Skin is the outermost organ and acts as a barrier between the organism and environment. Skin protects the organism from environmental insults, such as chemicals, pathogens, and UV light. Much of the protective function of skin is dependent on the epidermis, a multi-layered epithelium that is composed of various cell types such as keratinocytes and melanocytes. Keratinocytes produce protective components through a sophisticated differentiation process. Disturbance of keratinocyte differentiation is related to several skin diseases such as psoriasis and atopic dermatitis. In this study, we prepared extract of combined medicinal plants (ECMP) consisting of Taraxacum platycarpum H. Dahlstedt, Heartleaf Houttuynia, Glycyrrhiza uralensis Fischer, and root bark of Ulmus davidiana. We demonstrated that ECMP enhanced keratinocyte differentiation and barrier functionality using an in vitro cell culture system and in vivo animal test. Treatment of cultured keratinocytes with ECMP resulted in induction of keratinocyte differentiation, as evidenced by increased differentiation markers such as involucrin, loricrin, and filaggrin. In line with these results, ECMP decreased proliferation of keratinocytes cultured in vitro. ECMP applied topically to tape-stripped mouse skins accelerated reduction of transepidermal water loss (TEWL), indicating fast recovery of barrier function. Immunohistochemistry showed that ECMP increased the filaggrin level in tape-stripped mouse skins. These results suggest that ECMP may be applicable for keratinocyte differentiation-related skin diseases.

Oral squamous cell carcinoma (SCC) is one of the leading causes of cancer mortality worldwide. It is generally thought that adjuvant chemotherapy provides modest prolongation of survival in various carcinoma. Docetaxel (Taxotere, TXT) play a significant role in the treatment of various solid tumors of epithelial origin. CsA (immunosuppressive drug) was widely used as adjunct for the treatment of cancer. Thus, it is important to pursue the apoptosis of IHOK and oral SCC induced by TXT combined with CsA related to the pathogenesis of oral SCC. But TXT combined CsA effect on IHOK and oral SCC remains unclear. After cultured IHOK and HN 22 oral squamous cell carcinoma cell line treated by 10 nM TXT and 1 μM, and caspase inhobitor, respectively, apoptosis index, cytochrome c and caspase-3 -8, -9 mRNA expression by RT-PCR, and procaspase-3 protein amount by immunoslot blotting was prepared. The purpose of this study were to examine the TXT-induced apoptosis pathway via caspase activation by CsA enhancement, and to apply these results to an effective therapeutic treatment plan for oral SCC by TXT combined CsA . 10 nM TXT showed about 60%, 55% celluar apoptosis of IHOK and HN 22, cell line, respectively, while CsA alone did not induce apoptosis in IHOK and HN 22 cell line. 1 μM CsA combined with 10 nM TXT increased apoptosis in IHOK and HN 22 cell line through caspase-3 and cytochrome c mRNA expression, while could not effect on caspase-8 and -9. Caspase inhibitor suppressed apoptosis of IHOK and HN 22 cell line induced by a combination of 1 μM CsA and 10 nM TXT. Immnoslot blotting showed procaspase-3 activation by a combination 1 μM CsA and 10 nM TXT, while caspase inhibitor inhibited activation. It suggested that a combination of CsA and TXT might induce increased apoptosis of IHOK and HN 22 oral squamous cell carcinoma cell line through caspase-3 activation. This treatment with a combination of TXT and CsA may be an effective therapeutic strategy for oral squamous cell carcinoma

Cyto kinc production by epiclerma l keratinocytes has been investiga ted ext ensive ly cluring the past decacle in the skm Furthermore‘ cyto kines produced by pidermal kerat inocytes may be regar decl as important regulators in inflammation, 1 nunune responses‘ and during wound healing, The associa tion of specific cytokine patterns in proliferative a ncl/or infl ammatory related cha nges in the s kin suggests a role in the pathogenesis of certain skin diseases, Although it is conceiva b1e that the pa ttern of cytokine express ion in o1'al kera tinocytes might be sirnilar t o tha t of epidermal origin, the1'e a re only sparse reports that have s hown t his experi menta lly, In addition, there is s ome evidence that there may be differe n ces in the proliferative capacity of oral versus epidermal keratinocytes, Since t his may be crucial for better un clerstanding the biologica l processes in the oral mucosa and how they may differ from the e pidennis, we a na lyzed the cyto kine expression pat tern of human oral kera tinocy tes, The purpose of this study were to investigate mRNA & protein express ion 0 1' va rious cy tokines between NHOK and NHEK by RT-PCR & immunoslot blotting, and to apply its results 1‘0 1' bet ter understancling t he pathological processes in the o1'al mucosal d1seases Cultured NHEK showecl larger a rea of cel lula r s tratil'icat ion tha n cul tu ++ 1'ed NHOK in 0 05mM Ca concentra tion, 1L - 1α , IL- 6 mRNA expression 0 1' cult ured NHOK we1'e hi gher than that of NHEK, TNF- mRNA expression of NHEK was about 1, 2 folds than that 0 1' NHOK, ICAM- 1 mRNA expression of NHOK was a bout 13 folds t han that of NHOK, while NHEK was weakly detected, 1L- 1a , IL-6 pro tein expression of cul tured NHOK were hi gher t han t ha t of NHEK TNF-a protein expression of NHEK was about 1, 2 fo lds than that of NHOK, 1CAM- 1 protein expression of NHOK was about 40 folcls than that of NHOK, whil e NHEK was weakly detectecl , mRNA express ion was associa ted wi th prot ein expression in cul tured NHOK ancl NHEK, It s uggestecl that lL- 1a ‘ 11-6 and ICAM-1 mRNA and protein be highl y expressecl in cultured NHOK, Especially, ICAM- 1 would be a useful ma rker for the pathogenesis of oral mucosal di sease,

Many researchers are interested in wound healing in the t reatment of burns, prevention of post surgical adhesions and cosmetic s urgery by excess collagen production and scar formatlOn Synthetic epidermal substi tutes with cultured epi thelial cells seem to be an attractive strategy since keratinocytes have been demonstrated to modulate fibroblast growth and collagen synthesis. Bioa bsorbable and biocompatible chitosan structurally mimics hyaluronic acid. Recently, a bio compatible synthesi zecl ch itosa n-PVP(polyvinyl pyrrolidone) hydrogels demonstrated in vitro biocompat ibi li ty for bio medical applications . However. there is no re port on this hydrogeJ"s ability to modulate human gingival fibroblast growth. The purpose of this study were to investigate different growth modulation between human gingival fibroblast and normal human oral keratinocyte by chitosan- PVP hydrogel, and to apply this biocompatible synthetic polymer to oral and maxillofacial wound healing. We have synthesized a hydrogel from chitosan-PVP and examined its effect on human gingival fibroblast growth modulation in vitro. Non-toxic and biocompatible hydrogel with human gingival fi broblasts and epithelial cells was tested by MTT assay. HGF showed a higher growth proliferation than that of NHOK after cell seeding. In MTT assay, 30% hydrogel leach out products showed a higher cellular viability in NHOK than that of any other products. In MTT assay, 30% hyclrogel leach out products showed relatively lower cellular viability of HGF ln growth profile, NHOK showed about 7 fo lcls higher than HGF after 1 day, while about 2 fo lds higher after 5 days. And also NHOK showed above about 70% cell ular via bility from 1 to 7 days. It suggested that Chitosan-PVP hydrogel would inhibit relatively the growth of HGF and s timulate the growth of NHOK_ This phenomenon may prove to be of use in wound management 0 1' oral and maxillofacial area as epitheli al substitutes.

Cytokines play a vital role in the host immune response by regulating the development and function of im munocompetent ce11s One immunomodulatory agent that has received attention in oncology research recently is interleukin - lO(IL-lO). IL-IO inhibi ted tumor antigen presenta tion and induced energy in T lymphocytes that had been s timu lated by autologous MHC class II positive tumor ce11s Patients with head and neck cancer have been shown to exhibit profound irnmunosuppression. The mechani sm by which tumor ce11s alter immunological function in the host is poorly understood. Recently. production of biological active IL- IO was confirmed in ovar‘ian cancer, melanoma, skin cancel‘ & head and neck cancer, suggesting that IL- lO reduces the function of tumor infiltrating lymphocytes and contributes to the tumor growth. IL-IO expression has not been examined extensively in human oral cancer and has not yet been cla rified. The purpose of t his study were to investigate IL-IO mRNA and protein expression in NHOK, IHOK and oral squamous ce11 carcinoma(OSCC) ce11 line by RT-PCR and irnmunoslot blotting, and to apply its results to examine its thera peutic significance for oraJ cancers. Cultured NHOK showed a lower level of IL-IO mRNA and protein expression than cultured IHOK and HN 22 OSCC cell line under pre and postconfluency. HN 22 OSCC cell line under pre and postconfl u ency. showed the highest level of IL-I0 Cul tured IHOK showing a intermediate expression of IL- IO could be as a vaJ u a bJe marker for oral carci nogenesis ste p. During the terminal differentiation of a11 the ce11 lines, IL- IO ex pression was significantly unchangeabl e. IL- IO mRNA expression of a11 the ce11 lines was consistent with IL-10 protein expression. It suggested that IL- lO expression might play an important role in oral carcinogenesis and IHOK could be a valuable marker for oral carcinogenesis step. And aJso IL- 10 related gene may be future targets for gene discovery and possi bJy therapeutic intervention

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.

Annexin I plays an important role in the process of keratinization as a compont of the cornified envelope of skin epithelium. The effect of annexin I on the terminal ifferentiation of normal human oral keratinocyte(NHOK) have remained to be defined. To understand the role of annexin I on the terminal differentiaiton of NHOK, NHOK and NHEK cells were primarily cultured in KBM bullet kit. When the cells reached confluence, terminal differentiation was induced by switching the medium to KGM bullet kit containing 1.2mM Ca2+. Preconfluency of NHOK under 0.05mM Ca++ conc as control group was used. The cells was examined with inverted microscope. Under 0.05mM Ca++ conc(Precon, Postcon), and 1.2mM Ca++ conc(Postcon), RT-PCR for annexin I mRNA measurement, and immunoblotting for annexin I protein measurements in triplicate, respectively. The purpose of this study were to study differential mRNA & protein expression of annexin I between NHOK & NHEK by using RT-PCR & immunoslot blotting during terminal differentiation, and to apply these results to study a role of annexin I on epithelial differentiation of oral mucosal diseases in the future. Cultured NHEK showed larger area of cellular stratification than cultured NHOK in 1.2mM Ca ++ concentration. Annexin I mRNA and protein expression of cultured NHOK showed higher than that of cultured NHEK in higher calcium concentration. Annexin I mRNA and protein expression of cultured NHOK showed about 2-2.7 fold higher in 1.2mM Ca++ conc. than in 0.05mM Ca++ conc. Although annexin I was involved in the terminal differentiation of cultured NHOK & NHEK in higher calcium concentration, annexin I play an important role in the terminal differentiation of cultured NHOK in higher calcium concentration. From the aboving results, It was suggested that annexin I would play an important role in the terminal differentiation of NHOK in higher calcium, which be helpful to study epithelial differentiation of oral mucosal diseases.

Pro-inflammatory cytokines are important mediators of cutaneous cellular activities during many oral mucosal diseases. IHOK culture model transfected by E6/E7 genes provide further evidence for the role of HPV in tumorogenesis. It is interesting to investigate cytokine expression of immortalized human oral keratinocyte(IHOK). The purpose of this study were to analysis cytokine mRNA expression levels of NHOK and IHOK by RT-PCR. IHOK showed about 5 fold increases of IL-6 compared with NHOK, while TNF-α was the lowest. It suggested that immortalization of NHOK with E6/E7 could result in elevated expression of IL-6, and IHOK be in the intermediate stage of oral carcinogenesis.

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.

Cultured normal human oral kera tinocyte(NHOK) & inunortalized human oral keratinocyte(IHOK) provide a valuable model in ce llular proliferation and differentiation after proper stimulation , And it is interesting to study these estab lished cell lines esca ping normal control on their growth and differentiation, SPRR1 is induced during t erminal differ entiation 0 1' human epiderma l kerat inocytes but is rarely in anaplastic cells of keratinocyte origin, But SPR1 expression has not yet been explained during differ entiation uf NHOK and Lransformed oral keraLinocyLes , The purpose of this study were to examine mHNA and protein expression of SPR1 in response to a known differentiation signal, calcium conc in NHOK, lHOK a nd oral SCC ce ll line(HN 4) , and to apply these results for investigating the molecular mecha nisms of tra nsformed cellular differentiation , Primary cultured NHOK, established IHOK and HN 4 cell line were cul tured in KBM bullet kit Preconfluency of NHOK as control group was used Under O, 15mM Ca++ conc(Precon, Postcon) , and 1, 2mM Ca++ conc(Pos tcon)‘ the insoluble final pellets were measured fo1' cornified cell envelope measurements, and RT- PCR for SPRR1 mRNA meas urement, and immunoblotting for SPRR1 protein measurements in tripli cate , resp ectively , The terminal different ia tion of cu ltured NHOK and IHOK was depend on calcium concentration, while HN4 cell line was not SPRR1 mRNA and protein expression of cultured NHOK showed the highest among cultured IHOK & HN 4 cell line in hi gher ca lcium condition , SPRR1 mRNA and protein expression of cultured IHOK showed higher‘ than tha t of HN 4 cell line in hjgher cacium condi tion , SPRH1 was expressed in differentiation of NHOK and IHOK t ransfected by E6/E7 genes but ra rely expressed in malignant oral keratinocytes , It suggested that SPRR1 ex pression as kera tinocyte terminal diff‘erent ia tion marker involved in cellular cornification would be differentially effected by immorta li zation and ca rcinogenic transforma tion

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

We studi ed the difTerential elTect of vitamins A, C, U. and E on normal human 이 al keratinocyte(NHOK) , HPV-16 E6E7 immor talized human oral keratinocyte(1HOK) , Oncogene transfected HPV-16 immortalized ce1ls(OTOK) , and two ol'al sq ua mous cell line(HNSCC30‘ HNSCC31) according to carcinogenesis stage. The vitamin effect was evaluated by morphology. ce ll viabi lity. a nd orgnaotypic culture Vitamin A has a greater negative effect on growth for all NHOK IHOK HNSCC. es pec ially N-Ras t rans fected IHOK, Vitamin D & E revealed no significant cell activity on NHOK lHOK, ad OTOK Vitamin C was found increased cell viability to IHOK and OTOK 1n primary oral squmaous cell ca rcino ma (HN30 ). vitam in 0 and C showing increased cell growth , but vitamin E showing no effect 1n metastatic oral squamous cell ca rcinoma(HN31), vitamin C has prol iferative effect , but vitamin 0 & E has anti-proliferative effect Vitamin A t reated normal a nd ma lignant ce1ls by organotypic cu lt ure. showed reduction of epithelial layer and in vasion to connective tissue. , especia lly in 1HOK & oncogene-transfected 1HOK, 1n conclusion. three-dimensional culture sys tem may be useful as a model to acess the efficiency of agents such as a1l trans retinoic acid can preventing progression of these premaligant lesion to maligant oral carcinoma(ch emopreventive agent) .

Cervical carcinoma is the 1st most common malignancy in korean females. HPV have been strongly linked to progression of cervical carcinoma. E6 and E7 as a small DNA virus encoding two major oncoproteins of HPV can act together to produce efficient immortalization of primary human epithelial cells, providing further evidence for the role of HPV in tumorogenesis. It is important to pursue the development of Immortalized human epithelial keratinocyte(IHEK) culture model which could be related to the pathogenesis between cervical and oral carcinoma. If we establish IHEK transfected by E6E7 gene, IHEK will be accepted as a model system for HPV-linked cervical carcinogenesis. The purpose of this study were to culture primarily normal human epithelial keratinocyte(NHEK), and to establish IHEK for applying these results to cervical and oral carcinogenesis in the future. The obtained results were as follows. 1. After 7-9 passages, cultured NHEK was almost senesce and disappeared, but cultured IHEK showed most basal cell and monolayer of polyhedral cells under 0.05mM Ca++, while small area of stratification and flattened epithelial cells with irregular border under 1.2mM Ca++. 2. The cultured IHEK showed relatively resistant growth to high calcium condition. 3. The mRNA E6E7 in cultured IHEK by RT-PCR was detected. 4. During the terminal defferentiation in cultured NHEK and IHEK, increase of insoluble cornified cell envelope formation was accompanied with induction of TGase 1 activity. But the cultured IHEK showed less CEM and TGase 1 activity than those of cultured NHEK. 5. Cultured IHEK showed non-tumorogenecity, but week anchorage independence. From the aboving results, we have developed technique to transform NHEK into IHEK by transfecting cells with E6E7 gene. Cultured IHEK was established as intermediate stage cell for studying the pathogenesis of human cervical carcinoma.

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,

be involved in the development of oral SCC. If we compare the morphologic features of NHOK to IHOK according to calcium concentration by TEM, IHOK have been gained wide acceptance as a model system for HPV-linked oral carcinogenesis. We already have established immortalized oral keratinocytes(IHOK) transfected by E6 and E7 gene. The purpose of this study were to examined the ultrastructural features of cultured NHOK, IHOK, and HN4 oral squamous cell carcinoma cell line, and to apply these results to oral carcinogenesis in the future. NHOK from healthy retromolar pad was primarily cultured at 37oC and 5% CO2. IHOK, and HN 4 cell line which were cultured under 0.15 and 1.2mM Ca++ of KBM bullet kit. For transmission electronmicroscopy(TEM), under preconfluency, and after 3 days of postconfluency under 1.2mM Ca++, cultured NHOK, IHOK, and HN4 cell line were immediately fixed in 2.0% glutaraldehyde in 0.1M cacodylate buffer(pH 7.4) at 4OC for 1h. The ultrathin sections were stained with uranyl acetate and lead citrate, and examined by TEM. The obtained results were as follows. 1. TEM of cultured NHOK under 1.2mM Ca++ showed increased tonofilaments, and vaculated ovoid cells with cornified envelope, while cultured IHOK showed prominent microvilli, unilateral desmosome in microvillus, and tonofilaments. 2. TEM of HN 4 cell line sowed numerous microvilli, increased N/C ratio, and lateral desmosome in microvilli under 0.15mm, while under 1.2mM well forming desmosomes. From the aboving results, under high calcium cultured IHOK showed less tonofilaments than that of cultured NHOK, while cultured IHOK, and HN 4 cell lines showed more increased desmosomes under high calcium. It was suggested that the ultrastructural changes of cultured IHOK would be accepted as intermediate stage cells for studying oral carcinogenesis.