| In recent years,autologous fat transplantation(AFT)has been widely used to remove scars,repair after burn,and repair and rebuild the whole body[1,2].Compared with other filler material,AFT have material availability,rich source,and with good appearance,high plasticity,and there is no immune rejection,etc[3-5].We could accept early satisfactory after the operation,but the long-term results were not satisfactory,mainly due to the high absorptivity(20% to 80%)after the fat transplantation and the low survival rate of the transplanted fat cells[6].The loss of volume and capacity after autologous fat transplantation leads to the loss of volume and capacity.It caused the low postoperative satisfaction and requires multiple operations.It also occurs with nodular calcification,liquefaction and cysts,which restricts the extensive use of fat transplantation.Therefore,how to improve the survival rate of AFT is the key to successful fat transplantation.Adipo-derived Stem Cells(ADSCs)is a mesenchymal Stem cell derived from Adipose tissue,which has the potential of multiple differentiation and is highly proliferative.At present,more studies have shown that adding ADSCs to AFT can improve the survival rate of transplanted fat cells[6-9].However,it is not clear how to improve autologous fat transplantation after ADSCs.At present,there are no definite conclusions on this issue in the academic circle.There are three main views: host replacement theory,adipose cells survival theory,and ADSCs alternative theory,and the substitute theory of stem cell of fat source.Neuhof believes that autologous fat transplant will slowly die after the recipient area,then replaced by the receptor tissue,that is,the host substitution theory;Peer and Brucker think that some fat cells will die after the fat transplantation,and that part of the fat cells can survive,that is,the fat cell survival theory;and Japanese scholars Kotaro think the fat after transplantation is fine.The cell soon dies and is replaced by the next generation of ADSCs-derived next generation adipocytes,who believe that ADSCs plays an important role in reconstructing adipose tissue.Therefore,it is urgent to explore how ADSCs can improve the survival rate of AFT.In recent years,with the development of different tracer methods,we can further explore the causes by marking ADSCs.The tracer refers to the cell or tissue that is used to mark the desired study by means of a special method,showing the migration,survival,proliferation and differentiation of the marked material by means of color rendering or other corresponding methods.In recent years,research in the field of transplantation is mainly concentrated in the transplanted cells in vitro after the tag will inject this living,its distribution in the body such as migration,proliferation and differentiation,survival outcome for detecting biological function,to provide accurate analysis of transplanted cells survive[10-12].In AFT,we mainly study the survival and proliferation of grafts by marking adipose stem cells.The commonly used methods include fluorescent dye markers,molecular cell markers,imaging technology markers,etc.In this study,ADSCs were extracted by isolating the inguinal fat of SD rats.The first part of this study through a variety of different markup ADSCs tracer method,discussing different markup in vitro experiments in ADSCs marked effect if there is a difference,and the different marking method of ADSCs appreciation ability,biological activity,differentiation and outcome biology function whether there is any influence.At the same time to further in-depth analysis of the results,in the second part of this research,simulating process of AFT,grown fat subcutaneous transplantation tumor in nude mice subcutaneous further explore different markup method of ADSCs marked effect on whether there is a difference,and the fat transplantation after adding ADSCs and different dye marker whether ADSCs can promote the survival of fat transplantation and whether there is a difference.Based on the above results,we can in order to select a better ADSCs marking method,which can be more widely used in basic research and clinical trials to investigate the causes of autologous fat transplantation survival rate.PART ONE A in vitro comparative study of the tracer effect and biological function of a variety of tracer markers in ADSCsProjective This part intends to explore whether there are difference in effect of different markers in vitro experiments,and whether the different labeling methods have influence on the biological function of ADSCs.Methods In this study,ADSCs were extracted from the groin of SD rats.The specific surface antibody was verified by flow cytometry(FCM).The GFP labeling method,the PKH26 labeling method and the Brd U labeling method were respectively used to mark ADSCs.CCK-8 was used to detect the value added ability of each marker group and negative control group.The apoptosis and cell cycle of each marker group and negative control group were detected by FCM.The lipid differentiation and maturation capacity of the cells in each marker group and the negative control group were detected by oil red O after the induction of ADSCs 21 days.After 21 days of induction of ADSCs with osteoinduction,the differentiation and maturation of the cells in each marker group and the negative control group were detected by ALP and alizarin red.Results We successfully extracted the cells from the groin of SD rats,and FCM identified the expression of the surface markers: CD29+/CD31-/CD34+/CD44+/CD45-.We use three methods of tag are successful marking ADSCs,through a fluorescence microscope showed different fluorescence,GFP method marking efficiency and stable genetic,PKH26 original tag efficiency is good,but as the rate of cell mitosis mark it down to the third generation has disappeared,the Brd U method the initial rate compared with other two kinds of mark is not high,at the same time as the rate of cell mitosis mark it also gradually fell to no.In the CCK-8 experiment,it was shown that there was no significant difference in proliferation ability of the negative control group(P>0.05).There was no significant difference in FCM detection of cell apoptosis rate and cycle change in each marker group and the negative control group.ADSCs were successfully induced by either lipid or osteogenic induction,and there was no statistical difference in the detection of different markers by oil red O,ALP and alizarin red(P>0.05).Conclusion We successfully extracted ADSCs and identified the cell by cell surface markers.ADSCs can be marked with different labeling methods,but the efficiency and stability of marking are different.There was no significant effect on the biological function and differentiation ability of ADSCs.PART TWO A in vivo comparative study of the tracer effect of a variety of tracer markers in ADSCsProjective This part intends to explore the tracer in vivo,the method for computing the different effect of ADSCs markers on whether there is a difference,and the fat transplantation after adding ADSCs and different dye marker whether ADSCs can promote the survival of fat transplantation and whether there is a difference.Methods By the first part of this study successfully labeling ADSCs,separation of SD rat groin again and the separation of cut up into the fat particles,mixed composition of each transplanted into nude mice after subcutaneous transplantation tumor.The size of the transplanted tumor was measured at a fixed time per week,and the rats were removed and weighed in the 4th and 8th weeks.At the same time,the transplanted tumor was frozen section,and the fluorescence cell and fluorescence intensity were calculated by different staining methods.Results After 4 weeks and 8 weeks of observation,we found that the fat tumor successfully constructed the subcutaneous tumor model.Weekly measurement transplantation tumor length to diameter tip,initial each team leader diameter has no obvious difference(P > 0.05),to the last groups of data are: 7.27± 0.29 mm blank control group,negative control group 13.83± 0.44 mm,GFP method group 11.96± 0.69 mm,PKH26 method group 11.95± 0.50 mm,Brd U method group 12.03± 0.65 mm.Each method group internal comparison,two compare the tumors had long diameter has no significantly difference(P > 0.05),compared with no mark negative contrast group size comparison,the tumors had the length to diameter smaller statistics have difference(P < 0.05),while the less cell blank control group,the tumors have obvious growth length to diameter,statistics have difference(P < 0.05).In week 4 we measured the mass of each group,and the data of each group was: 6.87±0.59 mg blank control group,negative control group 21.12±1.56 mg,GFP method group 13.51±0.77 mg,PKH26 method group 12.70±0.66 mg,Brd U method group 12.68±0.43 mg.In week 8 we measured the mass of each group,and the data of each group was: 6.63±0.15 mg blank control group,negative control group 24.41±1.88 mg,GFP method group 15.11±1.16 mg,PKH26 method group 14.19±1.26 mg,Brd U method group 16.47±0.77 mg.The results are consistent with the long diameter results.Fluorescence cell count and fluorescence intensity measurement results showed that the number of markers in each group was as follows: GFP method group 30.80±4.55,PKH26 method group 21.00±5.96,Brd U method group 36.20±3.90.Between two groups of comparison to each other,there was no statistically significant difference between GFP method group and PKH26 method group(P = 0.078),GFP method group with Brd U method group and method group with PKH-26 method group was statistically different(P < 0.05).In the 8 week of detection,the number of fluorescent markers in each group decreased significantly,and the difference was statistically significant(P<0.05).The number of fluorescent cells in each group was as follows: GFP method group 14.20± 4.76,PKH26 method group 5.40 ±3.65,Brd U method group 10.60± 4.45,there was no statistical difference between each of two groups.Fluorescence intensity was detected,and we defined fluorescence intensity of the GFP method group was 1,and the fluorescence intensity of the PKH26 method group and the Brd U method group was 0.52±0.13 and 2.71±0.31 in 4 weeks.The differences between each group were statistically significant(P<0.05).However,the relative fluorescence intensity of each group decreased significantly during the 8 week test,and the differences in each group were statistically significant(P<0.05).At the same time,we still used GFP method group strength as 1,and compared the relative fluorescence intensity between each group,the PKH26 method group was 0.63 ±0.08,and the Brd U method group was 0.61±0.10.There was no significant difference between the GFP method group and the other two groups(P<0.05),and there was no significant difference between the PKH26 method group and the Brd U method group.Conclusion The addition of ADSCs can promote the survival of transplanted fat,and different labeling methods may affect ADSCs to promote the survival of transplanted fat.In vivo experiments,the GFP method group marked efficiency and result stability,which can be considered as the first choice of marking method. |
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