Studies On The Activation And Migration Of Amelanotic Melanocytes In Outer Root Sheath Derived From Human Hair Follicles And The Inhibitory Effects Of Icariin On Melanogenesis Of Melanoma Cells And Epidermal Melanocytes

In human embryos, melanocytes originate from the neural crest cells of the neural tube and migrate lateroventrally to the dermis, where they invade the epidermis at around weeks 10 of gestation. Epidermal melanocytes migrate into the follicles along the surface of basement membrane under the outer root sheath (ORS) during hair follicle morphogenesis between about weeks 11 and 12 of gestation. At the end of follicle morphogenesis, these melanocytes presenting in ORS of the middle and lower follicles stop to differentiate, and serve as a subpopulation of precursor melanocytes. These cells express premelanosomal antigens, recognized by NKI/beteb. They can not be indentified by antibodies to mature melanosomes or the functional proteins tyrosinase (TYR), tyrosinase related protein-1 (TRP-1), or tyrosinase related protein-2 (TRP-2). It has been demonstrated that theses cells are identifiable by toluidine blue staining but not by DOPA staining. They are named after amelanotic melanocytes (AMMC) because they cannot produce melanin. AMMC may provide a reservoir of melanocytes for repigmentation of the epidermis in vitiligo, burns, and dermabrasion, and for hair bulbs during early anagen, during hair regrowth in alopecia areata. Thus the study about the biology of AMMC can play an important role in treatment of depigmenting diseases.Repigmentation of vitiligo is usually seen in a perifollicular manner. During this process it is demonstrated that inactive AMMC become activated, proliferate, and migrate into depigmented epidermis. Little previous work has been performed to evaluate activation and migration of AMMC induced by usual drugs for treating vitiligo. The main obstacle to performing further investigation is lacking in availability of reliable methods of growing AMMC in cluture. Our studies consist of three parts. In part I, the methods of culturing AMMC were improved, and lots of AMMC with similar biological traits with these in vivo can easily be gotten. In part II, I observed the effect of activation of usual drugs for treating vitiligo on AMMC, next then the mechanism of activation were investigated in detail. In part III, the studies were focused on migration of AMMC induced by cytokines which showed apparent chemotaxis to epidermal melanocytes. Part I: Improvement of the methods for culturing AMMC derivedfrom ORS Tobin DJ et al first reported successful culture of AMMC in 1995.According to their method, the occurrence of fibroblasts contaminating the culture was permanent. It is difficult to obtain the purification and proliferation of AMMC. In addition, there were no detail illustration about the effect of components in cluture medium on the proliferation and morphology of AMMC in their study. Thus we investigated elimination of fibroblasts in cultures of hair follicle melanocytes from human scalp, and found out the effect of components in culture medium on AMMC. The method using collagenase V was employed to culture cells. Geneticin of different concentration was used to eliminate the contaminated fibroblasts. The transformation of cells were observed and the proliferation was tested after using different concentraton TPA and K-SFM including BPE. Our resluts showed the treatment with 100ug/ml geneticin for two days eliminated a majority of fibroblasts. Rate of AMMC was more than 90 per cent at 7 days after treatment. 50 ng/ml TPA stimulated proliferation of the cells continuously and had no difference from 100 ng/ml TPA (P>0.05) . 100 ng/ml TPA induced rapid dendrite formation. Proliferation resulting from K-SFM including BPE depended on the concentration of BPE. So we concluded treantment with lOOug/ml geneticin for two days can remove contaminated fibroblasts conveniently, and it can be used repeatedly. 50 ng/ml TPA is appropriate concentration and it can stimulate proliferation of cells, but can't cause transformation of AMMC. K-SFM including BPE stimulates theproliferation of AMMC in concentration-dependent manner.AMMC located in ORS keep in undifferentiared condition because of no melanogenesis...