The author evaluated the optimal concentration of 3 compositions of TIC medium which has used as the melanacytes culture medium. The concentrations of placental extract and bovine pituitary extract, which have the ability to promote proliferation of melanocytes, were evaluated also. Modified TIC medium with above 5 components of evaluated concentration was very effective in melanocytes culture. The results were as follows : l. 12-0-tetradecanoyl-phorbol-13-acetate (TPA) showed effective melanocytes proliferating activity at the concentration of 30ngml (p(0.05) 2. Isobutylmet:hyl xanthine (IBMX) showed effective melanocytes proliferating activity at the concentration of 0.3mM (p(0.05) 3. Cholera toxin (CT) showed effective melanocytes proliferating activity at the concentration of )OnM (p(0.05) 4. Two percentages of placental extract in culture medium showed effective melanocytes proliferating activity. S. Two percentages of bovine pituitary extract in culture medium showed effective melanocytes proliferating activity. 6. Placental extract and isobutylmethyl xanthine proved to have high melanocytes proliferating activity. 7. Melanocytes proliferated rapidly on modified TIC medium (Proliferation doubling time . about 43 hours) 8. The peak time of melanocytes proliferation (7.2 X 10/cm) was observed on the seventh day of culture, From this data, this culture system can be recommended as a new melanocytes culture.
Cholera Toxin ; Humans* ; Melanocytes* ; Tics ; Xanthine

In humans, the major stimulus for cutaneous pigmentation is ultraviolet radiation. Little is known about the mechanism underlying this response, in part, because of the complexity of the interactions involving the whole epidermis. The present stucy was undertake to evaluate the effects of a single exposure of UVB on cultured normal melanocytes. Melanocytes were exposed to UVB from 5.1 mJ/cm to 203 mJ/cm. The results were as follows : 1. The main morphologic changes in UVB-exposed groups w re larger sized cells, more blunted dendrites, and shorter dendrites than in the control group. These cells increased sized according to the increased doses of VVB, but above 101.5 mJ/cm, the melanocytes shrunk and were destroyed. 2. From 20.3 mJ/cm of UVB, the proliferation of melanocyte was decreased, Especially, there was statistical!y significant difference above 50.8 mJ/cm (p<0.05, p<0.01). 3. The antiproliferativo effect increased with the passage of tirie after UVB exposure. So, cell count could not be done in 101.5 mJ/cm and 203 mJ/cm on the third day, and in 50.8 mJ/cm, 101.5 m J/cm and 203 mJ/cm on the seventh day. 4. Statistically the melanin content per well was significantl dicreased to 11-28% of each control group with dose above 50.8 mJ/cm (p<0.05, p<0.01). The melanin content per cell was increased to 107-128% of each control group when doses were below 20.3 mJ/cm and decreased to 49-79% of each control group when above 0.8 mJ/cm on the third day, but there was no statistically significant difference. In summary, when melarocytes were exposed to UVB, morphclogic changes progressed to cell differentiation. The results also suggested that a low or dose of UVB has an antiproliferative arid mild melanogenic effect, and a higher dose of UVB has a direct cytotoxic effect.
Cell Count ; Cell Differentiation ; Dendrites ; Epidermis ; Humans ; Melanins ; Melanocytes* ; Pigmentation

To evaluate the effects of kerationocytes on the growth of melanocytes, keratinocyte conditioned media (K-CM) with different molecular weight obtained by dialysis were added to melanocyte growth medium (M-GM). In addition, K-CM only, and K-CM mixed with each component of M-GM, such as TPA(12-tetradecanoyl- phorbol-13-acetate), IBMX(isobutylmethylxanthine) and CT(cholera toxin), were used for the culture of melanocytes. 1) The proliferation of melanocytes was incresased to 2.86 x 10(5)+/-0.87 x 10(5) cells/ well and 2.87 x 10(5)+/-0.71 x 10(5) cells/well in 25% K-CM with a cut-off molecular weight of 2,000 and 25% K-CM with a cut,-off molecular weight between 6000 8000 respectively, as compared to 1.88 x 10(5)+/-0.45 x 10(5) cells/well in the control group (p < 0.05). 2) The amount of melanin was increased to 0.2987+/-0.0830ng/mlin 25% un- dialyzed K-CM, as compared to 0.2264+/-0.0643ng/ml in the control group, but this differnce was not statistically significant. 3) Maximum proliferation of melanocytes was observed in 35% concentration of K-CM with a cut-off molecular weight of 6000 8000. 4) Maximum of melanin production was observed in 35% concentration of undialyzed K-CM 5) As compared to 7.86 x 10+/-1.74 x 10(5) cells/well in M-GM,proliferation of melanocytes in 35% K-CM with a cut-off molecular weight of 6000 8000 was de- creased to 1.38 X 10(5)+/-0.97 X 10(5) cells/well. 6) There was no difference in melanocyte proliferation between 6.81 x 10(5)+/-2.19 x 10(5) cells/well in 35% 6,000 8,000 M.W. cut-off dialyzed K-CM, with IBMX only, and 7.86 x 10(5)+/-1.74 x 10(5) cells/well in M-GM. 7) Compared to 0.2303+/-0.0700ng/well cell in M-GM, the amount of melanin was increased to 0.3227+/-0.0900ng/cell, 0.3624+/-0.0900ng/cell and 0.2928+/-0.0500ng/cell, respectively, when TPA, IBMX, CT was added to 35% undialyzed K-CM. It also increased to 0.3176+/-0.1100 in 35% undialyzed K-CM(p<0.05). In summary, the results proved that cellular activating substances released from keratinocytes affect the proliferation of melanocyte and the synthesis of melain. It is also expected that methods used in this study can be clinically utilized because melanocyte culture is possible on K-CM without adding tumor promotors.
1-Methyl-3-isobutylxanthine ; Culture Media, Conditioned ; Dialysis ; Keratinocytes* ; Melanins ; Melanocytes* ; Molecular Weight

The authors investigsted the effects of azelaic acid on human melanoma cells (G 361 melanoma cell line) and cultured normal melanocytes obtained from the prepuce of newborn. The results were as follows : 1. The proliferation of melanoma cells was decreased, but not in a dose-and time- ependent fashion. The cell population of melanoma cells after 2, 4, and 6 days of culture was decreased to 3.80 x 10(5)cells/well, 4.55 x 10(5)cells/well, and 4.30 X 10(5)cells/ well, respectively, in the presence of 10(-2)M azelaic acid. The proliferation of mormal elanocytes of normal melanocytes was decreased in a dose-dependent, but not in a time-dependent fashion. The cell population of normal melanocytes after 2, 4, and 6 days of culture was significantly decreased to 6.38 x 10(5)+/-1.37 x 10(5) cells/well, 5.33 x 10(5)+/-0.73x10(5) cells/well, and 7.20x10(5)+/-1,11 x10(5)cells/well, respectively, in the presence of 10(-2) M azelaic acid(p<0,05, p<0.01). 2. A dose-and time-dependent inhibition of DNA synthesis was not found in either group. However, DNA synthesis in melanoma cells was decreased to 92710+5188 CPM, 16268+/-15S5 CPM, 8518+/-996 CPM, respectively, after 2, 4, and 6 days of culture, and in normal melanocytes was decreased to 9398+/-2279 CPM(p>0.05) and 6953+/-1217 CPM (p<0.05) after 4 and 6 days of culture in the prensence of 10(-2) Mazelaic acid, 3. There was no difference in melanin content per well at various concentrations in either group, but the melanin content per individual melanocyte of the melanoma cells was increased to 0.0303 ng/cell, 0.0253 ng/cell, and 0.0377 ng/cell, respectively, and that of normal melanocytes was signifieantly increased to 0.0754+/-0.0215 ng/cell, 0.0719+/-0.0144 ng/cell(p<0.05), and 0.1089+/-0.0185 ng/cell(p<0.05), respectively, after 2, 4, and 6 days of culture in the presence of 10(-2) M azelaic acid.
DNA ; Humans ; Infant, Newborn ; Melanins ; Melanocytes* ; Melanoma

BACKGROUND: In the skin, the major stimulus for cutaneous pigmentation is ultraviolet radiation. The most important physiologic role of melanin is protection against harmful UV radiation to skin. It is known there are some differences in melanization between a single and multiple exposures of UVB, in vivo. Little if known about the functions of the melanocyte alone in cutaneous pigmentation after ultraviolet exposure, because of the complexity of interactions in the whole epidermis. OBJECTIVE: To investigate the effects of multiple exposures at various dosages of UVB, and to compare the effect of UVB in multiple divided exposures with a single exposure at the same total dosage of UVB on proliferation and melanization in cultured human melanocyte. METHODS: Melanocytes were cultured by modified TIC medium. The melanoctes were exposed daily for three consecutive days to UVB at 2, 4, 8 and 16 mJ/cm2and a single exposure at 24 mJ/cm2. The morphologic changes were examined by phase contrast microscopy. The melanocytes were counted by hemocytometer and melanin contents were assayed by spectrophotometer. RESULTS: 1. The effects of multiple UVB exposures: 1) The morphologic changes were as follows: With three time exposures at a dosage of 8 mJ/cm2, themelanocytes enlarged in size, and elongated their dendrites slightly; with three time exposures at a dosage of 16 mJ/cm2, enlargement in sized and elongation of dendrited were more significant. 2) With three time exposures at dosages of 2 nd 4 mJ/cm2, the proliferation of melanocytes was stiumlated significantly(p<0.05). However, with three time exposures at dosages of 8 and 16 mJ/cm2the proliferation was inhibited(p<0.05). 3) With three time exposures at dosages of 2 and 4 mJ/cm2, the melanin contents were decreased. However, with three tiem exposures at a dosage of 16 mJ/cm2, the melanin contents were highly increased(p<0.01). 2. The comparison between multiple divided exposures and a single exposure at the same toal dosage of UVB: 1) There were no morphologic differences of dendrities between with three time exposures at a dosage of 8 mJ/cm2 and with a single exposure at a dosage of 24 mJ/cm2. However enlarged melanocytes were more numerous with a single exposure. 2) The proliferation of melanocytes was more inhibited with a single exposure than with multiple divided exposures(p<0.05). 3) The melanin contents were more increased with a single exposure than with multiple divided exposures(p<0.05). CONCLUSION: With multiple exposures at lower dosages of UVB, the proliferation of melanocytes was stimulated, and melanization was decreased. However, with multiple exposures at higher dosages of UVB, the proliferation was inhibited, and melanization was increased. At the same total dosage of UVB, the proliferation was more inhibited, and the melanization was more increased with a single exposure than with multiple divided exposures.
Dendrites ; Dermatitis, Irritant ; Epidermis ; Humans* ; Melanins ; Melanocytes* ; Microscopy, Phase-Contrast ; Pigmentation ; Skin ; Tics

BACKGROUND: Human growth hormone(hGH) plays a central role in linear bone growth and body metabolism. Its mitogenic effect in human tissues is mediated via direct and indirect actions. As proposed by the "somatomedin hypothesis", many circulating GH-mediated effects are exerted indirectly and systemically via stimulation of hepatic synthesis of insulin-like growth factor 1(IGF-1). Given additional evidences for the expression of growth hormone receptor(GH-R) and IGF-1 receptor(IGF-1R) on many target tissues including keratinocytes, melanocytes, and fibroblasts, it is now evident that the GH can act via systemic IGF-1 secreted by the liver and locally produced IGF-1, as well as directly through the GH receptor. OBJECTIVE: The purpose of this study was to investigate not only the effect of IGF-1 on the morphologic changes, proliferation, and melanization of cultured human melanocytes but also on its signal transduction pathway through the IGF-1R. METHODS: Melanocytes were exposed to IGF-1 at 10, 25, 50, 75, 100ng/ml and we examined the changes of cell morphology, number of cells, [3H]-thymidine incorporation, MTS assay, and melanization according to the concentrations and exposure times of IGF-1. Also, the activity of p44/42 MAPK/ERK according to the various exposure times of IGF-1(25ng/ml) was examined using the Western blotting method to find out about the signal transdution pathway of IGF-1. RESULTS: The results were as follows: 1. There were no significant morphological changes of cells between the control and experimental groups according to the concentrations and exposure times of IGF-1. 2. The effects on melanocytes according to the concentrations of IGF-1 5 days after adding IGF-1 : 1) The number of cells, [3H]-thymidine incorporation, and MTS assay were significantly higher than those of control group in all experimental groups(p<0.05). 2) The melanin content showed an insignificant decrease in all experimental groups. 3) The melanocytes responded independent of the IGF-1 concentration in the assay of cell number, [3H]-thymidine incorporation and MTS. 3. The effects on melanocytes according to the exposure times(3 days, 5 days, 7 days) of IGF-1(25 ng/ml) : 1) The number of cells, [3H]-thymidine incorporation, and MTS assay increased as time went by, and was significantly higher than those of control group at all exposure times(p<0.05). 2) The melanin content decreased after exposure of IGF-1, especially that of 3 days exposure group showed a significant decrease(p<0.05). 4. The activities of p44/42 MAPK/ERK increased suddenly at 5 minutes with a peak at 60 minutes and then abruptly decreased at 120 minutes after adding IGF-1 CONCLUSION: In summary, this study demonstrates that IGF-1 has no effect on the morphology, but it does increase the proliferation and slightly decrease the melanization of cultured human melanocytes. In addition, it is suggested that IGF-1 plays a role in regulation of proliferation of melanocytes via the receptor PTK pathway with activation of p44/42 MAPK/ERK.
Blotting, Western ; Bone Development ; Cell Count ; Fibroblasts ; Growth Hormone ; Humans* ; Insulin-Like Growth Factor I* ; Keratinocytes ; Liver ; Melanins ; Melanocytes* ; Metabolism ; Signal Transduction

The authors investigated the antiproliferative effect and expression of HLA-DR an- tigen by recombinant gamma-interferon (r-IFN-y) on cultured human keratinocytes (KC). The results were as follows, 1. From 10l.J/ml of r-1FN-p exposure, the proliferation of KC decreased in a concentration dependent fashion. But there was little difference of antiproliferative effect above 30U/ml of r-IFN-y exposure. 2. The expression of HLA-DR antigen on KC increased in a concentration and time dependent fashion of r-IFN-p exposure. E3ut t,here was little difference of HLA-DR antigen expression on KC above 30tJ/ml and most of HLA-DR antigen were expressed within 48hr. 3. The opt,imal condition for HLA-DR antigen induction on KC by r-IFN-p was likely t,hat HLA-DR KC was observed at 48hr under the our exposure of 30U/ml of r-IFN p. 4. After 4hr exposure of 30U/ml of r-IFN-p, KC expresed HLA-BR. antigen, reaching a maximum intensity at 3 days. At, 7 days, the loss of HI A-DR KC showed over 90% of maximum intensity.
HLA-DR Antigens* ; Humans* ; Interferon-gamma ; Interferons* ; Keratinocytes*

BACKGROUND: Psoralen has been used in the treatment of certain hypojigmentary disorders with UVA or solar irradiation. However trecent report proposed the actions of psiralens are direct and do not require the presence of ultraviolet light. The report also suggested that tze specific receptors other than DNA would be present. OBJECTIVE: This study was done ta identify the effects of 8-methoryporalen(8-MOP) on the proliferation and melanization of cultured normal human melanocytes without UVA. METHODS: Melanocytes were cultured in melanocyte culture medium neluding 16% or 5% FBS. We added 8-MOP by their concentrations from 10 M to 10 M. After 8 hours treatment, we investigated the melanocytes proliferation and Lhe melanin contents. RESULTS: We could not detecet any significant differences of melanoytes proliferation and melanin contents between the control end experimental groups. CONCLUSION: There were no effect on the proliferation and the milanization of cultured normal human melanocytes with 8-MOP only.
DNA ; Ficusin ; Humans* ; Melanins ; Melanocytes* ; Methoxsalen ; Ultraviolet Rays

BACKGROUND: The main function of melanocyte is known to protect the skin from hazardous sunlight. But, some investigators have claimed lately that melanocytes are also related to the immunologic role in the epidermis because these cells produce IL-1 activity and IL-1beta convertase activity, in vitro. OBJECTIVE: Our purposes were to investigate the effects of rIFN-gammaon the proliferation of melanocytes, melanin content, and the expression of HLA-DR antigen on melanocytes after a rIFN-gammaexposure. MEHTODS: The number of melanocytes, the melanin content, and the expression of HLA-DR antigen were evaluated on cultured human melanocytes according to a time sequence and various concentrations of rIFN-gamma. RESULTS: Antiproliferative activity on melanocytes was dependent on the exposure time and the concentration of rIFN-gamma. According to the exposure time and the concentration of rIFN-gamma, melanogenic acivity was inhibitd or stimulated. Normal melanocytes didn't express HLA-DR antigen, but when normal melanocytes were exposed to rIFN-gamma, the expression of HLA-DR antigen increased in a time-and concentration-dependent fashion. After the removal of rIFN - gammafrom the culture media, the expression of HLA-DR antigen on melanocytes also disappeared. CONCLUSION: In our study, melanocytes seem to be related to the immunologic role in the epidermis because these cells expressed HLA-DR antigen after rIFN-gammaexposure and we think that study could help to investigate between melanocytes and immunologic mechanisms in various inflammatory skin diseases.
Culture Media ; Epidermis ; HLA-DR Antigens* ; Humans* ; Interleukin-1 ; Melanins ; Melanocytes ; Research Personnel ; Skin ; Skin Diseases ; Sunlight

Human epidermal cells were obtained from suction blisters of 14 healthy individuals, and were cultured for 24-96 hours st a concentration of 1x 10(7)/ml, 5 x 10(6)/ml, 1 x 10(6)/ml, 5 x 10(5)/ml. Cells were also cultured with or without stimulants such as phorbol myristic acetate(PMA), muramyl dipeptide(MDP), and endotoxin. Then, cell-free supernatants of cultured epidermal cells were tested for ETAF by a thymocyte prolifera.tiom assay. The results were as follows : 1, The highest activity of ETAF was produced by fresh epidermal cells(EC) at a concentration of 1 x10(7)ml. Its highest 3H-TdR was 4928+/-2480cpm. The highest activity of ETAF was produced by cultured EC at a concentration of 5 x10(6)/ml. Its highest 3H-TdR was 13983+/-8045 cpm. 2. The highest activity of ETAF was produced by fresh EC with n culture time of 24 hours. Its highest 3H-TdR was 5357+/-3760cpm. The highest activity of ETAF was produced by cultured EC with a culture time of 72 hours. Its highest 3H-TdR was 11905+/-5327cpm. 3. The highest activity of ETAF was produced by both fresh and cultured EC at a titer of 1: 8 dilution of cell-free supernatants. 1ts highest 3H-TdR was 4928 +/-2480cpm in the fresh EC, and 11905+/-5327cpm in the cultured EC. 4. Alhen fresh EC was stimulated with PMA, MDP and endotoxin, higher activity of ETAF was found in the group stimulated with PMA or MDP compared with its control group. But lower activity of ETAF was found in the group stimulated with endotoxin compared with its control group. The 3H-TdR was 6000+/-1936 cpm in the group stimulated with PMA, 6945+/-3182 cpm in the group stimulated with MDP, and 36943+/-36861cpm in the group stimulated with endotoxin.
Blister ; Humans* ; Suction ; Thymocytes