Study On Basic Mechanism(s) Of Development Of Sweat Glands And Induction Of Epidermal Stem Cells To Directly Differentiate Into Sweat Gland Cells

It is irrevocable that sweat glands do not regenerate to form their three-dimensional organization via the division and terminal differentiation of sweat gland cells when the entire epidermis is lost, such as in full-thickness burn wounds. Due to the lack of sweat glands in the healed scar, the function of body temperature regulation is impaired when the deep burn is very extensive, thus markedly affecting the patient s quality of life. With the aim to allay this common suffering after an extensive deep burn injury, it behooves us to study the problem of reconstruction of sweat glands during wound repair. With current progress in the study of tissue engineering, investigations have shown that it might be possible to find a way to induce the epidermal stem cells to directly differentiate into sweat gland cells through in-depth study of the developmental mechanism(s) of sweat glands. However, the morphogenesis of sweat glands is a very complex process, and the developmental mechanism(s) of sweat glands have not been completely clarified at present. The epidermal stem cells, histo-specific stem cells, are considered as bearing a close relationship for the development, repair and reconstruction of skin and skin appendages. Nevertheless, the exact pathway of transformation of the epidermal stem cells to sweat gland cells is uncertain. It is surprising that there has been few research work in this respect. With full cognizance of intricacies and difficulties in the solution of this problem, we studied the development of sweat glands in the fetus as the first step.There are at least two most important fundamental links between the organofaction and its regulation during embryogenesis. one is the activity of matrix metalloproteinases which are regulated by a series of growth factors, and another is the cellularmotion.Therefore, our objectives in this study were as follows: 1) to determine when epidermal stem cells first appear in the developing sweat gland promordias by immunocytochemisty in human fetal skin preparation: 2) to observe the distribution pattern of epidermal stem cells in relation to the development of sweat glands;3) to find out the correlation between the epidermal growth factor (EGF) and matrix metalloproteinases (MMPs) during the development of sweat glands in human fetal skin by observing the expression of EGF, MMP-2. MMP-7, laminin (LN) and fibronectin (FN) with immunohistochemical methods and in situ hybridization;and 4), to establish the culture method for epidermal stem cell in vitro. In view of the above mentioned objectives, we attempted to look for the experimental evidences pertaining to the induction of epidermal stem cells to directly differentiate into sweat gland cells.In addition. The healing of an adult skin wound is a complex affair of considerable clinical interest. The majority of investigations on cutaneous wound healing have concentrated their efforts on dermal repair and wound bed remodeling, with little emphasis was paid on epidermal regeneration. To elucidate the roles of epidermal stem cells in wound repair in vivo, we dynamically investigated the distribution of epidermal stem cells in rat full-thickness wound tissues during the wound healing process.The major research contents and conclusions were as follows: 1. Human skin specimens of embryos obtained from spontaneous abortions atgestational ages from 11 to 31 weeks were used in this study. P 1 integrin and keratin 19 (K19), which are the characteristic markers of epidermal stem cells, were employed to identify the epidermal stem cells, and the developing sweat gland buds or cells were identified by cytokeratin marker K7 or K8 with SP immunohistochemical methods. The developmental process of sweat gland was studied with routine histological methods of HE staining. Histologic studies revealed that at 16 weeks of gestation age. cells in primary epidermal ridge gathered together to take form of many hillocks, and from gestational weeks 18-20. the hillocks of cells that form buds in the embryonic epidermis migrated downward as cords into the dermis. The mature sweat glands were found in the epidermis of 24-week fetus. Immunohistochemical studies showed that P i integrin and keratin 19 immunoreactivity was respectively observed not only in developing sweat gland cords and buds but also in mature sweat gland cells. However, coexpression of P i integrin and keratin 19 was not present until 31 wk of gestational age in mature sweat gland cells. The cells with immunostaining property for K7 appeared in early sweat gland buds at 14-16 wk of gestation, and from then on, K7 was concentrated in developing sweat gland cords or cells. We postulate that the morphogenesis of sweat gland in human fetal skin begins at 14-16 wk of gestational age, and essentially completes by 24 wk. Epidermal stem cell is considered as the key source for the formation and proliferation of sweat glands in developing skin.2. The epidermal cells were dispersed from rat epidermis by agitation in the presence of trypsin. The cells were gently centrifuged and resuspended in the culture medium, which constituted EMEM without calcium, and 50% fibroblast conditioned medium (CM ). The CM was collected from freshly isolated primaryneonate fibroblast cultures after 48h. and frozen stored at -20°C until use. 1 X 106/ml epidermal cells were incubated on dishes coated with collagen IV, then the adherent cells were grown to confluency. Cultures were observed for colony formation under a phase contrast microscope, and the structure of the rapidly adherent cells were observed with electron microscopy;the expression of P i integrin and K19 was detected in the rapidly adherent cells and colony cells with SP immunohistochemical methods. The cells selected by rapid adherence to collagen type IV formed large colonies at 7 days, and showed an immature feature under electron microscopy by manifesting P i integrin and K.19 expression. On the basis as above results, we believe that the epidermal stem cells could be cultured in vitro.3. 80 annular full-thickness wounds were produced on either side of the paravertebral region in 20 Wistar rats labeled 60 days previously with BrdU ( 4 wounds in each animal). The 80 wounds were randomly divided into 2 groups as follows: group A: with topical treatment of sulfadiazine silver- sulfadiazine zine cream (Ag-Zn cream) (n=40) and group B: no-treatment (n=40). BrdU, P i integrin and keratin 19 (K19) were employed to determine the epidermal stem cells with SP immunohistochemical methods, and the speed and quality of epithelialization were determined with routine histological methods of HE staining on the 3rd, 7th, 14th, and 21st days after operation. It was shown that no cells with positive immunostaining for P i integrin, K19 and BrdU was found in granulation tissues of any wound of both groups during the healing process. However, a few P i integrin and K19 positive cells, bearing no anatomic relation with the epidermal stem cells in the basal layer, were found scattering within the stratum spinosum and stratum granulosum of the epidermis on the wound edgeson the 3rd day post-injury. From then on. these positive cells gradually became more and more in number, and were mostly concentrated on the border of wound edges till the wounds healed up. In addition, the number of cells positively stained for (3 , integrin and kl9 on wound edges in the infected wounds was less than that in non- infected wounds. These cells which were positive for P i integrin and kl9 staining on the wound edge were also immunohistochemically stained with BrdU in the cellular nuclei. Based on above results, we propose that epidermal stem cells play a major role during wound epithelialization. and Ag-Zn sulfadiazine cream is an important agent to prevent local infection, and also is capable of stimulating the fast- wound epithelialization.