The Preliminary Study Of Bone Marrow Mesenchymal Stem Cells In Treating Wounds

PartⅠThe establishment of Pig burn modelObjective:According to animal experiments, explore a method to make a simple animal model of burn wounds, and to explore the depth of porcine skin burns, control of burn area, lay the foundation for the implementation of the subject, for the production of pig burn model offers a new ways and means.Method:3-4 month old pigs for 2, The corresponding burn wounds on both sides of the back of their own are divided into the histological test group and the general observation group. Pigs were anesthetized and the back skin and hair were removed with an electric shaver knife, fixed in the prone position on the operating table, prepared in advance window 3cm X 3cm of asbestos put over the back of pig shaved skin and hair area, covered with the same size of the openings of iron, assistant press tight metal, we start to jet fire in vertial to use gasoline blowtorch from the wound in 10cm. The first pig, According to injection duration, is divided into 2 seconds,4 seconds,6 seconds,8 seconds,10 seconds,12 seconds,14 seconds 7 wound group, There are four wounds in each group. Burn wound skin changes were observed after 30min and each of the four wounds are observed in histological examination in 12 hours after burn. According to histological examination results of the first pig to determine the required duration time of burn depth to the shallowⅡ, deepⅡdegree,Ⅲdegree burn, The second pig, according to injection duration, is divided into 2 seconds, 6 seconds,14 seconds wound group of three, There are four wounds in each group, To verify the burn time onece again.30min after burn wound skin changes were observed every other day. We observe the wound healing by the wound dressing, We observe and record the wound healing, respectively,1 week,2 weeks,3 weeks, 4 weeks in general observation group. burns 12 hours after, we cross-derived lines corresponding on both sides of the burn wound histological examination group. There are 2 wounds in each group for histological examinationResult:30min after, burn wound was saw in general observation:2 seconds, there is skin, dry, is purple and slightly white, and the wound healed within 2 weeks; 6 seconds, there is skin, carbide and the wound was coke yellow, the wound healed in 3 weeks; 14 seconds, The part of the epidermis are carbonation demyelination, the basement was wax white,4 weeks, The wound was still covered by necrosis crusts.12 hours after for burn pathology result showed:2 seconds, most of the cell nucleus of epidermal longer, level disorder, the majority of the germinal layer of cells have lined nucleus, showing that there is the accumulation of fluid in skin and the exudation outer skin; 6 seconds, the majority of basal cell nucleus become length, some superficial dermal cell have condensation nuclei, disorganized, and there is liquefied aggregation between the basal layer and a shallow layer dermis; 14 seconds, part of epidermis drop out, dermal cell have nucleus condensation and cell level disorders, skin adnexal is necrosis, fat cell have nuclei of normal. In 4 seconds, the wound is close to wound of 2 seconds in general observation and pathology result; 8 seconds,10 seconds,12 seconds, the wound are close to wound of 6 seconds in general observation and pathology result.2 seconds for the lightⅡdegree; 6 seconds-14 seconds for deepⅡdegree; 14 seconds and above for the grade Conclusion:The method of making use of gasoline blowtorch to make pig burn model is simple, reliable, affordable. At the same time, This is also a simple equipment, easy operation and safe method of making pig burn model.PartⅡThe Preliminary Study of Bone marrow mesenchymal stem cells in treating woundsObjective:Based on animal experiments to explore the feasibility, safety and effectiveness of the bone marrow mesenchymal stem cells (B-MSCs) for treatment of burn wounds. Explored the method, cells, monitoring tools, which provide a theoretical basis and practical experience for clinical applications, and thus for the treatment of skin defect of caused by large areaⅢdegree burn to supply new ways and methods.Method:Buy one male southern Yunnan pig a small ear, the wound of itself back into the corresponding parts were divided into the experimental group and the control group, each of the two groups have four wound, A. particles Paper, B. small skin graft, C. large sheet of paper, D. allogeneic skin, the wound were cut into deep fascia shallow area of 5cm×5cm. Take 5ml bone marrow mesenchymal stem cells of female Diannan Small ear pig put 10%α-MEM medium,10% fetal bovine serum and 1% of the pairs of resistance (penicillin and streptomycin) into sterile culture flask, add 95% saturated humidity,37℃CO2 to culture the third generation, microscopic observation of cell morphology, we detect stem cell marker anti-CD29-, CD44-anti-, CD45-resistant by flow cytometry and detect ultrastructure by electron microscopy. Experimental group, dropped bone marrow mesenchymal stem cells in a density of 2×106./ml,0.5 ml in the wounds, control group wound were dropped the same amount of saline. Wound dressing was changed every other day after transplantation, and were recorded time of wound healing and general changes. On 30 days after skin graft, we drawn to observe epidermal thickness, layer by the histological examination of HE staining and DNA comparison testing.Results:The results of this experiment is concerned. (1). Flow cytometry found that our culture mesenchymal stem cells do not express hematopoietic cell-specific CD45, but expression of stromal cells and stromal cells specific to CD44, CD29. The results of electron microscopy showed oval-shaped nucleus, abundant cytoplasm, a large number of microfilaments, single nucleus, more larger, less autosomal, more different chromosomes. Organelles, There are microvilli structures, Relatively larger nucleus and plasma, indicating cell protein synthesis and secretion is strong, to a large number of intake of nutrients, active function, shows the naive characteristic of stem cells. (2) Wound healing has not significant difference in the experimental group and control group. The experimental group, the wound healing process in the basement more fresh than the control group, healing wounds in experimental group was similar to normal skin than the control group. (3). The results of HE staining, the experimental group were markedly thickened epidermis, stratified obvious than the control group.Conclusion:(1) According the local wound stimuli, secretion-stimulating factor, catalysing local vascularization, bone marrow mesenchymal stem cells may be improving the wound base environment in which the skin is easier to survive, so It may have some role in promoting in the wound healing process. (2) bone marrow mesenchymal stem cells may have some influence in wound repair process of structural adjustment on the skin, scar formation and the size and thickness of scar. (3) completely by bone marrow mesenchymal stem cells in wound repair epidermal cells directly into the wound, but also difficult to achieve.