Effects of supernatants of cultured keratinocytes on melanocyte proliforation and melanization

Suh, Woong Suck; Lee, Mu Hyoung; Park, Jai Kyung; Haw, Choong Rim

Abstract

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.