Study On The Repair Of Chronic Skin Ulcer Wound By

Purpose:To observe the effects of different factors on chronic skin ulcer model in mice, explore the compound factors modeling methods which accord with the characteristics of “yin syndrome” skin ulcers model in mice。Through experimental research to explore how to be close to the clinical symptoms of chronic skin ulcer models, and TCM syndrome types corresponding to the animal models is very necessary, also provide conditions for further experimental study. This experiment by comparing three kinds of ulceration, trying to provide an ideal for clinical and experimental research of small animal models.Chronic skin ulcer is to point to in the expected time cannot heal, or for a long time can not heal the wound, its definition, there is no uniform standard, clinical used to more will be after a month the regular treatment failed to heal, also do not have obvious tendency to heal the wounds become chronic skin ulcer. Experiment 2 used a medium skin defect superposition hormone injections and foreign material composite factors building method, to shrinkage, the granulation tissue of the wound wet weight, histological examination and immunohistochemical method and other methods for observation methods, discusses the effect creams to promote the role of chronic skin ulcer healing. Experiment 3 through Real- time PCR, immunohistochemical and histopathological methods VEGF wounds in different time points, FGF, TGF-β, CD34 expression level and the change of FB count, further illustrates the effect of promote chronic skin ulcer wound repair mechanism. Material and method:Experiment 1: clean level, kunming mice to 50, are male, 20 to 22 g weight. Random number table method were randomly divided into five groups: skin excision group、skin excision + hormone group、skin excision + foreign material group、skin excision + hormone+ foreign material group,10 mice in each group. Skin hormone + + foreign bodies with skin + hormone group mice for 3 consecutive days shares of intramuscular injection of hydrocortisone injection 4 ml/kg, and the rest of the amount of normal saline was injected into groups of mice. In building 4 days with 4% chloral hydrate anesthesia, cut back, in the central lumbar is the drapes on the shop, with iodine and alcohol after disinfection, minus the part of the skin, is about 1.2 cm in diameter circular full-thickness skin incision. Skin hormone + + foreign group, the skin of mice + foreign body in the skin incision after skin tissue separation at the same time with vascular clamp to muscle fascia, then use tweezers gently pull open incision, buried plastic ring edge of disinfection incision, finally will be close to the edge of the plastic ring skin suture needle, in case the ring fall off. After the success of the building to observe the general status in mice, wound form, discharge, regularly weigh weight in mice, measure the wound area and calculate the wound healing rate, building 14 days executed after mice, cut the wound granulation tissue and new gen weeks group, HE dyeing observation wound tissue capillaries and fibroblasts proliferation.Experiment 2: clean level 40, kunming mice were randomly divided into blank group, model group, a cream group, the rehabilitation of new liquid group, each group of 10, only except the blank group, each group of mice by adopting the method of skin hormone + + foreign group building, the treatment group, with the corresponding drug model group with normal saline smear wound, groups are daily switching time, with sterile gauze to cover the wound, blank group shall not interfere. General situation regularly macroscopic observation wound in mice after treatment, measure the wound area and calculate the rate of wound healing, 21 days after treatment executed mice, plastic ring granulation tissue, using electronic balance granuloma wet weight, and then fixed save 10% formalin solution, HE staining for morphological observation, immunohistochemical method to detect the BCL- 2, bax protein expression in wound tissue level.Experiment 3: clean level 120 kunming mice, were randomly divided into blank group, model group, a cream group, the rehabilitation of new liquid group, each group 30, on the basis of each group to its divided into group a week, two weeks, three weeks 3 subgroups, 10 mice in each group. Groups of mice in the observation time is over, after death, plastic ring granulation tissue, a third in 10% formalin fixed, for histological observation and immunohistochemical method to detect. Two-thirds of tube into the cryopreserved cryogenic refrigerator, for Real- time PCR detection. Observation indexes: Vascular endothelial growth factor(VEGF) : Real- time PCR hair and immunohistochemical method to detect gene and protein expression; Fibroblast growth factor(FGF) : Real hair- time PCR and immunohistochemical method to detect gene and protein expression; Transforming growth factor(TGF- beta) : Real hair- time PCR and immunohistochemical method to detect gene and protein expression; CD34 antigen: immunohistochemical method to detect protein expression; Fibroblast(FB) count: HE staining to observe the wound granulation tissue fibroblasts counting. Results:Experiment 1:1. The general condition: building was detected after 1 week + foreign body, pure skin lesions group activity state, movement sensitive, eating good, stool forming, body slightly thin compared with the blank group. Skin hormone + + foreign bodies with skin + hormone group posture apparent angular, food intake, less thin stool forming, activity decreased significantly, curled up, cold limbs and the tail.2. The wound: pure skin lesion group wound within 3 days after the building namely appear bright red granulation tissue, wounds moist, a small amount of serous effusion, wound flat, no obvious scabby, fast healing. Clear skin wound color dim + hormone group, secretion, less amount of granulation tissue and not fresh, slow wound healing is pure skin lesion group. Skin + foreign group of the early has large thick yellowish-white discharge, discharge wound basal organization under the bright red color, late to dark red, wound scab thicker, healing trend is not obvious. Early skin hormone + + foreign groups, there is a lot of clear fluid, middle ulcers form loose, flat collapse, dark purple color and granulation pale and covered by scabby, healing trend is not obvious.3. Weight: made no difference between the groups of mice weight on the same day, 1 week after building groups compared weight, pure skin lesion group weight, the fastest growth was detected the slowest + + group foreign body weight growth hormone. Factorial design analysis of variance showed that the hormone injections and foreign material on body weight in mice were two factors.4. Building groups of mice after wound healing rate, pure skin lesion group, fastest heal skin hormone + + foreign group of the slowest.5. Histological observation: pure skin lesions are seen in group more neat rows of newborn blood capillary, accompanied by a large number of fibroblasts, small amounts of fibroblast and atrophy of inflammatory cells, no obvious edema. Skin + hormone group section visible newborn capillaries and fibroblasts is relatively sparse, inflammatory cells. Skin + foreign body section shows a large number of shrinking set of inflammatory cells, macrophages and plasma cells, may be seen occasionally size vessels arranged disorderly and part is broken, and overflow scattered red blood cells in tissue clearance, accompanied by edema. Skin + + hormone group section visible capillary generated and fibroblasts scarce, arranged disorderly, inflammatory cells. Blank group of subcutaneous tissue section visible muscle fibers arranged orderly, no inflammatory cell infiltration.Experiment 2:1. The general observation: the trauma of mice a effect paste medicine 3 to 7 days after the color of the wound by the dark red, granulation tissue, wounds moist, more thick secretion, no obvious scabby, in a soft, healing trend is obvious; Rehabilitation group new solution in mice after 3-9 days is a small amount of granulation tissue, co-author of colour and lustre, the surface is relatively dry, secretion is clear, a little scabby coverage, union trend more obvious; Model group mice wound tarnished, ulcers form loose, flat collapse, no obvious granulation tissue healing early, to later period is visible a little pale granulation, the surface is covered by scabby, hit hard, no obvious trend of healing.2. The wound healing rate: 5 days treatment, the treatment group compared with model group, the effective cream group, the rehabilitation of new liquid group wound healing rate increased significantly(P < 0.05). Treatment for 10 days, the treatment group compared with model group, the effective cream group, the rehabilitation of new liquid group wound healing rate increased significantly(P < 0.05); A new fluid effect creams group and rehabilitation group, there was no statistically significant difference. 17 days treatment, model group, the effect paste new fluid, rehabilitation group wound healing rate increased significantly(P < 0.05).3. The wound granulation tissue wet weight: after 21 days, the treatment group compared with model group, a new fluid effect creams group and rehabilitation group mice wound plastic ring granulation tissue wet weight increased significantly(P < 0.05).4. Wound the BCL- 2, bax protein expression: the treatment group compared with model group, a new fluid effect creams group and rehabilitation group wound granulation tissue of mice the BCL- 2 expression significantly increased(P < 0.001), and the expression of bax significantly decreased(P < 0.001).5. Histological observation: normal group of subcutaneous tissue section visible muscle fibers arranged orderly, no inflammatory cell infiltration. Model group is not obvious, granulation tissue hyperplasia capillary number sparse, arranged disorderly, endothelial cells and fibroblasts were rare, inflammatory cells, obvious scar hyperplasia. A cream group of granulation tissue hyperplasia more apparent, capillary generates significantly more than the model group, neatly, with a large number of fibroblasts, and a small amount of fibroblasts, I have a small amount of inflammatory cells, no obvious scar hyperplasia. New liquid recovery set of capillary generation and fibroblasts with a similar effect to cream group, is a small amount of scar tissue.。Experiment 3:1. The fibroblasts count: Surface tissue fibroblasts count HE staining method monitoring results: the treatment group compared with model group, 7, 14, 21 days after medication, a new fluid effect creams group and rehabilitation of fiber cell count increased significantly(P < 0.05), a new effective cream group and rehabilitation is compared between fluid way, there was no statistically significant difference(P > 0.05).2. CD34 results: Wound tissue CD34 test results: the immunohistochemical method monitoring results: each point in time, CD34 protein expression level in model group were significantly higher than the blank group(P < 0.01); Compared with model group, the treatment group CD34 protein expression level obviously improved(P < 0.01), a paste is compared between group and rehabilitation of new fluid way, there was no statistically significant difference(P > 0.05).3. The VEGF results: Wound tissue VEGF test results: the immunohistochemical method to detect results: VEGF protein expression level of each point in time, model group were significantly higher than that of blank group(P < 0.05). Compared with model group, each time a level of VEGF protein expression cream group were improved, including 7, 14 days is compared between two groups was statistically significant difference(P < 0.05), 21 days there was no statistically significant difference is compared between two groups(P > 0.05). Compared with model group, each point in time recovery new VEGF protein expression liquid group were improved, which day 14 comparative differences between the two groups was statistically significant(P < 0.01), 7, 21 days there was no statistically significant difference is compared between two groups(P > 0.05). A new fluid effect creams group and rehabilitation group comparison, comparison between 7, 21 day group, the difference was statistically significant(P < 0.05), 14 days there was no statistically significant difference is compared between two groups(P > 0.05). The test results: the m RNA expression model group at each time point VEGF m RNA expression levels were significantly higher than that of blank group(P < 0.05). Compared with model group, treatment group at each time point VEGF m RNA expression level increased, the difference was statistically significant(P < 0.05). A new fluid effect creams group and rehabilitation group, there was no significant difference.4. The FGF results: Wound tissue FGF test results immunohistochemical method to detect the result: each point in time, FGF protein expression level of model group were significantly higher than the blank group(P < 0.001). Compared with model group, each time a cream group, all FGF protein expression level increased, which day 14 comparative differences between the two groups was statistically significant(P < 0.001), 7, 21 days there was no statistically significant difference is compared between two groups(P > 0.05). Compared with model group, each point in time recovery FGF protein expression of new liquid group were improved, but there was no statistically significant difference is compared between two groups(P > 0.05). A new fluid effect creams group and rehabilitation group comparison, 14 days is compared between two groups, the difference was statistically significant(P < 0.05), 7, 21 days there was no statistically significant difference is compared between two groups(P > 0.05). MRNA expression test results: each time point FGF m RNA expression model group were higher than that of blank group, among them 7 days comparative differences between the two groups was statistically significant(P < 0.05), 14, 21 days is compared between two groups has no statistical significance(P > 0.05). Compared with model group, each point in time, the FGF m RNA expression effect paste group were improved, comparing differences between the two groups was statistically significant(P < 0.01). Compared with model group, each point in time recovery new fluid way FGF m RNA expression were improved. A new fluid effect creams group and rehabilitation group, there was no statistically significant difference 7, 21 days(P > 0.05), 14 days difference was statistically significant.5. TGF-β results: Immunohistochemical method to detect the result: each time point, TGF- beta protein expression level of model group were higher than that of blank group, comparing differences between the two groups was statistically significant(P < 0.05). Compared with model group, each time a cream group of TGF- beta protein expression level increased, among them 7, 14 days is compared between two groups was statistically significant difference(P < 0.05), 21 days there was no statistically significant difference is compared between two groups(P > 0.05). Compared with model group, each point in time recovery TGF- beta protein expression of new liquid group were improved, but the 7, 14 days is compared between two groups was statistically significant difference(P < 0.05), 21 days there was no statistically significant difference is compared between two groups(P > 0.05). A new fluid effect creams group and rehabilitation group, the time difference had no statistical significance(P > 0.05). MRNA expression test results: each time point TGF- beta m RNA expression model group were higher than that of blank group, comparing differences between the two groups was statistically significant(P < 0.05). Compared with model group, each time a cream group of TGF- beta m RNA expression level increased, comparing differences between the two groups was statistically significant(P < 0.05, P < 0.01). New liquid group compared with model group, rehabilitation TGF- beta m RNA expression level increased, 14, 21 days difference was statistically significant(P < 0.05, P < 0.01). A new fluid effect creams group and rehabilitation group, there was no statistically significant difference(P > 0.05). Conclusion: 1. Superposition of skin defect hormone injections and foreign material composite factors building method, can well reflect in line with the "Yang" of traditional Chinese medicine syndrome type of overall state. Compared with other groups at the same time, no matter from the aspects of symptoms primary symptom, or the objective indicators, this method is the most can effectively maintain the state of chronic skin ulcer wound, the surgery of traditional Chinese medicine(TCM) at the same time, to a certain extent, can reflect more Yin disease ulcers syndromes characteristics, can provide relatively simple for clinical and scientific research of traditional Chinese medicine, economy and conform to the characteristics of TCM syndrome type of small animal models. 2. Compared with the model group the effect rate of wound healing after treatment cream group 5, 10 and 17 days tomorrow improve(P < 0.05), suggesting an effect creams to chronic skin ulcer to promote wound healing in mice. To a certain extent, simmer pus, the production method is verified with moist wound environment of chronic skin ulcer healing. 21 days after treatment, compared with model group, the effect of mice cream wound granulation tissue wet weight increased significantly(P < 0.05), histological examination revealed a cream mice wound tissue section number of capillaries and fibroblasts significantly more than the model group, the paste with wound granulation tissue prompts a effect and the formation of granulation tissue based material(fibroblasts, newborn capillaries) has a role in promoting. Each treatment group after the wound granulation tissue in mice in the BCL- 2 expression significantly lower compared with model group, and bax protein expression level increased significantly. The results can reflect to some extent drug treatment can influence the expression of apoptosis related gene, suppressing cell apoptosis, and promote the healing of chronic skin ulcer wound. 3. A effective cream set in wound tissue VEGF and CD34 expression level increased at various time points, prompt a effect creams to promote chronic skin ulcer wound healing is related to angiogenesis. Its effect in promoting angiogenesis, can improve the microcirculation of local wound, thereby promote wound granulation tissue growth, action may be a valid paste the manifestation of "promoting blood circulation to remove blood stasis" effect; Newborn blood capillary, on the other hand, as an important part of granulation tissue, its growth speed can directly promote granulation tissue freshmen, this effect could be a valid paste "production" role. According to the results of this study, compared with model group, the group effect creams TGF- beta m RNA and protein expression level increased, can prompt a effect creams by raising wound tissue TGF-β horizontally, FGF fibroblast proliferation, accelerating wound granulation tissue of the freshmen, which are beneficial to the chronic skin ulcer healing of the wound.