| Objective: For diabetic patients with the skin vulnerable to injury, there are delayed wound healing after injury or non-healing. In diabetic patients, the incidence of foot ulcers is among 2%, and 15% of them will suffer from this complication in the whole life. With poor prognosis of diabetic ulcers, its 3-year survival rate is at 50%. Past studies suggest that diabetic patients with impaired wound healing factors mainly concentrated in the following aspects:①glucose metabolism disorders and advanced glycation end products increase;②microvascular changes;③matrix metalloproteinases (matrix metalloproteinase, MMPs) in patients with diabetes-induced Abnormal expression of pre-and post-injury;④epidermal growth factor receptor changes;⑤obstacles with epidermal cell proliferation. At present, the mechanism based on the above for the treatment of diabetic ulcer drugs are a failure, only recombinant PDGF (recombinant platelet-derived growth factor) show that there is some effect. Even though the wound healing process of any one aspect of poor coordination or dysfunction may also lead to loss of control wound healing. At present, however, about the mechanism for impaired wound healing in diabetes has yet to clarify. Wnt signal transduction pathway is a key way for regulating cell growth, development and differentiation. The stability ofβ-catenin, the key factor of this signal pathway, and its location of cells, are regulated by a series of positive and negative factors which may lead to abnormal Wnt signaling pathway inhibition or abnormal activation, thus resulting in gene expression, cell adhesion, a series of developmental abnormalities and cancer metastasis. There are complex crosstalk systems among Wnt signaling pathway, PGE2/EP2 signal, EGF/EGFR/PI-3K/AKT and E-cadherin E-cadherin signal-regulated channels. The latest study found that, Wnt andβ-catenin expression can promote epidermal cell proliferation, differentiation and migration capacity, and speed up wound healing; In skin keratinocytes, Wnt signaling pathway affect the thickness of post-traumatic and skin pigmentation; Wnt signaling pathways involved in protection of high glucose caused endothelial cell damage; Abnormal regulation ofβ-catenin can promote or inhibit apoptosis; Chun-LiangLin such reports, Wnt /β-catenin signaling can enhance the viability of mesangial cells caused by high glucose ; High glucose inhibit endothelial cell migration and proliferation and vascular changes through the PI3K-Akt signaling pathways, but also can lead to the upward of adhesion molecule E-cadherin; Our preliminary study suggests that in endothelial progenitor cells, the expression ofβ-catenin, EGFR and cyclin D1 are reduced by high glucose treatment. From these indirect sources reflect the impaired wound healing in diabetes with the Wnt /β-catenin signaling pathway-related disorders. Therefore, we designed this experiment, to prepare for this preliminary study. Methods : 1. Refractory diabetic wound model in rats:①1 diabetes rat model set up: select the range of 3-month-old weighing 201 ~ 240g of clean grade SD male rats, after 12h fasting 50mg/kgSTZ (streptozotocin) intraperitoneal injection of liquid disposable, 1,3,14d after injection of glucose to the animals was determined. 3d after injection of animal blood glucose concentration> 16.65 mmol / L is determined for type 1 diabetes animal model.②the production of rat back skin defect: check the above-mentioned artificial reproduction of 60 diabetic rats and normal rats 20 to 3% pentobarbital sodium (25 mg / kg) intraperitoneal injection of anesthesia, back shaving, conventional disinfection. In sterile conditions with a special hole puncher in rat dorsal spine on both sides of the central hole of the one hit deep to the subcutaneous, the diameter of 1.8cm, the wound area of 2.54 cm2. Sterile gauze to stop bleeding after dressing, the dressing was changed 1 times / day, single-cage feeding, quantitative drinking water and feeding. All experimental animals are completed in a clean laboratory; 2. Deal with the control group and experimental groups: the experiment is divided into normal group, diabetes group, diabetes + lithium chloride group, diabetes + EGF group, a total of four groups, each of the 20 rats,①normal group and diabetic group, normal feeding, records of daily water intake, food intake, urine output and body weight change;②diabetes + lithium chloride group, with the exception Ibid treatment, the diabetic rats from the back skin defect model of success given day 1/20LD50 chloride Lithium (gavage administration, LD50: 526mg/kg), 1 times / d;③diabetes + EGF group, taken with sterile syringe dropwise at rhEGF 10ug on sterile gauze and cover the wound, Vaseline gauze, sterile gauze and bandage fixed daily by the same way. The dressing was changed everyday;④injury 3d, 7d wound bacteriology Detect Change in each group (per square cm of wound colonies of the common logarithm) in order to rule out the possibility of bacterial factors on delayed wound repair role ; 3. Observation of the back wound healing situation:①wound morphology description: moisture, color, secretions, shape, fate and 50% of invasive wound healing time;②wound area: immediately after injury and 3,7,14 d with hyaline membrane coverage of the wound along the creators draw fate film, cut film, home dividing the value of 0.1mg analytical balance weighing converted into space, and calculate the rate of wound healing;③injury cavity volume: immediately after injury and 3,7 d with 1 ml syringe intraluminal infusion of normal saline to the injury to the skin surface with a fate similar level until the volume injected record;④histopathological examination, 14 d after injury, wound tissue from HE staining to fibroblast proliferation, capillary proliferation, Epidermal hyperplasia was observed as indicators; 4. Detection ofβ-catenin expression (how strong or weak, the distribution of the region): the use of SP immunohistochemical method. Criteria: l0×20 times at under a microscope to observe a random 10 field of vision, each vision count 100 cells, immunohistochemical positive signals for the brown-yellow granular, observe signal-positive staining intensity, location and calculate the number of positive cells. Maruyama, etc. in accordance with the method, observation ofβ-catenin at the cell membrane, cytoplasm, nucleus of three parts to determine the expression of strength, the membrane> 70% positive for the normal expression, contrary to express missing, cytoplasm or nucleus> 10% positive for ectopic expression, regarded as a normal cell membrane expression ofβ-catenin-negative, membrane expression deletion or ectopic expression of β-catenin seen as positive. Results: 1. An animal model of stability, blood glucose levels in diabetic rats monitoring showed that blood glucose levels in rats has been maintained at 21.23-24.60mmol / L, before and after injury at different times of diabetes among the three groups no significant difference with the model production standards; 2. normal rats daily water intake, food intake and urine output were less than that in three groups of diabetic rats, whereas, the weight gain changes were more obvious. The difference has statistical significance; diabetic rats in each group no statistical difference; 3. after injury in rats to observe the wound and no signs of infection, injury 3d, 7d wound bacteriology Detect Change in each group, no significant difference, exclusion of bacterial factors on delayed wound healing effect; 4. The normal group, diabetes + lithium chloride group and diabetes + EGF group, rats wound dry, ruddy, secretions less invasive fate irregular shape, shrink significantly, 50 percent of normal rats wound healing time for the 5.5d, diabetes + lithium chloride group 5.9d, diabetes + EGF group was 5.7d, the three groups no significant differences, in diabetic group was significantly shorter than those (P <0.05), to 14 days after injury, the three groups the majority of wound have completely healed; diabetic rats wound moist, wine, there is some secretion, oval shape for many, depression wound, skin contraction was not obvious, as time after injury, the wound area of a slight narrowing, but the 50 % wound healing time in about nine days after injury to 14 days after injury, there are still unhealed wound 6; 5. injury after 3,7,14 d, the normal group, diabetes + lithium chloride group and diabetes + EGF group , the wound healing rate was no significant difference, but the diabetic group than those in the high, the difference has statistical significance; injury after 1d, four groups of wound healing rate was no significant difference; 6. injury after 3,7 d, the normal group, Diabetes + lithium chloride group and diabetes + EGF group, the wound cavity volume was no significant difference narrowed, but the diabetic group were significantly narrowed (P <0.05); injury after 1d, four groups wound cavity volume change was no significant difference; 7. 14 d after injury, wound tissue from HE staining, normal group, diabetes + lithium chloride group and diabetes + EGF group, epidermal hyperplasia and wound coverage, maturing granulation tissue, fibroblasts are abundant, dense collagen, with neatly wound capillaries with vertical, inter-group no significant difference; diabetic group to varying degrees of epidermal hyperplasia, the majority did not fully cover the wound, granulation tissue osteoporosis, newborn subepidermal capillaries more, a small amount of inflammatory cell infiltration, and on three groups obvious differences; 8. injury after3,7,14d, the normal group, diabetes + lithium chloride group and diabetes + EGF group,β-catenin membrane staining relative decrease in thickness ranging from emerging cytoplasm, varying the number of positive granules in brown shading,β-catenin positive cell rates among the three groups no significant difference, but with the diabetic group compared to the high rate of positive cells, the difference significant. In wounds of diabetic rats,β-catenin was continuous thread-like brown staining around the cell membrane with positive cells less obvious. Conclusion: 1. In diabetic rats wound,β-catenin-positive cells are lower than those in normal rats wound, suggesting that wound healing in diabetic rats at the process of Wnt / beta-catenin signaling pathway function is relatively low, the existence of Wnt / beta-catenin signal pathway dysfunction; 2. In the Wnt / beta-catenin signaling pathway to promote the role of agents, the rate ofβ-catenin-positive cells of diabetic rats wound increased significantly, the wounds of diabetic rats improved, obviously exhibited in clean wound, rich granulation tissue, wound healing rate and healing time, thus, to provide a new way in theory for the treatment of diabetic wounds; 3.EGF can promote wound healing in diabetic, Wnt / beta-catenin signaling pathway may be involved in. |
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