Regulation Of Tyrosinase MRNA In B16F10 Murine Melanoma Cells By Retinoic Acid

Skin pigmentary disorders are common in the clinic of dermatology, such as Vitiligo, Chloasma, Melanosis et al. These diseases can always lead to questions of personal features, so as to make the patient feel very unpleasant. Research has found that abnormal activity and quantity of melanocvtes as well as abnormal activity of tyrosinase are the main causes to this kind of disease. Therefore, the study of melanocyte and the synthetic mechanisms of melanin has already been one of the most important subjects in recent years. Tyrosinase is the key enzyme essential for melanogenesis and determines the synthesis and quantity of melanin, so many researchers regard the activity and expression of tyrosinase as the most important subject.Retinoic acid has various of complicated pharmacological activity. In the clinic of dermatology it can be used to treat Acne, Psoriasis and some Keratotic skin disorders. During the treatment, some doctors observed that topical using retinoic acid could result hypomelanosis on the skin of the patient. Then some researchers began to investigate the mechanism involved. But because of the limit of technology, their research only based on cell and molecular level. These years molecular biochemistry has progressed greatly, it provided a kind of useful and powerful tool for researchers to study the subject further.In this experiment, we used reverse transcription-polymerase chain reaction (RT-PCR) to detect the regulation of tyrosinase mRNA expression of B16F10 murine melanoma cells treating by retinoic acid and hydroquinone, and get insight on the mechanisms involved. First of all, according to the experiment design, we cultured the B16F10 murine melanoma cells in culture medium which contains different concentration of drugs; after treating for 48-96 hours, the cells Were collected from their culture-bottles into preparation tubes; then total RNA of the cells was extracted using the TRIzol kit; and using it as a template for reversetranscription to complementary DNA (cDNA); making the acquired cDNA as pedals for PCR assay; the PCR products were electrophoresed and were photographed under UV light; finally using computer to analyse the result.The results showed that retinoic acid exhibited an inhibitory effect on the expression of tyrosinase mRNA. Furthermore, when drugs concentration and treatment time changed, the inhibitory effect changed as well. As the concentration of retinoic acid is 100u mol/L, treating 72h, the expression of tyrosinase mRNA decreases 30.13%. The results partly indicated that retinoic acid inhibitted the synthesis of melanin based on the regulation occured at mRNA level. We also detected hydroquinone has no effect on tyrosinase gene expression at mRNA level. Furthermore, in this experiment retinoic acid showed another activity. It can inhibit the upregulation of tyrosinase mRNA expression induced by cholera toxin. It indicated that retinoic acid can influence expression of tyrosinase mRNA through cAMP/PKA way. So, we took the conclusion that retinoic acid regulated the expression of tyrosinase mRNA through not only direct but also indirect ways.These experiment results provided the theory evidence for using retinoic acid to treat pigmentary diseases; and the parameters obtained from the experiment contributed to produce appropriate topical medicine in clinic.The details of retinoic acid regulation on expression of tyrosinase are still needed to be studied further.