| Objective: In vitro specific conditions, the granules mixed with fatty humanadipose stem cells (ADSCs) amplified were transplanted in nude mice at theback of the fascia, the ability of ADSCs to adipocyte differentiation andpromote angiogenesis in the fat transplantation is researched, simultaneouslyexplore the best stem cell grafts proportion of fat particles in human adiposestem cells to provide a theoretical basis for further application in fattransplantation.Method:1Extracting adipose stem cells: aseptically extracted from humanadipose tissue15g, phosphate buffered saline (PBS) to remove residual bloodrinse, ophthalmic scissors and tweezers ophthalmic tissue removing visible fattissue and blood vessels, cut into pieces to size fine particles of3mm, whichwas placed in PBS，in which the concentration of collagenase digestion Ⅰis0.075%, in the shock digested at37℃30min, moved it in the centrifuge tube,and centrifuged at800g centrifugal force of10minutes, filtered through a100the precipitate in the centrifuge tube, and the resulting cell extract wascultured with DMEM (containing10%fetal calf serum,100U/mlpenicillin,100U/ml streptomycin,5mg/mL heparin and2ng/mL acidicfibroblast growth factor) and resuspended in an appropriate culture flaskswere seeded in sterile, at37℃, CO2volume fraction of5%, saturatedhumidity incubator culture, the half of medium was changed after48h, afterthe medium was changed every3d, to be the primary cells of80%confluence with0.25%trypsin-EDTA digestion the adherent cells weredigested liquid separation for2-3minutes, passaged1:3, inoculated in sterileflasks, three days for liquid1, then passaged again when the cells grow fullof bottom.. Flow cytometry cell culture surface antigen expression. 2Brdu mark adipose stem cells: when the adipose stem cells spread tothe fourth generation, added to a final concentration of10μg/mL of Brdu, thendetect the rate of marking after48h. The remaining cells digested with EDTAwere mixed with fat particles, they are used in the animal experiments.3fat particle separation: adipose tissue extracted from discarded humanclinical surgery, PBS wash solution three times to remove residual blood，cut into pieces to size approximately0.5~1mm3with scissors，then put thefat particles into a50mL centrifuge tube, added amount of DMEM/F12medium,1200r/min centrifuged3min, get fat particles superstratum.4weighing the transplanted fat particles: the fat particles after cuttingand centrifugation was placed in a centrifuge tube, the fat particles areweighed, the experimental group was divided into Group A, Group B, GroupC, D groups, were added to the fourth generation adipose stem cells whichwere labeled by Brdu, the proportion of stem cells and fat particleswere105:500mg,3×105:500mg,6×105:500mg,9×105:500mg, the controlgroup was pure fat particles. extracted500mg fat particles with1mL syringeafter weighed.5fat particles transplantation:1mL syringe equipped with a16-gaugeneedle, the fat particles containing500mg of fat stem cells injected into nudemice under the left rear fascia,500mg pure fat particles injected into nudemice under the right side of the back fascia.6detected the transplanted fat particles:28days after transplantation themice were sacrificed and get out the transplanted fat particles, peel off theenvelope surrounding the graft, placed on electronic weighing scales, andrecorded. Then placed in4%paraformaldehyde, paraffin sections of the graftwere stained with hematoxylin-eosin staining, detection of markers ofendothelial cells and BRDU antibody and CD34markers.Results:1adipose stem cells (ADSCs) were isolated, cultured and identified: cellextract after resuspended in DMEM properly seeded in sterile flasks, training3-5days observed polygonal cells and spindle cells, later polygonal cells gradually transformed into spindle cells.2expression by flow cytometry:the results revealed ADSCs expressedCD29, CD44, CD90, andCD14, CD45, CD146expressed hardly, proveisolated cultured cells to a more purified mesenchymal stem cells.3ADSCs transplanted fat particles affect weight:4weeks after transplantgrafts experimental group the average weight of the group A to group D were(401.22±8.43) mg,(429.79±7.30) mg,(462.53±5.18) mg,(465.42±3.87)mg a weight retention ratio were80.24%,85.95%,92.51%,93.08%.The averageweight of the control group (272.37±9.73) mg, maintain weight ratio54.47%.Statistics showed that in addition to C, D groups no significant difference inweight, as any other two groups were statistically significant.4graft microvessel quantity detection: take4high power field on eachgroup of central and peripheral zones respectively, after counting out thenumber of microvascular each view. The single endothelial cells dyed brown,each endothelial cell clusters are regarded as1vascular count unit, thencalculated per square millimeter area the number of microvessels, andcalculate the average. The number of vascular in the center in A-Dexperimental group were53.43±8.64,65.74±7.07,76.79±5.36,80.34±4.83, control group was38.04±9.10. The number of vascular around in theA-D experimental group were65.20±8.14,77.55±7.37,88.30±6.33,91.89±5.61, control group was47.42±9.96.Statistical analysis was performedusing SPSS13.0statistical software. Both the central and peripheral zone,there was significant difference between the experimental group and thecontrol group (P <0.05), the experimental group both vascular density ofcentral or peripheral zone was higher than the control group; the4experimental groups were compared, both central and peripheral zone, inaddition to the C group showed no significant difference with D group, theother two two were statistically significant (P <0.05), shows that thetransplanted stem the existence of an optimal ratio between the cell and fatparticles, which then increase the number of stem cells transplantation will notincrease fat blood supply; each experiment group and control group for comparison within group, there was significant difference (P <0.05), that boththe experimental group or the control group, the number of peripheral vesselsare higher than the central vessels.5immunohistochemical staining in the4experimental groups weredetected in BrdU antibody positive, proved that the ADSCs transplanted hassurvived. While the control group was negative for BrdU antibody detection.Conclusion:1In vitro extraction of adipose derived stem cells (ADSCs) and mixedtransplantation of fat granule, part of the fat stem cells transformed into fatcells, which made up a normal fat tissue.2Cells in a paracrine manner to promote angiogenesis transplantation ofadipose stem, and speed up the formation of new vascular transplantation ofadipose tissue, improved the transplantation of adipose tissue survival rate,and then improve the survival rate of transplanted fat.3Fat stem cells can promote angiogenesis, and the formation of bloodvessels in turn to promote the survival of transplanted fat.4Cells can be transformed into a new generation of fat cells4fat dry,mature adipocyte apoptosis existing, new generation of fat cells will becomepersistent, Everfount, dynamic force.5Fat stem cells mixed with fat granule transplantation, there is anoptimal proportion, the future in the clinical application the adipose derivedstem cells can be maximized.6This experiment confirmed that adipose derived stem cells can be usedas seed cells, play an important role in adipose tissue transplantation. |
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