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.

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 ,