| BackgroundThe physiological behaviors of epidermal cells play an important role in the re-epithelial and structural remodeling during skin wound healing. However, it is not very clear about the regulatory mechanism and influencing factors of the epidermal cell behaviors. Therefore, elucidation of the influencing factors and related molecular mechanism of the proliferation, differentiation, and cell division orientation of epidermal cell, could help for guiding the wound repair orderly, or providing a theoretical basis for forming the substitutes of skin tissue with normal structure and function, and achieving the perfect skin wound repair finally.Materials and methods1. Normal skin (n= 7), wound edge (n= 8) and scar (n=7) tissue samples were obtained from patients with surgical operations. The differences in morphology among the three tissues were observed through hematoxylin and eosin (HE), masson trichrome, and methenamine silver staining. In further, Immunofluorescence staining was used to detect the differences in the expression of components of basement membrane, Collagen Ⅳ, Laminin, Laminin-5 and its receptor, Integrin-β4, and detect the expression of CK10, CK14, CK5 and Integrin-β1 to identify the differences in differentiation lineages of epidermal cell among the three tissues.2. The model of epidermal cell differentiation was established by the Ca2+ stimulation in primary human epidermal keratinocyte (HEK) and human immortalized keratinocyte (HaCaT) in vitro. After using MaxGelTM ECM to mimic the epidermal basement membrane, the expression of Integrin-β4 was detected through Immunofluorescence staining and Western blot, and then the expression CK10, CK14 and CK19 was also detected through Real-time qPCR and Western blot to identify its effect on the proliferation and differentiation of HEK and HaCaT.3. The confocal laser scanning tomography combined with 3D structure reconstruction was used to detect the expression of PAR3, a cell polarity protein molecule, and then identify the cell division orientation by centrosome and spindle midbody (marked with Pericentrin and Survivin antibody respectively) to analyze the differences of epidermal cell division orientation among normal skin, wound edge and scar tissue.4. Immunofluorescence staining was used to detect the expression of PAR3 and Integrin-β4 in normal skin, wound edge and scar tissue to identify distribution relationship of them, and also detect the expression of a-SMA to identify the differences in distribution of human dermal fibroblast (HDF) among the three tissues. After in vitro using MaxGelTM ECM to mimic the epidermal basement membrane, and combining with the supernatant of cultured HDF, the spatial relationship of PAR3 and Integrin-β4 in HEK and HaCaT was identified through the confocal laser scanning tomography combined with 3D structure reconstruction, and the expression of PAR3 and Integrin-β4 was detected through Real-time qPCR and Western, which help to explore the molecular mechanism of epidermal cell division direction orientation. Moreover, Micro-contact printing combined with Time-lapse phase-contrast microscopy was used to observe and analyze the role of extracellular matrix (ECM) and HDF in regulating the mitotic cell division orientation of HEK.Results1. The tissue morphology of normal skin, wound edge and scar tissue was different during the skin wound healing. Compared to normal skin, the epidermis showed proliferative in wound edge. However, in scar tissue, the epidermis showed high differentiation, and the basement membrane was abnormal. These results indicated that basement membrane may regulate the differentiation lineages of epidermal cell, and then interfere with the outcome of skin wound healing.2. In vitro using MaxGelTM ECM to mimic the epidermal basement membrane could increase the expression of Integrin-β4 in HEK and HaCaT, and suppress the differentiation of HEK and HaCaT after the Ca2+ stimulation. These results illustrated that basement membrane could induce the adhesion of epidermal cells, and then regulate their behaviors of proliferation and differentiation.3. The expression of PAR3, a cell polarity protein molecule, and the epidermal cell division orientation were different among normal skin, wound edge and scar tissue. The epidermal cells showed oriented cell division in normal skin and wound edge, whereas disordered cell division orientation in scar. These results indicated that epidermal cell division orientation may affected by the epidermal cell-basement membrane adhesion, was closely related to the outcome of repair during skin wound healing.4. The expression of PAR3 and Integrin-β4 showed co-localization in normal skin, wound edge and scar tissue. In scar tissue, the expression of PAR3 and Integrin-β4 was abnormal, and the HDF marked with α-SMA in the basement membrane nearby were reduce, which indicated that the regional absence of HDF may be related with structural abnormality of basement membrane in early wound repair. In vitro using MaxGelTM ECM to mimic the epidermal basement membrane, and combining with the supernatant of cultured HDF also could affect the spatial distribution and expression of PAR3 and Integrin-β4 in HEK and HaCaT, which may refer to the ERK pathway. Besides, the supernatant of cultured HDF could regulate the cell division orientation of HEK by affecting the adhesion of ECM and HEK in vitro. These results illustrated basement membrane could induce the adhesion of epidermal cells, and then regulate the epidermal cell division orientation, which play an important role in skin wound healing.ConclusionDuring the skin wound repair, basement membrane could induce the adhesion of epidermal cells, affect the micro-environment of epidermal cells, regulate the epidermal cell behaviors, such as proliferation, differentiation and cell division orientation, and thus interfere with the re-epithelial and structural remodeling. The regional absence of HDF may be one of the cellular basis of abnormal formation of basement membrane. Therefore, exploring the factors that influence the formation of basement membrane, and theirs roles in regulating epidermal cell behaviors and related mechanisms, could provide a theoretical basis for achieving perfect skin wound healing. |
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