Observation of HaCaT cells and MV3 human melanoma cells under inverted microscope The HaCaT cells were in triangular or polygonal, and the cells were arranged tightly in cobblestone and connected into pieces with higher nucleus; the cytoplasm contained many particles, which similar with keratinocytes. There was a small amount of black dot cells with a small amount of low activity of the black dot cell, surrounded aperture shadow and surrounded with aperture shadow (Figure 1A). The MV3 human melanoma cells were in long spindle and polygonal and irregular in size and shape; the MVs human melanoma cells were large in cell body with abundant cytoplasm, prominent nucleoli and common karyokinesis (Figure 1B). "

  Histological observation with hematoxylin-eosin staining In the de-epidermized dermis without inoculation of HaCaT cells and MV3 human melanoma cells, there was no cell growth in the lumen (Figure 2A). Observation of hematoxylin-eosin stained sections with the ratio of MV3:HaCaT=1:10 showed the thin epidermis, the spinous layer cells were arranged like paving, the keratinocytes in some areas showed significant atypia, and showed pathological mitosis figures (Figure 2B). The observation of hematoxylin-eosin stained sections with the ratio of MV3:HaCaT=1:3 showed there was cell apoptosis and pathological mitosis figures in the superficial epidermis, the keratinocytes in some areas showed poloidal disorder with mild-moderate atypia, and the atypia was more significant in superficial epidermis (Figure 2C). Observation of hematoxylin-eosin stained sections with the ratio of MV3:HaCaT=1:1 showed there was no parakeratosis and hyperkeratosis in epidermis, cell apoptosis and pathological mitosis figures could be observed in the superficial epidermis, the keratinocytes in some areas showed poloidal disorder with mild-moderate atypia, and the atypia was more significant in superficial epidermis (Figure 2D)."

  Immunohistochemical staining results Negative staining of cytokeratin 10 and cytokeratin-pan in the surface of de-epidermized dermis of the control group indicated that there were no expressions of cytokeratin 10 and cytokeratin-pan (Figures 3A and 4A). Immunohistological staining of cytokeratin 10 in the surface of de-epidermized dermis inoculated with two kinds of cells showed brownish yellow, which indicated that there was expression of cytokeratin 10, and suggested that HaCaT cells could differentiate into epidermal cells when growing on the surface of de-epidermized dermis (Figures 3B-3D). Immunohistological staining of cytokeratin monoclonal antibody showed the superficial cells were stained into brownish yellow completely, indicating there was expression of cytokeratin-pan (Figures 4B-4D). When the ratio of MV3:HaCaT=1:10, the positive cells of cytokeratin 10 and cytokeratin-pan were observed on the superficial layer, and distributed layer-by-layer. With the increasing of the ration of MV3:HaCaT, the positive cells of cytokeratin 10 and cytokeratin-pan were downward from the superficial layer to the middle and lower layer, and changed from layer-by-layer distribution to mass distribution. When the ratio of MV3:HaCaT=1:1, the positive cells of cytokeratin 10 and cytokeratin-pan were closely attached on the basement membrane of de-epidermized dermis.    The staining of S-100 on the de-epidermized dermis of the control group was negative (Figure 5A). The protein staining of mixed inoculated S-100 specimen was positive (Figures 5B-5D). When the ratio of MV3:HaCaT=1:10, the number of MV3 melanoma cells was decreased and only formed 2-3 layer and all located on the surface of de-epidermized dermis; there was no significant positive stained cells in the de-epidermized dermis. With the increasing of the ratio of MV3:HaCaT, the number of MV3 melanoma cells was increased. When the ratio of MV3:HaCaT=1:1, the number of MV3 melanoma cells was increased, and the MV3 melanoma cells could be observed in the de-epidermized dermis."

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