| Color of human skin and hair is affected by structural and functional coloration. The structural coloration is a genetically determined and independent of outside influence. Then functional skin coloration changing with the environment.At present, the study determined that the family of tyrosinase was the main rate-limiting enzyme of synthesize the melanin. Tyrosinase is a copper binding glycolprotein, while the tyrosinase-related protein -2 is a glycoprotein combine with iron and zinc. In the course of melanogenesis, tyrosinase catalyzes three different reactions:the hydroxylation of tyrosine to 3,4-dihydroxy-phenylalanine (dopa), the oxidation of DOPA to DOPA-quinone,the oxidation of 5,6-dihydroxyindole to indole-quinone. All reactions from tyrosine to eumelanin can progress slowly without the participation of tyrosinase. The change of tyrosinase activity may lead to the incidence of vitiligo, while the mutation of tyrosinase gene is an important reason of albinism. Melanogenesis is submitted to further control by tyrosinase-related protein 1(TRP-1) and tyrosinase-related protein 2(TRP-2), which catalyze the subsequent step in the mlanocyte. The TRP-1 gene located in the brown and the TRP-2 gene located in the slaty,they mutation will change the skin color. The TRP-1, TRP-2 and TYR are similar in structure and have more than 40% amino acids homology. Above of all, the three have the same original gene, and composed of complex play a role in the process of melanin synthesis.In recent years, the transcription factor microphthalmia-assciated transcription factor(MITF) with HLHzip (basic-helix-loop-helix-leucine zipper) as new melanogenic regulatory factor has been paid more attention. The study confirmed MITF has a certain role in the course of melanocyte growth, differentiation and function regulation. It binds to conserved consensus elements in gene promoters-M-BOX, then activates many enzyme involving in melanogenesis,such as tyrosinase, tyrosinase related protein-1,tyrosinase related protein-2. Moreover, many transcript factors will regulate the expression of MITF. Meanwhile, the melanin synthesis signal transduction is one of hot spot in the research of melanocyte biology. Previous research determined PKA (Protein kinase A) and PKC (Protein kinase C) pathway make important role in the melanin biosynthesis. Recently, it was reported that activating PKA may activate MITF transcription, and enhance the melanin synthesis, which are related to activating PKA, then increasing CREB phosphorylation and modify MITF promotor.Effects of estrogen on physiological processes of keratinocytes, fibroblasts, melanocytes and appendant such as sebaceous glands have different degrees of regulation, and have played an important role to the aging of the skin, hair growth, sebum secretion and even skin cancer. It is confirmed that estrogen receptor is also expressed in melanoma cells and nevus cells. Early studies have shown that estrogen cause human skin pigmentation, and found many clinical reports that it is effectivity of using hormone therapy to vitiligo cases. As for the endogenic bioacive compound--estrogen which is easily oxidized inactivation, but synthetic estrogen diethylstilbestrol has more easily play a role with estrogen receptor binding in vivo than natural estrogen, and has all of the pharmacological effects of estradiol. So we chosen a synthetic estrogen-diethylstilbestrol as the subject of study drug.The study planned to observe diethylstibestrol effect on melanogenesis of melanoma cell and further discussion relationship with its role and cAMP-MITF-tyrosinase pathway. This topic is divided into three parts.Part I Diethylstibestrol on mouse melanoma cell B16 melanogenesisMethod Reference to physiological concentration of estrogen, we selected different concentration 10-9μM,10-8μM,10-7μM,10-6μM,10-5μM,10-4μM,10-3μM on melanoma cell 24h,48h,72h respectively, and determined cell proliferation activity with MTT. According to the result of MTT, we chosen different concentrated diethylstibestrol into melanoma cell and detected its melanogenesis after 24h by NaOH dissoling method. At the same time, we determined melanin content at one of drug concentrations after 24h,48h and 72h, respectively.Result Diethylstilbestrol promoted melanoma cell proliferation in the 10-8μM-10-3μM(p<0.01),which is obvious effection at the 10-4μM, and it was not significant statistical difference comparing with 10-3μM group(p>0.05). But diethylstilbestrol promotion proliferation to melanoma cell has no obvious different significantly at 24h,48h and 72h(p>0.05). After intervention with 10-6μM, 10-5μM,10-4μm,10-3μM diethylstilbestrol,result showed that it has promotion to melanin content in a dose dependent manner and the diethylstilbestrol effect on melanogenesis has no obvious statistically significant difference at 24h, 48h and 72h time points(p>0.05).Conclusion The diethylstilbestrol effect on melanogenesis has up-regulation in concentration dependent manner.Part II The diethylstilbestrol promotion melanogenesis is related to tyrosinase family. Method With 10-6μM,10-5μM,10-4μM,10-3μM diethylstilbestrol effected on melanoma cell respectively, tyrosinase activity were detected after 24 hours. Meanwhile, the semi-quantitative RT-PCR and Western blot methods detected diethylstilbestrol on mRNA transcription of TYR, TRP-1, TRP-2 and the corresponding protein under different concentrations.Result The diethylstilbestrol has up-regulation effect to tyrosinase activity and different degree up-regulation to mRNA transcription of TYR, TRP-1, TRP-2 and their protein expression.Conclusion The diethylstilbestrol promotion melanogenesis is related to raising tyrosinase family expression and increasing tyrosinase activity.Part III cAMP-MITF pathway is involved in diethylstilbestrol regulating tyrosinase and melanogenesis.Method After the intervention of different concentrations diethylstilbestrol, ELISA detected intracellular cAMP. Meanwhile RT-PCR and Western blot methods detected mRNA transcription and protein levels of MITF. Then we used specific PKA inhibitor H89 intervention melanoma cells for 1h, then different concentrations of diethylstilbestrol effected on melanoma cells for 24h, measured the melanin content, the changes of tyrosinase activity and the mRNA levels of tyrosinase and MITF. Result Diethylstilbestrol enhance the intracellular cAMP concentration(p<0.01)and the mRNA of MITF and protein levels has also increased(p<0.01). The effect of diethylstilbestrol in promoting melanogenesis, increasing tyrosinase activity and mRNA were was partly reversed by the PKA inhibitior (H89), the same as the up-regulation of MITF(p<0.05).Conclusion Diethylstilbestrol on melanogenesis is probable regulated by cAMP/MITF/TYR pathway.In surmmary,diethylstilbestrol has a certain promotion to melanogene-sis and possible play a role by cAMP/MITF/TYR. The study provides a theoretical basis and experimental evidence to diethylstilbestrol used to the depigmentation disease therapy at clinical.
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