| Background: Currently,skin and soft tissue expansion technique is widely used in Plastic repair and scar treatment after burn,it has gradually become a basic tissue repair technology.At the same time it is also accompanied by the expansion of a long cycle,high retraction rate and other shortcomings.If emphasis on the rate of expansion too much,it can lead to a higher rate of retraction and a poor quality in the expended skin.So,how to effectively reduce the expansion cycle and reduce the rate of expansion of the flap retraction is particularly important.ADSCs is an kind of Adult stem cells.Since Zuk reports,as the cells easily obtained,multi-source,Strong differentiation and other feathers,it has gradually become the focus in research of stem cells and tissue engineering.In recent years,many studies have shown that ADSCs can increase the angiogenesis,thereby promoting tissue regeneration,In this experiment,with rabbits as animal skin expansion model,By local injection of ADSCs,to explore preliminary experimental study on expanded flap vascular ization,tissue regeneration and effective in reducing the rate of expansion of the skin retraction,Playing a guiding role In clinical applications.Objective: To explore the possibility in the skin tissue expansion by injecting ADSCs that promote new sk in tissue regeneration and reduce the expanded skin retraction rate,observe the outcome of adipose stem cells after transplantation,flap vascularization and the expression of C D31,VEGF.Methods:(1)ADSCs extraction and identification: 2-3 month old New Zealand white rabbits,weighing about 2K g,were purchased from the animal center of Weifang Medical University.After the success of anesthesia from the inguinal fat tissue with trypsin and collagenase digestion,separation,the cells were seeded in culture flask(5% CO2,37 ℃)were cultured,cell morphology was observed under microscope.MTT measured the growth curve,cultured to the third generation,for flow cytometric detection and immunocytochemical staining and the expression of CD29,CD44,CD34 detection and CD106,based on ADSCs identification to induce chondrogenic differentiation induced by liquid cartilage,staining results and judgment induced by toluidine blue,the epidermal cells induced by experimental expression detected by immunofluorescence and CK19 induction for judgment.EdU labeled ADSCs to trace the distribution in vivo.(2)The establishment of animal model: the random number table method is divided into experimental group and control group,each of the 10.After anesthesia 4% isoamyl barbital sodium 1.5ml/K g skin preparation,on the side of the spine flap design.In the flap center to the out a 1.5cm * 1.5 cm square and regular disinfection,shop towels.Vertical opening in the direction of the spine,in the text box is below the deep layer of superficial fascia insertion of a 30 ml round dilator a gold and inject 25 ml of physiological saline,strict skin suture incision.Will the edu incubation of the third generation of ADSCs by trypsin digestion and separation will digest down the ce lls with serum-free DMEM is formulated as a concentration of 5 * 106 / ml cell suspension,the experimental group expansion of subcutaneous injection and edu labeled ADSCs suspension 1ml.With the method of control group only injected with serum free DMEM medium 1ml,wound healing after a week stitches,stitches later began filling,twice weekly injection,hydraulic meter water injection pressure is maintained at 5.3 kPa(40 mmHg),4 weeks inject water.(3)Area expansion,injection volume,back shrinkage rate and skin specimens collected: respectively labeled by the expanded skin after 1,2,4 weeks measurement area;water injection volume calculation: Weekly twice injection frequency on rabbit skin tissue expansion according to the recorded amount of water every time,and to observe water injection to 120 ml control group and the experimental group are needed time.Before remove the skin expander,a marker of 3cm * 3cm in the top part of the expanded skin flap is the central square,and the camera.Then the expanded skin after complete resection of the skin,to be free to return to its full after the image processing,with the image analysis software to expand the area of skin tissue contraction before and after the measurement.After the end of the expansion,the tissue was implanted into 4% to prepare the tissue section for further observation.The observed indexes:(1)the specimen thickness and histological examination.(2)ADSCs were edu labeled in expanded skin distribution.(3)immunohisto chemistry to observe the endothelial cell marker CD31,the microvessel density(MVD)judge.(4)ELISA method for the detection of EGF and VEGF expression.(5)Western blot was used for detection of CK19 in the epidermal cells.(4)A software SPSS17.0 was used to to calculate the statistics,there was statistically significant while P<0.05.Results:(1)ADSCs morphological observation and identification: cell 6-8h began adherent,24 h after adherent cells fully extended,to the third generation,cells covered the bottom of the bottle arranged more tidy and gathered together presented long fusiform shape distribution.(2)MTT method measured growth curve showed S type,the first two days of cell growth is relatively slow,from third days from the cell gradually into the growth period,seventh born Wanlinda peak,and then enter the platform period.(3)the expression of ADSCs,CD29,and,CD106(hematopoietic system associated antigen)was negative expression,which was consistent with the expression of the surface markers of CD44,and the expression of and CD34 was negative.(4)ADSCs by EGF to epidermal cells induced and detection of CK19 expression,indicating that ADSCs can be induced to differentiate into epidermal cells;chondrogenic induction medium to induce ADSCs showed intracellular matrix of homogeneous Aizen,that induced cells to produce the cartilage cell marker factor matrix glycosaminoglycan successfully induced differentiation into chondrocytes.(5)EdU labeled ADSCs: under the fluorescence microscope,it was found that the nucleus of Ed U stained red,Hoechst stained back ground cells were blue,after the overlap between the two orange red,indicating the success of EdU markers ADSCs.(6)Animal experiment: two groups of skin expansion the 1,2,4 weeks the area was increased,skin in the experiment group and the amplificat ion of the area was significantly greater than that of control group(P < 0.05).In the amount of water to achieve 120 ml.The control group took 3.5 weeks and the experimental group only 2.5 weeks,the experimental group significantly faster in the contrast group.Two groups of expanded skin in vitro have different degree of retraction,through the calculation of visible experimental group expanded skin retraction(29.6 2.01%)was significantly less than that of the control group(49.62±3.25%)(P < 0.05).(7)ADSCs in the expansion of the skin and the distribution of the indicators to check: fluorescent microscopy of the experimental group EdU labeled red fluorescent cells involved in the formation of the skin structure.Histological examination revealed two groups of loosely arranged collagen fibers in the skin expansion,the fracture part of collagen fibers,the experimental group than in the control group,the number of blood vessels increased,thickening of the skin is more obvious,especially the epidermal layer(P< 0.05).Masson staining showed that two groups of expanded skin dermis fiber was dyed blue,in the experimental group compared with the fiber tortuosity the control group was significantly thicker,fibroblast density also increased significantly(P<0.05).Immunohistochemical staining showed that: compared to the control group,the experimental group of vascular endothelial cell specific markers CD31(P<0.05),microvessel density(MVD)also increased(P<0.05).ELISA method measured the experimental group the expression level of EGF and VEGF were significantly higher than control group(P<0.05).Conclusion:(1)In this study,we successfully isolated and extracted adipose derived stem cells from rabbit inguinal adipose tissue and cultured stably and rapidly in vitro.(2)The fat stem cells in the inducer cartilage induced liquid conditions successfully induced and differentiated into cartilage cells;the epidermal cell growth EGF induced differentiation into epidermal cells which once again confirmed that the adipose derived stem cells have multilineage differentiation capacity.(3)In the process of skin and soft tissue expansion in animal experiments,adipose derived stem cell transplantation can promote the regeneration of skin tissue,and partic ipate in the formation of new blood vessels,improve the quality of skin expansion.(4)Cell transplantation significantly reduces the expansion of skin tissue retraction 4 fat stem,to more effectively improve the efficiency of expansion.(5)Adipose derived stem cell transplantation can accelerate the rate of skin tissue expansion,thereby reducing the time of skin tissue expansion. |
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