| As to research of wound healing is both original and classic topic. Cutaneous wound healing is a very complex biological process involving both keratinocyte migration and proliferation from the wound edge under the influence and direction of growth factors. During the process, keratinocyte migration and proliferation will depend on their interaction with dermal fibroblastes and with the extracellular matrix and on a variety of growth factors and cytokines present in wound fluid. It is known that wound repair comprises 4 stages during the process, i.e. granulation tissue formation, re-epithelialization, cicatrix formation and re-modeling. The majority of early investigations on cutaneous wound healing have focused on dermal repair and wound bed remodeling, with little emphasis on epidermal regeneration. With the current progress of study on various kinds of stem cells, it has been shown that epidermal stem cells (ESCs), a specific stem cell of skin tissue, play an important role not only in self-renewing of epidermis but also in wound closure. However, detailed knowledge is not available to assess the role of ESCs in wound healing at present. Thus, one of the main questions still to be answered is how the ESCs are stimulated to migrate into a specific direction and how the reorganization of the cell network necessary for the motion is induced. Our recent in vivo study showed that during wound healing process there was relation between ectopia of ESCs situated in the basement membrane of the wound edge and wound re-epithelialization, but little is known about the exact mechanism. In this study, we explored the feature of proliferation and differentiation of cultured ESCs that treated with wound harvested effusion (WHE) of full-thickness wound in vitro, and preliminarily investigated the molecule mechanism of proliferation anf differentiation of cultured ESCs in wound fluid, in order to provid experimental grounds for in-depth studies on the relationship between the ESCs and wound healing.The major research contents and conclusions were as follows:1. Wound fluid was harvested from the 80 full-thickness wounds produced on both sides of the back in 40 adult Wistar rats. ESCs were isolated, purified from neonatal Wistar rats by referring to the formerly records and binding our ideas. When the cultured cells showed up clone growing, they were influenced with the wound fluid. Then,β1 integrin, keratin19 (K19), keratin14(K14) and keratin10(K10) were employed to determine the ESCs differentiation status with immunohistochemistry staining methods and flow cytometry technology at 0, 6 th,12 nd, 18 th,24 th, 30 th,36 th,42 nd,48 th, 54 th,60 th,72nd hour post- influence. During the treating process with WHE, it was found that the cultured ESCs can still keep on the growth state of like-clone. There are a number of cell populations with positive immunostaining for K14 to be found, and in addition, these positive cells populations gradually increased in number as treating time goes by. However, a few positive of K10 stained cells were found scattering within the K14 positive cell populations. These findings suggest that WHE can directly induct ESCs to become committed to the differentiation pathway during which the differentiated cells'phenotype displays the feature of transient amplifying cells.2. To observe the effect of WHE on intracellular free Ca2 + of ESCs in vitro, and investigate the relationship between mitogen-activated protein kinases(MAPKs) signal pathways and Ca2 + mobilization, the ESCs, showed up clone growing, were divided into five groups as follows: group A: control group (no-treatment) ; group B: only treatment with WHE; group C: treatment with WHE and PD98059; group D: treatment with WHE and SB203580; group E: treatment with WHE, PD98059 and SB203580. Then, the cells were incubated with fluorescence Ca2 + dye fluo-3/AM at 37℃for 30min, and measured by using laser scanning confocal microscope. The results showed that the fluorescent intensity of group B was higher than that of group A. There was a phenomenon of calcium oscillation to be found in group C and group D. Furthermore, a rapid decrease of fluorescent intensity was observed in the cells that were preincubated with PD98059 and SB203580 at the same time. Based on above results, we propose that WHE can directly induce an increase of cytoplasmic and intracellular free Ca2 + concentrations of ESCs. MAPKs signaling pathway has an important function of feedback regulation for free Ca2 + mobilization of ESCs, and also is capable of affecting the biological behaviour of epidermal stem cells.
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