Application Research And Of The Adipose Derived Stem Cells In Plastic Surgery

Objective:To investigate the methods of isolating and culturing rabbit adipose—derived stem cells (ASCs), and to identify and analyze the biological characteristics of rabbit ADSCs and To explore the optimized condition for adipose-derived stem cells(ADSC) culture in vitro. To observe the result of mouse's ASCs autotransplantation subcutaneously. To solve the problem of congestion in the expanded flap by use of the ASCs. To observe the therapeutic effect of ASCs assisted adipose transfer for soft tissue defectMethods:①The ASCs was derived from the inguinal fat tissue of Vistar mouse and was cultured after the fat tissue was degested by collagenase. Then ASCs was labeled with eGFP transfection at second or third passage. The ASCs of experimental control group was killed by G418 after labled by eGFP. The blank control group normal saline.They were injected into the auricle of mouse subcutaneously seperately and the results was observed at 2nd,4th,6th week with frozen section.②Adipose was collected from the inguinal region of News Zealand rabbits and the ASCs were isolated with adherent method and collagenase method and cultured in vitro. The morphology of ADSCs after passage 2 was analyzed. The growth curve and doubling time were drawn. The collagenase with different concentration and duration and FCS with different concentration were used.③The ASCs was derived from the inguinal fat tissue of rabbit and was cultured after the fat tissue was degested by collagenase. Then ASCs was labeled with Dil at second or third passage. The blank control group normal saline.They were injected into the expanded flap with congestion after transplantation. The result was observed at 2nd week with pathologic section and CD34 immunohistochemistry.④12 patients diagnosed as soft tissue defect received the ASCs assisted adipose transfer for soft tissue atrophy after radiotherapy, surgical treatment and aging. The patients want to received the treatment after consultation. The ASCs was isolated with collagenase I from the adipose in patients abdomen or thigh under local anesthesia Then the ASCs(2×106/ml) was mixed with the adipse for half an hour before being injected to the soft tissue defect region. Moderate pressure should be given after the mixture transplantation delicately.Results:①Formation of primary and passage of ADSCs:ADSCs had spindle and polygon shape adherent growth, and their growth and differentiation were active.②Growth curve and doubling time of ADSCs:The growth curve was like "S" shape. The doubling time of ADSCs was about 55 hours.δThe ADSCs adipogenic differentiation was induced and the rate of positive rate was 65% through the Oil Red O stain and positive cell counting.4 Based on the collagenase concentration, cells were divided into four groups, i.e.,0.5mg/ml, 1mg/ml,1.5mg/ml and 2mg/ml groups. When the duration was 1 hour,2mg/ml group had the most total cell number(TCN) and the living cell rate(LCR) was statistically different from other groups. When the duration was 2 hours, TCN increased in every group and there were no statistical differences among 1mg/ml,1.5mg/ml and 2mg/ml groups. LCR was the highest in 1mg/ml and 1.5mg/ml. 2.Based on FBS concentration, the cells were divided into five groups, i.e.,0,5%,10%, 15%and 20%groups. The growth character was the best in 15%and 20%groups.⑤The new formed tissue labled by GFP was found in the subcutaneous tissue.⑥The survived length of flap with ASCs injection was longer than the flap in the control group. The CD34 immunohistochemistry showed more vessel formation in the flap section with ASCs injection than the flalp in the control group.⑦No obvious absorbtion was found after follow-up for half year.Conclusion①The ASCs of rabbits isolated in this trial are characterized by stable growth and quick proliferation in vitro. The optimized conditions for human adipose-derived stem cells culture are collagenase concentration of 1mg/ml, digestion duration of 2 hour and FBS concentration of 15%.②Autotransplanted ASCs can survive without scaffold.③.ASCs injection could improved the survived length by enhance the vessel formation.④The ASCs could improve the survive rate of adipose transfer.