The transcription factor nuclear factor-kB (NF-kB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-KB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Numerous evidences showed that over expression of TGase 2 might be linked with constitutive activation of NF-KB. To understand the pathways responsible for constitutive activation of NF-kB is important for rational design of NF-kB inhibitors for cancer therapy. Human salivary gland adenocarcinoma has been most aggressive solid tumors. Current therapy does not significantly improve survival rates. Thus, to investigate new therapeutic modalities against this adenocarcinoma is necessary. The purpose of this study was to study a constitutive activation of NF-kB with the expression of TG2 in SGT cell line origianted from human salivary gland adenocarcinoma by TGase 2 activity, RT-PCR and immunoslot blotting method. SGT cell line showed the highest TGase 2 activity and NF-kB activation than any other cell line. All the cell lines showed increased NF-kB mRNA activation after A231027 treatment than that of control. In immunoslot blotting, SGT cell line showed NF-kB activation correlated with TGase 2 expression after A231027 and BSA. It suggested that there might be a direct correlation between TGase 2 expression and NF-kB activation in SGT cell line.

Numerous bone cell culture models have been presented by the development of isolation and culturing techniques of cells. The culture of osteoblast-like cells of human origin with a specific osteoblastic phenotype has become an important experimental model in bone biology. Recently, it has become increasingly popular to utilize bone marrow cultures because these cultures are therefore thought to represent earlier stages of the osteoblast differentiation pathway. There is no report about culturing normal human osteoblast from oral and maxillofacial area. Primary cultured cells from oral and maxillofacial cancellous bone were analyzed by morphologic features, total DNA contents, ALP, osteocalcin and von Kossa staining positivity. The purpose of this study were to culture the cell population from oral and maxillofacial cancellous bone and to analyze the phenotypic expression of cultured normal human osteoblast by the bone marrow isolation technique. Growth curve of NHost showed about 45hrs of doubling time and about 70μ g/well of total DNA content. NHost showed spindle shaped cytoplasm with ovoid nucleus under preconfluency and after cellular differentiation, they formed irregular numerous nodules from stratified cellular layers under D medium. ALP activity was about 2 folds higher under control medium with 10nM 1,25(OH)2D3 than that under control medium. Osteocalcin expression was about seven folds higher under control medium with 100nM 1,25(OH)2D3 than that under control medium. Scattered mineralized nodules stained by von Kossa method were seen on the cellular layer under D medium. It suggested that NHost might be established from oral and maxillofacial area by characteristic cellular shape, ALP activity, osteocalcin expression and numerous mineralized nodules.

was first described by Pindborg in 1955. they occur as intraosseous(94%) and extraosseous variants. Although the prognosis of CEOT was regarded as ameloblastoma in the past, contemporary accumulating data suggest that CEOT have better prognosis than ameloblastoma. But decisive evidences are lacked. Although CEOT is a rare odontogenic tumor, the histopathologic features have so much diversity. Especially interesting aspects are the being of amyloid and Langerhans' cells. Author classify 6 cases of CEOT to scanty, small, and lage as produced amount of amyloid and then perform immunohistochemical study about pancytokertin, cytokeratin8/18, vimentin, CD1a, and VEGF(vascular endothelial growth factor) for verifying the differentiation state of tumor cells and the comparative infiltrative potential with ameloblastoma. Author obtain several conclusion and presumptive facts through this study and previous researchs. Tumor cells of CEOT exhibited different differentiating features as amount of amyloid, presumably tumor cells of CEOT with scanty amount of amyloid represent enamel epithelium-like cells of presecretory stage in amelogenesis, tumor cells of CEOT with small amount of amyloid represent ameloblast-like cells of secretory stage in amelogenesis, and tumor cells of CEOT with large amount of amyloid represent reduced enamel epithelium-like cells after enamel formation. Epithelial-Mesenchymal transition phenomenon developed in tumor cells of CEOT with small amount of amyloid. Inflammatory reaction was not related with being Langerhansʼ cells. Author tentatively concluded that CEOT with Langerhans cells exhibited a tendency of non-calcification, scanty amyloid formation and frequently occurring at the maxillary anterior region through the previous studies and this study. Infiltrative growth potential of CEOT was lower than ameloblastoma regarding only VEGF expression.

It is not yet clear to know how normal human osteoblasts(NHost) from oral and maxillofacial area deposit, stabilize, and configure their extracellular matrix on dental biomaterial surfaces. Therefore it is necessary to design biomaterials with improved biocompatibility that will promote earlier bone differentiation and mineralization. There is now increasing evidence that TGase 2 may play a role in the initiation and regulation of the mineralization processes and serves to cross-link osteocalcin and osteopontin, which are predominant substrates for TGase 2 expressed during bone mineralization. But it is still unclear as to which TGase 2 is expressed by NHost in vitro bone formation. The purpose of this s tudy was t o determine the level of TGase 2 expression associated with collagen I , osteopontin and osteocalcin in NHost cell lines from oral and maxillofacial area in vitro. We will investigate whether TGase 2 has an active role in the biocompatibility of dental biomaterials during differentiation and mineralization of NHost. NHost cell lines were cultured under DMEM with 10% FBS at 37ﾟC and 5% CO2. Collagen quantification, mineralization and calcium assay, ALP activity assay, and RT-PCR analysis during bone differentiation and mineralization were done. ALP levels showed higher activity under AA+hGP t han under AA. I nhibition o f T Gase 2 by cystamine showed d ecreased Ca++ concentration, c ollagen I deposition and ALP level during 11 days of differentiation. TGase 2 mRNA expression of NHost was constant during mineralization stage. TGase 2 enzyme activity was increased during differentiation. Osteopontin mRNA expression during mineralization was higher than that of osteocalcin and collagen I . It suggested that TGase 2 associated with collagen I, osteocalcin and osteonectin might play a role in the differentiation of NHost from oral and maxillofacial area, but a little involved in mineralization.

Demineralized Freeze Dried Bone(DFDB) graft material have been used for reconstruction of large bony defects or augmentation of thin alveolar ridge during implantation of titanium fixtures. But at present osteogenic effect of DFDB do not overcome the capacity of autogenic bone graft. Thus many investigators had applicated various bioactive substance to DFDB to enhance the ability of osteogenesis of DFDB. In this study, mixture of grafting material was made from fibrin glue(F) and DFDB(D)(group 1: F+D), fibrin glue, DFDB and rhBMP-2(B) (group 2: F+D+B), fibrin glue, DFDB, polylactic- co-glycolic acid(PLGA)(P) and rhBMP-2(goup 3: F+D+B+P), fibrin glue, DFDB, PLGA, rhBMP-2 and autogenic osteoblasts( O)(group 4: F+D+B+P+O), and fibrin glue, DFDB, autogenic osteoblasts (group 5: F+D+O). During first surgical procedure, extraction of molar teeth was performed at male Biggle dog's mandible, and collected bone marrow tissue from tibia at same Biggle dog. Collected bone marrow tissue was cultured and differentiated into osteoblasts in vitro, and stored in nitrogen bottle. After four months, titanium fixture was implanted with prepared graft material to Biggle dog's mandible. After four and eight weeks respectively, experimental dog was sacrificed. Obtained tissues were prepared for examination by using resin embedded ground section method. Prepared sections were evaluated with transmitted and polarized microscope, and areas of osteoid and cacified bone were calculated with IPTK 5.0( image processing tool kit version 5.0). Resultant data was statistically analyzed by SPSS 13.0 software. Results of this study showed that autogenic osteoblats had more enhancing capacity of bone formation than rhBMP-2, but PLGA inhibited bone forming potential of bony tissue.

The prominent side effect of cyclosporin A, immunosuppressive agent, in oral tissues is gingival overgrowth(GO). It is characterized by the gingival enlargement with epithelial thickening, a large number of proliferating fibroblasts and overproduction of ECM components. Fibroblast accumulation in Cs A induced GO is caused by the Inhibition of apoptosis. But CsA effect on normal human oral keratinocyte(NHOK) remains unclear. Transglutaminase 2(TGase 2) which is expressed and activated in tissue where epithelial cells undergo apoptosis has been used as a marker for apoptotic cells. The purpose of this study were to study the effect of CsA on the proliferation and apoptosis of cultured NHOK by TGase 2 expression. After primary cultured NHOK was treated by 0.1, 1.0 and 10ug/ml Cs A, growth curve, MTT assay for succinyl dehydrogenase activity and RT-PCR for TGase 2 mRNA expression were done. The obtained results were as follows. MTT assay showed about 65% cell viability at 1.0μg/ml and 40% at 10μg/ml CsA. Growth curve showed normal S curve on control & DMSO, while decreased growth rate after 3 days of higher CsA tx. TGase 2 mRNA expression of cultured NHOK was the highest at 10μg/ml Cs A. TEM showed chromosome margination, and vacuole formation and clustered mitochodria in cytoplasm of cultured NHOK after CsA tx. It suggested that higher CsA might induce apoptosis of NHOK correlated with increased TGase 2 mRNA expression

AJthough salivary gland adenocarcinoma accounts for third prevalence rate of all salivary gland tumors. it is one of the most aggressive solid tumors. Current therapy does not s ignificantly improve survival rates. Thus‘ investigating new therape utic modali t ies aga inst sali va ry gland adenocarcinoma is necessary. Manumycin A. a natural product o{ Streptα7Jyces parvuJus‘ inhi bits farn esy l- transferase by competition with farnesyl pyrophosphate groups . Manumycin has shown antitumor activity in several ex per‘ imental systems through identifying the regulatory pathway of apoptosis. The hi erarchical relationship of caspase-8 to caspase-3 and caspase-9 in the drug-induced a poptosis pathway in antitumol effect is not clear. The hi erarchical relations hip between cytochrome c and the caspases and provided evidence to support the hypothesis that the release of cytochrome c was upstream of caspase activation in the enhanced apoptosis induced by manumycin A Manumycin A has not been examined extensively in human salivary gland tumor and has not yet been clarified. The purpose of this study were to investigate mRNA and protein expression of Bc l- 2 、Bax, Cytochrome C‘ caspase- 3 , 一8 and -9 in SGT cell line by RT-PCR and immunoslot blotting, and to a pply its results to exami ne iLs chemoprevention for salivary gland adenocarcinoma. MTI assay showed about 50% cellular viability of SGT cell line treated by 50μM manunycin A Bcl-2. Bax‘ and caspase-8 mRNA expression in SGT cell line were unchangeable after 50μM manu nycin A Cytochrome C‘ caspase-3 and -9 showed about 1.5-5 folds higher mRNA expression in SGT cell line than that of control a nd DMSO- t reated group a fter 50M manunycin A. Bcl-2, Bax, and caspase-8 protein expression in SGT ce ll line were unchangcable after 50μM manunycin A. Cy Lochrorne C, caspase-3 and -9 showed about 2-7 fo lds higher protein express ion in SGT cell line than that of control and DMSO-treated group after 50μM manunycin A. mRNA expression was assoc iated with protein expression in SGT cell line after 50μM manunycin A. It suggested that manumycin A would induce poptotic effect on SGT cell line by caspase-3 and - 9 activation through cytochrorne c release. And man umycin A will be a useful chemoprevention drug for human salivary gland carcinoma in future.

Nowdays, implant treatment became a branch of universal dental treatment and results in mOI‘ e success by surface con dit ioning and continuous improvements. Recently, a method to extract crystal shows sirnilar Ca/P ratio as HA has introduced which is anodizing electrolyte pure titanium anodize by electrolyte ß -glycerophosphate disodium salt hydrate and calcium acetic acid on pure t itanium before hydrothermal treatment ln t his study fixu res have divided in 3 group: Machined(Group 1), Anodized(Group 1I), implant whi ch has a surface containing Ca and P formed by anodization and hyd rothermal treatment( Group m). Total 18 fixtures were impla nted on rabbit which sacrifi ced on week 2 and week 4 for the histological specimens. By t hese specimens polarized microscopic view‘ ll1icro CT view‘ EPMA, ISQ value were ll1easured, cOll1pa red and a nalysed by each group to f igure out the possible clinical use of titanium implant containing Ca and P by hydrothe rll1al treatment and anodizing electrolyte. ISQ value had no s ignifïcance differences between each 3 groups, However in each group 4 , 8 weeks had hi gher ISQ value than 2 weeks 1n polarized rni croscope, calcification level was following Group 1I ‘ Group m, Group 1. 1n rnicro CT‘ formation of cancellous bone level was following ‘ Group 1I , Group m, Group 1. 1n EPMA, distribution and concentration of Ca was following : Group 1I was two t ill1es more higher than Group 1 , Group m. Group m were higher level t han Group 1. In distribution and concentration of P Group 1I was high er than the other group. but th e re were no statical s ignifi cances. Finally, anodi zed implant was the most exellent on the early bone for ll1a tion Containing Ca and P implant by anodizing and hydrotherma l treatment was more better than machined surface implant, but there is no effi cience at ear ly bone formati on

The characteristic of implant s llrface is the most i mportant factor in osseointegra t ion procesR , Prol i fera tion and rli ffel' entiat ion of osteoblast, a nd local factors concerning bone forma tion a l'e inflllenced by sUl' face cha racteri s tic and it al so con t l'ols bone reactlOn The purpose of this reseal'ch was to s tlldy a boll t ini t ial adhesion. prolife ration and acti vation of osteo blast to titanium surface treated wi th mac hined‘ hydroxyapat ite coating, resorba ble blast materi al blasting and a n odi zing method To attach and culture osteobl ast‘ titanium cylinder block wi th 5mm in di ameter and 5mm in height was mad e , After t l'eating the titanium surface of each bl ock with machined, hydroxyapatite coating, I'esorhable blast ma teri al blasting and anodi zed coating, i.mpllrities wel'e I'emoved and stenllzed The number of cells attached from cultul'ed osteobl ast of I'espective expel’imental groups were measured at 1, 4, 7. and 14day, Al ka line phosphatase, calcium. and inorganic phosphate concen tra ti on 0 1' cultured solution was measul'ed, Anodi zing group showed the hi ghest. and RBM treated grollp was foll owed, Machined grollp was the worst ra te of cell a ttachment and pl 미 ife ra ti o n activity, RBM t l'eated grollp s howed the high est in creasi ng on theil‘ alkaline phosphatase activi ty of 1 and " days in cultllred osteobJast to compnrc wi th othcr groups. Thc rc was no significant differe nce among other grollps , stati stically, RBM trea ted grollp showed the hi ghest rate 0 1' increa s ing on the ca lcillm apposition of 1 a nd 4 days in cllltured osteobl ast to compa re wi th other groups , The re was no s ignificant differ ence among other groups ‘ statistica lly, RBM t reated g roup showed the highest rate of inorganic phospha te apposi t ion to compare with other groups , The re was no s ignifi cant diffe rence among other groups‘ s tati stically, It suggested tha t surface modi fï cation of titani um would be profoundly effected on the attachment. proliferation and activation 0 1' osteoblast in init ial stage osseolll tegrat lOn

This r esearch was designed to find the specific and economic methods of diagnosis about malignant melanoma For this study, we selected a typical case that was ambiguous in diagnosis between malignant melanoma and simple mela notic pigmentation, Tissue sections was st ained by H&E method, and immunohistochemical analyses was performed about 8-100 protein and MART-1 molecule, This research showed that MART-l had a more specificity for melanocytes than 8-100 protein , Patterns of MART-1 molecule distribution was more helpful in estimation of malignancies than 8-100 protein distribution patterns, On the basis of diagnostic usefulness and economical aspects, 8-100 protein and MART-1 molecule showed synergis tic and complementary relationship in confirming of tumor origin and they would be much useful for accurate diagnosis of malignant melanoma

The purposes of this study were to estimate Brdu positive and apoptotic cells dis tribution in co ndyl ar art icular and proliferative layer. and hi stopathological evaluat ion during one sided mas t icatory condition. 15 of 30 adults Sprague Dawley male rats were experimental group, and 15 rats were control group. Experime nta l group were gen t ly extracted on all lower and upper molar teeth to make unilateral mastication. Experimental gl'Oup were divided into two group as extracted side and nonextracted side condyle. Diluted bromodeoxyuridi ne(Brdu) solution(5mg/Kg) was injected in peri toneum before sacrifice at inte rval 1 week, 2 week, 4 week Streptoavidi n-Biotin method for Brdu was used to evaluate cellular proliferat ive activity, and fluorescent TUNEL method was used to estimate the a poptotic activity. The resul ts of this experiment were recorded about anterior and posteri or condyle sepa rately On anterior condyle, contl'Ol group was sustained increased proliferative activity th roughout experiment. whi le cel lu lar proliferative activity of extracted and nonextracted s ide condyle showed more decreased than control grou p ‘ On posteri or portion of condyle‘ control group and nonextracted group showed dramatically decreased cellular pr。 liferat ive activity during all expel‘ imental period. On anterior portion of condyle, control grollp s howed decreased a poptotic activity with time passed. bllt expel'imental gl'oup(both ext l'acted and nonextracted) exhibi ted incrcased a poptotic activity. Es pecia lly extracted grollp showed prominent increased apoptotic activity. On posteri or portion of condyle. extracted group showed dec reased apoptotic activity wi th time progress. but co ntrol and nonextracted group exhibi ted increased apoptotic activity with time progress. Conclllsively. antel'ior portion of condyle on ex t racted s ide expressed hypoplasia by low cellllJar prol iferative activity and increased apoptotic activity. and poste ri or portion on extracted side showed condylar surface hyperplasia by continious proliferative cell ular activity and low a poptotic activity. a nd t hllS unmasticatol'y condyle had anLeroposLel'iorly shorter mo rph이 ogy and verti ca11y longer morphology than masticatory and nOl'mal functioning condyle

The aims 0 1' t his study we re to evaluate proliferative and apoptotic cell distribution pattern in condylar articu lar and pl'oliferative layer as two types of diet(soft and ha rd) we1'e supplied to growing 1'a ts, 30 male Sprague Dawley g1'owing l'ats(twenty-one c1ays fl'om bi l'th) were c1ivided into two group, Each 15 rats were supplied by soft and hal'd di et , After c1 iluted Bl'du solution(5mg/ Kg)was injected into pel'itoneum befol'c sacrificc. rats wel'e sacl'i f iced at 1. 2. 3 week llltel'vals St1'eptoavidin-Biotin method for Brdu was used to evaluate celluJar proliferative activity. ancl fluol'escent TUNEL method was used to estima te the apoptotic activity, The results about this expe riment wel'e recorded about ante1'io1' and posterior condyle separateJy, In anteriol' portion of condyle, soft diet group showed inc1'eased p1'olife1'ative celluJar activity tha n hal'd diet group dw'ing 1, 2 week but decl'eased than hal'd diet g1'oup at 3 weeks. while hal'd diet group showed constant p1'oliferative cellular activity during all experimenta l period , ln posterior pOl'tion of condyl e、soft diet group showed increased activity than hard diet group at 3 weeks, while ha rd di et gl'oup showed constant proliferative cellular activity during exper‘ imental period , In a nterior pOl'tion of condyle‘ soft c1 iet group showed increased apoptotic activity with time progress, but hard diet group showed continuous low level of activity during experimental period , In posterio1' porti on 0 1' condyle, hard diet group showed cons tant low level apoptotic activity, plthough showed the lowes t leveJ at 1 week, On the con trary soft diet g1'oup showed c1ecl'eased apoptotic activity wi th time progress, ConcJusively, in soft diet group an teroposterior direction was reduced in condyJar morphologic dimension because of decreased prol iferative cellular and increased apoptotic activity on anterior portion and vertical dimension 0 1' condyle was increased because of increased proliferative cellular and decreased apoptotic activity on posterior portion. but in hard di et group proli ferative cellular and apoptotic activity were comparatively constant. and thus harcl diet group s howed antet'opos teriorJy broad and flat condylar morphoJogy

Nowaday many researches has proved that glutaradehyde(GA) is more excell ent medicament in vital p띠 potomy practice than formocresol (FC) . But a number of dental practitioner prefer to use formocresol in vital pulpotomy procedure todays And thus author reeva luate proper ties of gluteraldehyde and formocresol through implantation into epidermis and trypsin digestion after f ixation at 2% buffered glutar aldehyde and Burkley's formocresol solution.. And then prepared t issues were s tained by H&E a nd Masson-TI’ ichome method. GA showed definite fixative zone and γery low diffusi ble property in epi dermis and pulp t issue as glutaraldehyde and formocresol were compared. GA r epresented exceedingly lower antigenici ty than FC GA fixation exhibited more resistibility to trypsin digestion than FC. As considering these results‘ it concluded that GA would be extremely supe ri 이 . medicament to formocresol in vital pulpotomy proce

Extensive oral mucosa loss from a variety of conditions is associated with significant functional morbidity and mortality. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-β1(TGF-β1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms expressed different levels of TGF-β1. When NHOK reached confluency in serum free medium(KBM), in test medium containing 1.2 mM Ca++ KBM NHOK were allowed to form multi-layers and differentiate. The purpose of this study were to investigate the mRNA level of TGF-β1, FGF-2, and TIMP-1 by RT-PCR analysis and also to evaluate the expression of TGF-β1 and involucrin in keratinocytes at different times of the onset of differentiation. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. Cultured NHOK in preconfluency under KBM medium expressed a significantly higher level of TGF-β1 relative to those grown in multi-layer forms, while the level of TGF-β1 mRNA gradually reduced to its lowest level at 7 days of growing cells in test medium. Cultured NHOK in preconfluency of KBM medium expressed a lower level of FGF-2 and TIMP-1 relative to those grown in multi-layer forms, while the level of FGF-2 and TIMP-1 mRNA showed the highest level at 3 days at gradually reduced to its lowest level at 7 days of growing cells in test medium. As a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. It suggested that the expression of TGF-β1 mRNA be consistent with the expression of FGF-2 and TIMP-1 mRNA in NHOK grown in high calcium medium during the terminal differentiation. But differentiated NHOK expressing higher involucrin mRNA could show constant espression of TGF-β1, FGF-2 and TIMP-1.

Adenocarcinoma NOS of salivary glands is characterized by a high rate of local recurrences and metastasis. Long-term survival rate of Adenocarcinoma NOS lis not promising. Thus, different chemotherapeutical approaches had been proposed for this neoplasm, including apoptosis induction by drugs. The current treatment of choice of adenocarcinoma NOS is controversible, and an effective treatment for them is not yet available. Chemotherpeutic agents that can be inhibit or reverse the tumor growth by targeting apoptotic pathways will be new candidates for cancer prevention and therapy. The purpose of this study were to study the effect of Brefeldin A(BFA) as apoptotic inducing agent in SGT cell line from human submandibular adenocarcinoma NOS and apply these results to make a plan of treatment and prognosis of salivary gland tumors involving adenocarcinoma NOS. SGT cells were treated with a 300μM BFA solution in serum-free medium during 18 hours. SGT cells were grown in DMEM with 10% fetal bovine serum served as controls. The growth curve and MTT assay for succinyl dehydrogenase activity were performed. For apoptotic analysis, fragmentation of genomic DNA was confirmed with gel electrophoresis. Transmission electron microscopy was assessed for the effect of BFA on SGT cells phenotype. Apoptotic cell recognition and counting were carried out with Annexin-V, caspase 3 and APo2.7 antibody through flow cytometry. Growth of SGT cell line was abrutply decreased after 1 day of BFA treatment. MTT assay for succinyl dehydrogenase activity of the cells showed about 55% after 300μM BFA treatment. Destruction of cellular organells, numerous vacuolation in the cytoplasm & nucleus, chromatin margination, & fragments of nucleus were seen with TEM after 300μM BFA treatment. DNA fragmentation of SGT cell line was induced by 300μM BFA treatment and confirmed by gel electrophoresis from genomic DNA extraction. Late apoptosis of the cells through flow cytometric analysis of Annexin-V staining as induced by 300μM BFA treatment. Early apoptosis of the cells through flow cytometric analysis of caspase 3 and Apo 2.7 staining was induced by 300μM BFA treatment. It suggested that early and late apoptosis of SGT cell line would be induced by Brefeldin A treatment in vitro study. This work evaluated the efficacy of BFA, a potent apoptosis inducer, on SGT cultured cell line. And BFA as chemotherapeutic agent will be used as the treatment choice for adenocarcinoam NOS, and be need to apply BFA to in vivo study & clinical approach in future.

Oral squamous cell carcinoma is the 1st most common malignancy in oral and maxillofacial area. HPV 16 has been strongly linked to progression of cervical carcinoma. E6 and E7 as a small DNA virus encoding two major oncoproteins of HPV 16 can act together to produce efficient immortalization of primary human epithelial cells. Thus it is important to pursue the development of Immortalized human oral keratinocyte(IHOK) culture model which could be related to the pathogenesis of oral squamous cell carcinoma. If we establish IHOK transfected by E6/E7 genes, IHOK will be accepted as a model system for HPV-linked oral carcinogenesis. The purpose of this study were to culture primary normal human oral keratinocyte(NHOK), and to establish IHOK for studying oral carcinogenesis in the future. NHOK was primarily cultured under normal culture condition, and transformed into IHOK by transfection of E6/E7 genes. After 100 passages depend on Ca++ condition, cultured IHOK was confirmed by growth curve, cornified cell envelope measurement, TGase 1activity, mRNA detection, tumorogenecity and anchorage independence assay. After 100 passages, cultured IHOK showed most basal cell and monolayer of polyhedral cells under 0.15mM Ca++, and small area of stratification and flattened epithelial cells with irregular border under 1.2mM Ca++. The cultured IHOK showed relatively resistant growth under high calcium condition. The E6/E7 mRNA was detected in cultured IHOK by RT-PCR. During the terminal differentiation in cultured IHOK, increased insoluble cornified cell envelope formation was accompanied with induction of TGase 1 activity. But the cultured IHOK showed less CEM and TGase 1 activity than those of cultured NHOK. Cultured IHOK showed non-tumorogenecity, but slight anchorage independence. We had developed a technique to transform NHOK into IHOK by transfection of E6/E7 genes. Cultured IHOK was established as intermediate stage cell to study the pathogenesis of human oral squamous cell carcinoma.

Since oral keratinocytes represent the natmal target for HPV(human papill omavi ruses) infecti on, HPV infection may be involved il1 the developmel1t of oral SCC. Through compaJ'ing the morph이 ogic featw-es of NHOK to 다fOK accorcling to calcium concentration by TEM, immortali zed oral keratinocytes(IHOK) transfected by E6/E7 gene of HPV 16 have been gained wide acceptance as a model system for HPV-linkecl oral carcinogenesis. The purpose of this study were to exami ned the ultrastructural fcaturcs of culturcd NHOK, IHOK, and HN4 oral squamous cell CaJ‘CI noma celJ line, and to apply these results to oral carcinogenesis in the future, Prima:rily cul tlU'ed NHOK, IHOK ++ and HN 4 cell line which were cu ltu red under 015 and 12mM CaTT of 1ιBM bulJet kit For tra nsmission electronmi crosco py(TEM). under preconfluency‘ and after 3 days of postconfluency uncler 1.2mM Ca ++‘ cultured NHOK IHOK, and HN4 cell line were immediately fixed in 2, 0% gluta:raldehyde in O.lM cacodylate buffer(pH 7, 4) at 40C 1'01' 1h TEM of cultured NHOK under 1. 2mM Ca ++ showed increased tonofi laments‘ and vaculated ovoid cells wi th cornifi ed envelope, whi le cultured IHOK showed prominent microvilli , unilateral desmosome in microvillus‘ and tonof t!amen ts Under high calcium cu ltured IHOK showed less tonofilaments than that of cultured NHOK, while cu ltu red lHOK a nd HN 4 cell lines showed more increased desmosomes under high calclUm It suggested that the ultrast ru ctura l cha nges of cultured IHOK would be accepted as the morphologic changes of intermediate stage aJl10ng oral carci nogenesis ,

It is well kwon that HPV have been strongly linked to progression of or al squamous cell carcinoma‘ Effici ent im mortalization of nonnal human oral keratinocyte(NHOK) should provid further evidence for the role HPV in tumorogenes is ‘ Because IHOK(I mmortali zed human oral keratinocyte) has been considered as a moclel syst em for study ing I-!PV- linkecl oral ca rcinogenes is , it is important to pursue the differenti ati al change of IHOK cul t ure moclel during t he culture passage, The purposes of this study were to examine the cha ra r’ ct eristic clifferential changes of cul turecl immorta lizecl human ora l keratinocytes during long term passage, and to apply these results to or al carcinogenes is in the future, NI-!OK was primarily incubated at 370C and 5% C02 under KBM bullet kJt IHOK was co ntinuously cul t ured towarcl 100th passage(two times per week) , Growth curve of NI-!OK and II-!OK clepend on clùture passage was taken For examining the cha racte ri s t ic clifferential changes of II-!OK, transrnission electron microscope, 1ì'ansgluta miase activity‘ E6/E7 mRNA detect ion, a ncl tumorogenecity were done 10th II-!OK showecl sl ight polygonal flattencl cells and sometimes apoptotic cells ‘ while 100th IHOK showecl increased polygonal cell s ‘ Cultu recl 100th IHOK showed r ela tively resis tant growth to high calcium than 10th II-!OK Microvilli from 10th II-!OK was not connect ecl with each other, ancl scatte red cytokeratin fil aments of 10th II-IOK. while decreased cytokeratin filaments in cytoplasm & prominent clesmosome of 100th IHOK. During the terminal differ entiation in cultured IHOK, induction of TGase 1 activity of 10th II-!OK was higher than that of100th IHOK mRNA E6E7 expresson was cletected and unchangable in both cul tured cells There was no tumorogenecity inclucecl by both culturecl cel ls. Although late passage IHOK showecl less r esemblance to NHOK, and lower TGase 1 acti vi ty than ea rly passage IHOK, it suggested that these cells should be 110t yet fully differ entiatecl to oral squ a mous ce ll carcinoma cells

Abnormal number of deciduous teeth causes esthetic and dental problems on infants or children, which has effect on articulation disorder and emotional development as well as their physical growth. Therefore, it is important to detect dental problems early and to provide comparable indications. The purose of this study was to find out the prevalence and pattern in abnormal number of deciduous teeth. The clinical and radiographic examination was undertaken for 200 at age from 1 to 10 years and statistical analysis was done. The result were as follows. Among the examined patients, congenital missing teeth 8.6% and supernumerary teeth 36.4% were seen(p<0.05). And abnormal number of deciduous teeth was prevalent in male. Most supernumerary teeth located on middle area(88.6%) was seen(p<0.05). The most frequently missing teeth was the mandibular primary lateral incisor and the mandibular 2nd. premolar. And these teeth were mainly located in maxilla and right portion. It suggested that this study would play an important role for the basis of the demographic research of abnormal teeth in oral pathologic field

The study of cornified cell envelope has been used to investigate the differentiation factors and to advance oral carcinogenesis, CE of human oral keratinocytes are in wet condition as saliva containing many proteases, growth factors, and many kinds of bacteria, The analysis of CE in Immortalized human oral keratinocyte(IHOK) derived from normal human oral keratinocyte(NHOK) will be used to study the pathogenesis of oral squamous cell carcinoma, The purpose of this study was to analyze the amino acid component derived from CE of cultured NHOK and IHOK, It will be helpful to study the role of transfected E6/E7 gene in forming CE, and to examine the pathogenesis of oral squamous cell carcinoma, After primry culture of NHOK, IHOK were cultured in KBM bullet kit at 370C under 95% C02 incubator, Growth curve according to calcium concentration, cornified cell envelope measurement(CEM), and protein chemistry for amino acid component of CE were done(Mena :f::SD) , respectively. The obtained results were as follows, lHOK showed small areas of stratification, more compact, with irregular border and tightly apposed cells in 1,2 mM Ca++, Cornified cell envelope exhibited an aggregated group of empty space surrounded by the remained cell membrane, During the terminal differentiation in cultured NHOK and IHOK, insoluble cornified cell envelope formation was increased, CEM of NHOK was about 4 folds than that of lHOK under high calcium, Amino acid component of both groups showed Pro/Glu(SPR) , Gln/Glu(lnvolucrin) , and Gly(Loricrin) in descending order, From the aboving results, ít was suggested that when the terminal dífferentiation in cultured NHOK and IHOK, major amino acid component of CE in cultured lHOK was the same to that of cultured NHOK, It was thought that E6 and E7 gene should be involved in preventing the differentiation and proliferation of IHOK from making CE,