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.