| Background:Human exposure to high heat materials,collectively referred to as the process of destruction of normal tissue Burns,including:hot gas liquid Burns,strong acid strong alkali corrosion,Burns,electric shock,fire or burns Burns.Burned skin is most common,the skin is the body's first line of protective barriers,injury of skin burn is an extremely complex process.After severe skin burns to large areas of early dehydration can occur,shock,infection,or sepsis,that life-threatening;late may cause serious functional impairment.Protect the wound of burn wounds,and to speed up the healing process can greatly reduce or even avoid the appearance of complications.For clinicians,and how to ensure faster and better on burn wound healing is a huge challenge.In recent years,the tissue engineering therapies in the treatment of tissue repair received very high attention,adipose-derived mesenchymal stem cells?adipose-derived stem cells,ADSCs?is a kind of adult stem cells,according to other related studies:ADSCs extracted fat tissue,easy to get,easy extraction process and ADSCs has the potential to differentiate into a variety of adult cells.Therefore,the ADSCs is increasingly becoming the current research in tissue engineering and cell therapy,the ADSCs experiment extraction and development,establishment of New Zealand White Rabbit animal model of skin deep burns.Burn wounds:local injection of ADSCs,observe the wound healing process,research related to its promotion mechanisms.Take ADSCs as seed cells of tissue engineering therapies in the treatment of burn wounds provided a theoretical basis,open up a new approach to clinical treatment of burn wounds.Objective:Extract fresh fatty tissues in rabbits under sterile conditions and isolation of high purity ADSCs,in burn wound in rabbits by local injection of ADSCs suspension must concentration,in order to accelerate the wound healing process of ADSCs and study the role of play in the healing process and its principles.Methods:?1?Extraction and identification of ADSCs:aseptic level of health of New Zealand White Rabbit?about 2-3 months old,weight control in 2Kg?,derived from Weifang Medical University laboratory animal Center,Select the appropriate ear vein in ear and ear vein anesthesia and sterilization,drape,Under sterile conditions to extract fresh groin Yellow Rabbit adipose tissue,in the presence of enzyme digestion gradually extracted fat stem cells,The extracted stem cells inoculated culture bottles?5%CO2,37??cultured cell growth under the microscope,by MTT test to describe its growth curve,Third generation of adipose-derived stem cells for immunofluorescence,detection-related antigen expression of CD44,CD29,CD34,CD106,in order to identify the extracted ADSCs,Induce induced them to differentiate into chondrocytes of cartilage using toluidine blue staining results and determine induction,using induced liquid into fat cells into fat cells,and oil red o stain test the lipid droplet formation induced by the identification of results;Will EdU ADSCs marks to trace its wound healing in rabbit distribution within an organization.?2?the establishment of an animal model:New Zealand White Rabbit37 clean,in which random wound depth identification only,the remaining 36,according to the randomization methods these animals into the experimental group and the control group?group n=18?.Choosing the right ear in rabbit ear vein,according to inject 1.5 ml per kilogram of 4%sodium amobarbital way Auricular vein anaesthesia,The rabbit back near the hip,hair removal,disinfection drape,prepared 35 layers of gauze put 99?constant temperature water bath pot,after 5minutes in the making of deep II-III degree burn wounds 20s?death wound wound depth identification identification of animals?,The next day,the use of pancreatic enzyme mark prior EdU ADSCs digest from the bottle down,and do not add Sera prepared by DMEM culture fluid of the cell in 5x106 number of levels per ml of cell suspension,injected into the prepared cell suspension evenly scald wound in rabbits in the experimental group,According to the same method in the control group injected DMEM culture fluid of the same content without added serum 1ml.?3?Observing and recording changes of wound,respectively after burns the 1th week,2weeks,3 weeks film indirect weighing method and recording of wound healing rate and air embolism death of 6 animals.Respectively cut wound repair organization,brings the Organization into 4%paraformaldehyde for tissue sections for further observation.Outcome measures:?1?Wound repair organizations collected in accordance with the procedure step by step production of paraffin and cut and slice tissue HE dyed,and observed under a microscope;?2?ELISA detection of burn tissue by Hyp?hydroxy-l-PROLINE?content;?3?Will be making these steps of slicing through MASSON stain,and under a microscope to observe and record the changes of collagen fibers;?4?ELISA detection of EGF and VEGF expression in the wound;?5?Immunofluorescence staining of Burns is marked by EdU ADSCs in 3rd week of specimen tissue distribution.?4?application software SPSS20.0 will collect data to import,on the statistical analysis of the data related to the treatment,P<0.05said there was a significant difference between dataResults:?1?Morphological observation and identification of ADSCs:under a microscope found in adipose tissue-derived mesenchymal stem cells in 7h in flasks around the wall attached,about a day after the wall of stem cells begin to stretch,the3rd generation of spiral stem cells gathered??2?MTT method renders the growth curve shows that the stem cells grow more slowly at the start,but from the 3rd day of cells begin to enter accelerated growth phases,a week after the cells to grow the fastest,grows slowly.?3?immunocytochemical staining:CD44,CD29 expression,expression of CD34,CD106,is negative,consistent with its surface marker expression.?4?the cartilage induced liquid ADSCs,intracellular matrix display a uniform purple dye,note chondrocyte cell induced by the iconic factor matrix glycosaminoglycan,successfully induced to differentiate into chondrocytes.ADSCs after into fat cells induced in the presence of oil red o show:cell culture expression throughout the fat droplets in the vision;?5?EdU mark ADSCs:ADSCs nuclei by EdU staining in fluorescence microscope view of look red,ADSCs by Hoechst staining cytoplasm appears blue,after the overlap between orange-red.?6?animal models of wound healing rate and the general situation:the 1th week both groups wound are kept dry,exudate is not obvious,redness and inflammatory symptoms gradually subside,the experimental group and the control group,there is no significant difference in the rate of wound healing significance;The 2nd week,the rabbit in the experimental group of scald wound inflammation significantly reduced wound scab Pison,wound callus in rabbit skin firmly in the control group,part of the granulation tissue,the experimental group in rabbits wound healing rate significantly higher healing rate;3rd week,the wounds are both obviously recovered last week,compared to the control group still has some wounds exist,and recover better in the experimental group,high healing rate of the control group.?7?ADSCs distribution in wound and the index check:HE was observed:1th week of wound tissue exists in a lot of circles of inflammatory cells;the 2nd week,this round has been largely disappear,spindle formation in cells in the experimental group than in the control group,and differentiation of epidermal layer;the 3rd week,the epidermis differentiation of meditation in the experimental group,control group is poor.MASSON wound staining:1th week,two groups of proliferation of collagen in the wound is small,2nd,3 weeks,the collagen fibers in the experimental group than in the control group expression to read more,and shape the law,closely spaced,3rd week is more obvious.Hyp contents:Hyp gradually increased in the two group healing wounds,Hyp increased significantly in the experimental group than in the control group.1th week of wound tissue content of EGF and VEGF in the experimental group with a similar control group?P>0.05?.2nd and 3 weeks after injury,content in the experimental group than in the control group?P<0.05?.EdU fluorescein-labeled tracking cell:can be found in experimental wound healing in rabbit tissues were marked by EdU ADSCs.Conclusion:?1?In this experiment,adipose stem cells can accelerate the healing of burn wounds by promoting the increase of VEGF and EGF in the wound.?2?Adipose derived stem cells can promote the proliferation of fibroblasts and the deposition of collagen fibers in burn wounds,so as to improve the quality of wound healing.?3?In the animal model of wound healing rate,the transplanted adipose derived stem cells can survive in the burn wound healing tissue,and directly participate in the repair of the wound through self differentiation. |
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