-2 생산능과 비교한 결과, IL-2 생산능이 정상 대조군에서 가장 높았고 다음은 관해상태 환자군이었으며 급성기 환자군에서 가장 낮았다. 세 군 사이에는 모두 유의한 차이가 있었다. (F=35.35, p<0.001). 결 론: 일부 한국인 정신분열병 환자에서 질병상태와 유의하게 관련된 세포성 면역기능의 저하가 관찰되며, 이는 정신분열병의 다양한 원인중 자가면역 기전에 의한 발병을 시사한다. Objects : This study was designed to examine the decreased cellular immune response and the change in cellular immunity related to to clinical status in Korean schizophrenic patients. Methods : The subjects were 19 acute schizophrenics and 21 remitted schizophrenics who met the DSM-IV criteria of schizophrenia and 40 healthy volunteers as the normal control. After the lymphocyte was separated from blood which had been drawn from the subjects and then T lymphocyte was stimulated by PHA and proliferated by cell culture, the examiner measured IL-2 productivity by IL-2 immunoassay kit. Results : IL-2 productivity of the schizophrenics was significantly lower than that of the normal controls. There was no significant difference between male and female in two groups. In the comparison by clinical status, acute schizophrenic group was the lowest, remitted schizophrenic group was next, and normal control group was the highest in the IL-2 productivity, with statistically significant difference among the groups(p<0.001). Conclusion : We suggest that there is a decrease in the cellular immune response which is significantly related to clinical status in some Korean schizophrenics. This finding supports that onset of schizophrenia may be the result of autoimmune mechanism, which is one of the various etiologic factors in schizophrenia.

정신분열병 환자의 인터류킨-2 생산능

김경용,김임,박주흥,은헌정,김동인,이선미 예수병원 1997 예수병원학술지 Vol.18 No.-

SMAGE1/2 단백질의 생산과 anti-MAGE-1 polyclonal 항체의 생성

이정희,김종진,박주홍 昌原大學校 基礎科學硏究所 1999 基礎科學硏究所論文集 Vol.11 No.-

MAGE-1, with was originally identified by reacting with cytolytic T lymphocytes derived from the blood of melanoma patients, is a member of a gene family consisting of twelve structually and expressionally related genes, called MAGE-1 to 12 located in the Xq28 region. The MAGE genes are expressed only in testis among normal tissues and in a variety of tumors. However, what biological functions it has is currently unknown. SMAGE genes were found in a study aiming at detecting mouse genes homologous to human MAGE genes. Because mice are generally easier than humans to work on in studies endeavored for functional analysis of genes, functional study of SMAGE may be very helpful in understanding biological functions of MAGE protein. In the present study we have tried to develop antibodies against SMAGEl/2 protein to better understand biological functions of SMAGEl/2. We cloned the SMAGE-1 cDNA spanning exon Ⅲ amplified by polymerase chain reaction using mouse hepatocyte genomic DNA into an Escherichia coli expression vector and were able to produce Ni-NTA resin-purified recombinant SMAGEl/2 protein of 43 kDa on SDS/PAGE which was subsequently used for immunizing mice. Sera collected from immunized mice were shown to specifically react with SMAGEl/2 antigen by ELISA in combination with Western blotting This polyclonal anti-SMAGE serum will be used for functional studies of MAGE protein.

상아모세포 관련 유전자, OD314의 발현과 기능 연구 : OD314

김두현,김흥중,정문진,손호현,박주철 大韓齒科保存學會 2004 Restorative Dentistry & Endodontics Vol.29 No.4

Odontoblasts are responsible for the formation and maintenance of dentin. They are known to synthesize unique gene products including dentin sialophosphoprotein (DSPP). Another unique genes of the cells remain unclear. OD314 was isolated from the odontoblasts/pulp cells of rats and partially characterized as an odonto-blast-enriched gene (Dey et al., 2001). This study aimed to elucidate the biological function of OD314, relating to odontoblast differentiation and dentinogenesis. After determining the open reading frame (ORF) of OD314 by transient transfection analysis using green fluorescent protein (GFP) expression vector, mRNA in-situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western analysis were performed. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were expressed in odontoblasts of developing coronal and root pulp. 2, OD314 was a novel protein encoding 154 amino acids, and the protein was mainly expressed in cyto-plasm by transient transfection analysis. 3. Mineralized nodules were associated with multilayer cell nodules in the culture of human dental pulp cells and first detected from day 21 using alizarin-red S staining. 4. In RT-PCR analysis, OD314, osteocalcin (OC) and DSPP strongly expressed throughout 28 days of culture. Whereas, osteonectin (ON) mRNA expression stayed low up to day 14, and then gradually decreased from day 21. 5. Western blots showed an approximately 17 kDa band. OD314 protein was expressed from the start of culture and then increased greatly from day 21. In conclusion, OD314 is considered as an odontoblast-enriched gene and may play important roles in odontoblast differentiation and dentin mineralization.