| Skin wounds can happen easily in diabetic patients and their healing process takes much longer time than in non-DM (diabetic mellitus, DM) population and may even never heal completely.Currently, it is still a big clinical challenge to treat chronic wounds in diabetic patients. Domestic and international studies suggested that impaired wound healing in DM were due to glycometabolic disorders and advanced glycation end products increase, vasculopathy and endothelial cell abnormalities, neuropathy, growth factor receptor and other changes, abnormal expression of matrix metalloproteinases, epidermal cell proliferation barriers and other factors. Therapeutic strategies derived from these targets achieved some results, but still not satisfactory. Recent studies have confirmed that epidermal stem cells (Epidermal Stem Cells, ESCs) as the specific stem cells in skin tissues, are considered as the key source of the occurrence, repairing, rebuilding of skin and its appendages, and play an important role in regeneration and wound healing of normal skin; the amount and activity of epidermal stem cells in diabetic wounds significantly decreased, there is the local specific "stem cell bank dried up" phenomenon. Thus suggest that characteristics of proliferation and differentiation in epidermal stem cell may be one of the important reasons in diabetic impaired wound healing.β-catenin is a cytoskeletal protein that is coded by the gene CTNNB1 located on chromosome 3p21. As an important differentiation regulator of epithelial cell, it maintains the structural integrity of epithelial tissue,mainly through the epithelial cadherin binding, involved in cell-cell adhesion and connection of cell to extracellular matrix. At the same time it is a critical part of the signal transduction, and plays a pivotal role in the Wnt signaling pathway which regulate the growth, development and differentiation of cell. When levels ofβ-catenin increased, Wnt signal pathway was activated, then activation of the downstream target gene cyclin D1, etc.,which promoted cell proliferation and differentiation. Research has confirmed thatβ-catenin can promote the proliferation and differentiation of intestinal epithelial stem cells and involve in repairing intestinal mucosa after injury. It indicates thatβ-catenin is closely related to proliferation and differentiation of adult stem cell. High expression of Wnt andβ-catenin enhanced proliferation, differentiation and migration of epidermal cell,and accelerated wound healing. However, expression ofβ-catenin and CyclinDl in epidermal stem cells and their role in diabetic skin wound repair is not clear.In this study, we investigate localization of epidermal stem cells and the expression ofβ-catenin and cyclinDl between diabetic rats model and normal rats skin; in vitro, study gene and protein expression ofβ-catenin and cyclinD1 in epidermal stem cells of diabetic rat skin; explore the effects and expression ofβ-catenin and cyclin Dl after transplanting the epidermal stem cell on diabetic wound healing, it will provide experimental basis and theoretical foundation in its further application to diatetic wound healing.Part I Localization of epidermal stem cell and and expression ofβ-catenin and cyclinDl in diabetic rats skinOBJECTIVE:To study localization of epidermal stem cell and expression ofβ-catenin, cyclinDl and its related proteins in diabetic rats skin, and their role in diabetic impaired wound healing.METHODS:Twenty SD rats were divided into DM group and control group randomly.The DM rats were induced by intraperitoneal injected 65 mg/kg streptozocin (STZ),4 weeks after injection, pancreatic tissue were taken for HE staining in two groups, then full-thickness skins were taken from the back of diabetic rats, and normal skin samples taken as controls, they were obtained for hematoxylin and eosin (HE) staining and immunohistochemical staining ofβ-catenin, cyclinDl, Wnt1, proliferating cell nuclear antigen (PCNA),keratinl9 (K19) andβ1 integrin. Then integral absorbance(IA) of positive cells of basal layer were measured with image analysis software.RESULTS:DM rats were monitored for 4 weeks.their blood glucose were more than 16.7 mmol/L, and little fluctuations in blood glucose. The achievement ratio of diabetic rat model was 90% and the models have good stability. HE staining analysis showed the amount of pancreas islet cells in DM group significantly reduced and appeared necrotic,but in control group islet cells have intact structure and no necrosis. Expression of K19 and (31-integrin,markers of epidermal stem cells, were visible in the basal layer of two groups, and positive cells ofβ-catenin, cyclinDl, Wntl, PCNA concentrated in the basal layer. The expression ofβ-catenin, cyclinD1, Wntl, PCNA,K19 andβ1 integrin was higher in DM skin than in normal skin (P<0. 01).There was significant difference in the epidermal thickness and integral absorbance of the positive cells ofβ-catenin, cyclinD1, Wntl, PCNA, K19 andβ1 integrin between DM rats and normal rats.CONCLUSION:Epidermal stem cells were mainly located in the basal layer of diabetic rat skin, expression region and signal intensity ofβ-catenin and cyclinDl is consistent with localization and positive expression of epidermal stem cells. It suggests that the reduced amount of epidermal stem cells and the decreased expression of (3-catenin, cyclinDl in diabetic rats skin may be closely related to the low repair capacity of the diabetic wound skin.PartⅡIn vitro characterization ofβ-catenin and cyclinDl in epidermal stem cells of diabetic ratsOBJECTIVE:In vitro, to explore characterization ofβ-catenin and cyclinDl and its related proteins in epidermal stem cells of diabetic rats, then to study the potential mechanism of difficult recovering wounds in diabetic skin.METHODS:Forty SD rats were divided into DM group and normal control group randomly (n= 20).The DM rats were induced by intraperitoneal injected 65 mg/kg streptozocin (STZ), The normal control group without treatment. after modeling of 4 weeks, full-thickness skins were taken from the back of two group rats for isolation,cultivation and identification of its epidermal stem cells, the cell cycle were measured by flow cytometry. Cell colony formation rate of the two groups were detected after 1 week. Meanwhile, they were obtained for immunocytochemical staining of K19,β1 integrin,β-catenin, cyclinDl, Wnt1 and PCNA. mRNA and protein expression ofβ-catenin, cyclinDl and Wnt1 were detected by RT-PCR and Western blot.RESULTS:Under the inverted phase contrast microscope, the amount of epidermal stem cells in DM group significantly reduced, and cell growth is slower, compared with control group. The clone forming efficiency of ESCs in DM group (6.43%,12.87±2.03/200%) was significantly lower than that of ESCs in the controls(11.37%,22.75±3.24/200%)(P<0.01). Flow cytometry indicated that 3.98% apoptotic cells and 88.89% cultured ESCs of the DM group were in resting state/pre-DNA-synthetic gap(G0/G1), as the controls were 91.50% and no apoptotic cells. Integral absorbance of positive expression of K19,β1 integrin, (3-catenin, cyclinD1, Wntl and PCNA in ESCs of DM skin were respectively 82.63±14.77, 21.59±4.71,76.49±6.58,69.35±5.82,32.57±6.13,90.77±12.44 than in that of normal skin, these in ESCs of the controls were respectively 151.24±42.83,54.48±17.43, 116.39±9.26,105.47±7.52,71.29±8.25,110.62±20.67), it showed significant difference in two groups(P<0.01). the mRNA expression level ofβ-catenin[(0.556±0.017),(0.850±0.059),P<0.01] and cyclin D1[(0.682±0.023), (0.887±0.038),P<0.01] were lower in DM group than in control group. Analysis of western blotting revealed thatβ-catenin and cyclin D1 were expressed in both groups, whereas the DM group showed weaker expression ofβ-catenin[(0.583±0.054) (1.128±0.077),P<0.01] and cyclinD1 [(0.843±0.037),(1.307±0.072), P<0.01] in comparison with control group.CONCLUSION:1. In vitro epidermal stem cells of the two groups showed clonal growth, high proportion of G0/G1 phase cells, the positive expression ofβ-integrin and K19. It indicated epidermal stem cells isolated and cultured in this experiment conditions have the biological characteristics of stem cells. However, ESCs in DM group showed slow growth rate and lower colony formation ability.2. mRNA and protein expression of (3-catenin and cyclinDl were significantly reduced in epidermal stem cells of diabetic rat skin. the less expression ofβ-catenin and CyclinD1 which causes the decreased amount and the reduced proliferation and differentiation capacity of ESCs may be one of the important mechanisms in diabetic impaired wound healing. PartⅢExpression ofβ-catenin and cyclinDl and its effct on epidermal stem cell transplanting in diabetic wound healingOBJECTIVE:To investigate expression changes of P-catenin and cyclin Dl and its effct on epidermal stem cell transplanting to wound healing of diabetic rats, and provide a new idea for clinical treatment of difficult healing wound in diabetic skin.METHODS:Twenty male SD rats were randomly selected to establish diabetic model. Ten male SD rats were selected to prepare epidermal stem cells.ESCs of SD rats were isolated, cultured, identified and labled with 5-bromo-2'-deoxyuridine (BrdU) in vitro. The wound model of diabetic SD rats were established,then divided into A,B,C group. Human amniotic membrane (HAM) loading labled BrdU ESCs were implanted to A group, Human amniotic membrane (HAM) were implanted to B group, but control group not. At 7,14 days after ESCs transplantation, general situation of wound healing, the healing rate of wound, hematoxylin and eosin(HE) staining, immunohistochemical staining of Brdu, K19,β1-integrin,β-catenin, cyclinD1,Wntl and PCNA in wounds of every group were investigated. Integral absorbance of positive cells were measured with image analysis software.RESULTS:Compared with the other two groups, collagen under the newborn epidermis evenly distributed, arranged in neat in epidermal stem cell trratment group(A), the wound healing rate of A group was significantly higher at different observation times (P<0.01). BrdU-positive cells in the wounds and newborn epidermis of A group were visible, while wound tissue of the other two groups had no BrdU-positive cells. K19.β1-integrin,β-catenin, cyclinDl,Wntl and PCNA-positive cells in wound tissue of each group could be seen, but integral absorbance of positive cells were significantly different in A group compared to that in B, C group (P<0. 01).CONCLUSION:Epidermal stem cells may accelerate wound healing of diabetic skin. And expression of K19,β1-integrin,β-catenin and CyclinDl were higher in the basal layer of newborn skin of epidermal stem cell treatment group.It suggested expression activity changes ofβ-catenin and CyclinDl may be one of the important mechanism in epidermal stem cells promoting diabetic wound healing. |
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