| ObjectiveThe diabetes mellitus (DM) is now a common illness and frequently occurring illness which harmed human health seriously. There are about 2% to 5% people of the world having this kind of disease. In 2000, the patients' number of our country had reached 3000 million, which showed this tendency increased year by year. So the prevention and cure of DM had become the important item of national tackle key problems in science and technology. Some reports said that the local application of insulin (INS) could promote the healing of the dermal ulcer in DM patients or the difficult healing wounds after surgery, but the specific mechanisms weren't clear yet. This study first constructed wound's difficult healing model of DM rats, and observed the dynamic change regularity of epidermal stem cells (ESCs) in different time points, then locally applied INS on wounds, and observed the change of ESCs and wound's healing rate in a certain time point, and furtherly explored the relationship and mechanism between the two, which supplied scientific base for the clinical study and curation of DM wound.DM patients easily complicated dermal ulcer or difficult healing of wound, which usually became one of the important reasons of causing death or multilation of DM patients. The existed studies considered that the mechanisms of diabetic wound's difficult healing mainly focused on the following aspects: First, glycometabolism disorder and the accumulation of its advanced glycation end products; Second, microvascular change; Third, the abnormal expression of matrix metalloproteinase; Fourth, the change of growth factor receptors; Fifth, the proliferated hindrance of epidermic cell. In the course of wound's healing, these factors didn't happen lonely, but with a style of high coordination and mutual effect. However, the ESCs are stem cells of tissue specificity, which have strong reproductive activity, and can undertake self-renewal to maintain the quantitative balance of ESCs as well as differentiate into epidermic cells of all phases to maintain skin metabolism. Especially when the skin developed trauma, ESCs were the key seed cells of skin reconstitution. Then, was the change of ESCs after trauma in DM state different from that in normal state? In addition, there were some experiences of local application INS solution for promoting wound's healing, and if it played a part through starting ESCs was still remaining further study.This experiment applied the methods of HE staining, immunohistochemistry staining and western blot to research the epidermis width of DM rats in different periods after trauma, the change of ESCs' quantity and distriuting character as well as wound's healing rate, then approached the relationship between ESCs and diabetic wound's hard healing. On this basement, further studied the location and quantity of ESCs and the change of wound's healing rate after local application of INS on DM wounds, then explored the correlationship between them, and furtherly identified if INS exerted its effect of promoting diabetic wound healing, and to observe and determine the location and quantity of ESCs, then analyzed that if. there had correlate relationship between them, and further identified. the mechanism of diabetic wound's difficult healing and the effect of ESCs. Besides, we carried out the isolation and purification of Wistar rats' islets, which would supply theoretical and experimental basement for the genic reconstruction of ESCs or the co-cultivation of ESCs and frame which aimed at promoting the diabetic wound's healing.Materials and methods1,Model building68 male Wistar rats weighing 180～200g were divided randomly into model group (n=44) and normal group(n=24). The former applied intraperioneal injection of streptozotocin (STZ, 65mg/kg) to make diabetic model. After 3 weeks of model building, all rats were created a pair of 2.54 cm~2 area full-thickness skin defects on the back, which were treated as wound hard healing model. Then 24 rats were picked randomly from model group conducted as DM group, while the healthy rats (n=24) in normal group were treated as contrast, and the influence of DM on ESCs after skin's trauma was observed. Then 12 rats were picked randomly from model group and were divided into control group (n=6) and experimental group (n=6), and the influence of local application of INS on ESCs of DM wounds was observed.2,Observing the influence of DM on ESCs of skin wounds after traumaAll wounds of rats in DM group and normal group were stopped bleeding and wrapped by sterile gauze, then the wounds were cleaned every day and gauzes were changed. All rats raised at the temperature of 18～25℃, humidity of 45%～65%, freedom hydroposia and foodintake. At the 3rd, 7th, 14th and 21st day after trauma, the information of wounds' healing and infection were observed, and the wounds' sizes were shoted by digital camera, and wounds' healing rates were calculated. At the same time, 6 rats in each group were sacrificed at each time point, and the granulation tissues of wound's edge were obtained and fixed by 4% paraform for normal paraffin imbedding and sectioning. The successive 3 sections were treated as a group, and were numbered as A, B and C. The group A was made HE staining to observe the histological change and measure the epidermis width. The groups B and C were made immunohistochemistry staining of K19 andβ1 integrin respectively to observe the change of ESCs' quantity and location.3,Observing the local application of INS on ESCs of DM woundsFrom the first day of trauma, the wounds of experimental group rats were given insulin treatment (8U for every rat), and other two groups' rats were given Sodium Chloride of same doses, once every other day. Before the first treatment and 1h, 2h, 3h, 4h, 6h, 12h as well as 24h after first treatment, the blood glucose concentration of all rats were measured. At the fourteenth day of wound creating, the wound areas were measured, and wound healing rates were calculated. At the same time, wound margin tissues were obtained, and partly of them were fixed with 4% polyoxymethylene for HE staining and immunohistochemistry staining of K19 andβ1 integrin, and others were cryopreserved at-70℃for detecting the relative amount of K19 andβ1 integrin.4,The isolation and purification of Wistar rats' islet cellsThe islets were digested and isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation. Then the purified islets were subjected to histological staining, electron microscopy and radioimmunoassay for identification of specificity and viability.5,Statistical analyzeAll the data were indicated by (?)±s, and SPSS 13.0 software was used for statistic analyze.Results1,The identification of modelAfter 72h of STZ injection, the random blood sugar and body weight as well as the quantity of drinking water were measured. The vena caudalis blood was taken for measuring blood sugar, and if the value was exceeding for 16.8mmol/L for successive 3 times, and typical symptoms of hyperdiuresis, polydipsia and polyphagia were accompanied, the DM rats could be definited. As a result, 39 rats were sured as DM models.2,The influence of DM on ESCs of skin wounds(1) The macro and micro observing results of wounds' healingThe wounds of rats in normal group were dry and ruddy, and had less excretion. The shape of wound's edge was abnormal and had obvious contraction. The time of wound's healing for 50% was the 7th day. At the 21st day after trauma, the average of wounds' healing rates was 99.4%. But the wounds of DM rats were wet and red. The shapes were oval mostly and the constraction of skin wasn't obvious. The time of wounds' healing for 50% was delayed, and between the 7th day to the 14th day. At the 21st day after trauma, the average healing rate was 85.4%. By HE staining, we could see that the change regularities of epidermal width in normal group and DM group were the same, both of them were thickening first and thinning after. But the peak of rats' skin in normal group occurred at the 7th day, while that in DM group occurred at the 14th day. The epidermis of the former had complete layers and the arrangement was in order. However, the epidermal layer of the latter had some absence and the thickness wasn't homogeneous, and the cells' morphous wasn't coincident or the arrangement was indiscriminate, and also accompanied with inflammatory cell infiltrating of different degree. By immunohistochemistry staining, we could see that the location of ESCs in experimental group changed after trauma, which migrated from basal layer to prickle cell layer and surface layer, even the whole cuticular layer. The positive cells in wound margin were more than that in other places. But the quantity and staining intensity of ESCs in control group were less than that in experimental group.(5) The detecting results of Western blotThe gray scale ratios of K19 andβ1 integrin in experimental group were higher than that in control group, which demonstrated that the expression quantities of the two proteins in experimental group were higher than that in the control group.4,The result of the isolation, purification of Wistar rat's isletsThe histological staining revealed the viability and purity of purified islets were above 95% and 85% respectively. Electron microscope showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules. Radioimmunoassay demonstrated the secreted insulin concerntration between low-glucose groups and high-glucose groups varied significantly, which justified the islets with good function.Conclusions1,The location and quantity of ESCs of DM rats' skin changed before and after trauma. The quantity of ESCs was few and the activity of ESCs was low, which might be the important reason for the delayed healing of DM wounds.2,After the application of INS, the ESCs in DM rats increased, which promoted the wound's healing of DM rats.3,The method of Hepatopancreatic duct perfusing with collagenase is a good method for obtaining more islet cells.
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