The Effects Of Calcium Signal Transduction Pathway On Hyperpigmented Human Skinautografts

BACKGROUNDSkin autografts is one of frequntly used operations of plastic surgery, which plays an important role in the reconstruction of wound, the improvement of physical form and the restoration of function. However, a diversified degree of hyperpigmentation on skin often accompany the success of autographts, thus may affect the cosmetic result. The previous study by our group found that a-MSH in the normal process of skin pigmentation play an important regulatory role, which bind to its specific receptor-MC-1R to promote melanin synthesis of melanocytes, and promote of melanosome diffuseion. Ca2+ as an intracellular second messenger can regulate cell metabolic activity. When Ca2+ combine with CaM can regulate gene transcription and cell secretion, mediated the interaction between the other signal systems and so on. Recent studies showed that calcium signal transduction pathway is involved in the process of skin pigmentation, such as:the synthesis of a-MSH, the supply of raw materials, synthesis of melanin, melanocyte proliferation and so on. Another research have found that a-MSH bind to MC-1R to can increase calcium influx,increase intracellular calcium levels, so activate of intracellular calcium signaling pathways. So how is calcium signal transduction pathway in the regulation of pigmentation and which realtionship between with a-MSH to stimulate melanin synthesis,it is worthy of further study.OBJECTIVE(1). To detection the expression of CaM,IP3RI and TYR in human skin autografts, with their normal skin as control, and to recognize the role of CaM, TYR and IP3R in hyperpigmented process of skin autografts.(2). To observate the role of verapamil on melanocytes intracellular calcium concentration, cell proliferation, CaM, TYR, IP3RI expression and melanin synthesis, and to elucidate the effect of calcium signal transduction in the melanin synthesis of melanocytes.(3). After verapamil interaction with a-MSH on melanocytes,observe the cell proliferation, CaM, TYR, IP3RI expression and melanin synthesis, and elucide the relationship between a-MSH and calcium signal transduction in the melanin synthesis of melanocyte.(4). To observe the role of verapamil antagonize eumelanin synthesis in guinea pig skin autografts,and elucidate the role of the calcium signal transduction further in the regulation of hyperpigmented process.METHODS(1). Immunohistochemistry and Realtime-PCR method were applied to detect the expression of CaM, TYR and IP3RI in the autologous skin grafts (full-thickness skin autografts, split-thickness skin autografts,medium thickness skin autografts autografts) and the normal skin respectively..(2). The cell proliferation, tyrosinase and melanogenesis of cultured melanocytes affected by verlapamil and a-MSH were measured by MTT,oxidation DOPA in vitro and NaOH fragmentation methods respectively. The expression of CaM, TYR and IP3RI were measured by western blot and Realtime-PCR.(3). Immunohistochemistry and Realtime-PCR were used to detect the expression of CaM and TYR in guinea pigs skin autografts before and after verapamil treatment and normal skin respectively. mRNA expression and compared with the control group.RESULTS(1). The expression of CaM was located in kytoplasm of melanocytes and keratinocytesk and was upward regulated in skin autografts. The expression of TYR was situated in kytoplasm and cell membrane of melanocyte. IP3RI expression localized in the epidermis basal melanocytes, keratinocytes kytoplasm. (2). Verapamil can significantly reduce the concentration of intracellular calcium in melanocyte, inhibition of cell proliferation, reduced the expression of CaM and TYR, at last decrease melanin synthesis.(3). Verapamil can antagonize the effect of a-MSH on melanocyte proliferation, reduced expression of CaM and TYR, reduced the activity of TYR synthesis at last decrease melanin synthesis.(4). The expression of CaM and TYR in skin autografts on guinea pigs after verlapamil treatment was downward regulated, as opposed to that of control skin autografts.(5). The content of melanin is obviously diminished in epidermal basal cell of skin autografts after ASIP treatment, as opposed to that of control skin autografts.CONCLUSIONThe expression of CaM was markedly increased in human skin autografts than in control samples and was correlated with the pigmentation of skin autografts Verapamil can antagonize the melanogenic effect of a-MSH and can inhibited the melanin synthesis in vivo.The results illustrates the calcium signal transduction pathway may play an important role in the hyperpigmentation process of skin autografts.