| Objective: To induce human umbilical cord mesenchymal stem cells(hUC-MSCs) committed differentiation to the adipose cells under thespecifically isolate conditions. The hUC-MSCs which subsequently exhibitedthe phenotype of adipose cell were mixed with the granule adipose tissueimplanted into abdominal fascia of nude mice, established the basic theory offat tissue transplantation.Method: Obtained the umbilical cord from the full-term pregnancy anddelivery the newborn under the sterile conditions, rinsing of umbilical cord toremove residual blood by physiological brine. Removed a umbilical vein andtwo umbilical arteries by toothed forceps to avoid the vascular endothelialcells pollution the organization. Teared Wharton's Jelly tissue which from theumbilical cord into cord-like, then cut the tissue to volume of1mm3,fragment of Wharton's Jelly tissue culture in the bottle of petri dish, obtainedhuman umbilical cord mesenchymal stem cells (hUC-MSCs) lastly, flowcytometry was used to determine the expression of surface antigens.Combinedusing four reagents (1.0×10-6mol/L dexamethasone,0.5mmol/L1-methyl-3-isobutyl xanthine,10mg/L insulin,0.2mmol/L indomethacin) to induce thehUC-MSCs differentiation to adipose cell, observed the instance of celldifferentiation and used the oil red-O immunohistochemical stainingdetermined.Gained fat tissue from adult male SD rat mesenteric and inguinal,then cut into the volume of0.51mm3fat particles and centrifugation.Immunohistochemical staining and scanning electron microscopy observedthe morphology of vascular and content of the great vessels in fat particles;30male nude mice were randomly divided into three groups,7days,14days,28days, with body control, left is experimental side, the right is controlside,divided into7days experimental group,7days control group,14days experimental group,14days control group,28days experimental group and28days control group.The final concentration of10μmol/L brdu antibodycalibration of hUC-MSCs which subsequently exhibited the phenotype ofadipose cells were mixed with the granule adipose tissue implant into liftabdominal fascia of nude mice,the same volume of granule adipose tissuewithout hUC-MSCs implant into lift abdominal fascia of nude mice. Aftertransplantation7,14,28days, taked out the transplanted adipose tissueweighing, analysis fat weight difference between the experimental and controlgroup in the same period.Histological observation:HE staining observed thetransplanted adipose tissue structure, cell morphology and microvesselcontent.Immunohistochemical staining:use the vascular endothelialcell-specific antibody CD34, immunohistochemistry showed the microvessels,fat graft according to different periods divided into peripheral zone and centralzone division, quantitative study of the microvascular in every group. Brduantibody was used in the experimental group to determine whether the stemcells marked with BrdU antigen subsequently exhibited the phenotype ofadipose cell exists, ensured the transplanted stem cells survived, anddifferentiation to adipose cell.Results:1Laboratory of early identification confirmed: flow cytometry identifyResults showedhUC-MSCs positive for expression of stem cell surface markerCD29, CD44and CD105, hematopoietic stem cell surface markers of CD14,CD34and CD45expression was negative.This results prove that ourobtained the purified mesenchymal stem cells.2Combined with dexamethasone,1-methyl-3-iso-butyl xanthine,insulin and indomethacin induced hUC-MSCs differentiation to adiposecell,conversion rate up to90%, oil red-O staining was positive,prove thatstem cells differentiation to adipose cell.3The infection of stem cell on the weight of fat transplantation:transplant400mg granule adipose tissue, experimental group graft averageweight7days is399.22±5.43mg,14days is380.79±7.30mg,28days is 362.53±9.48mg, the rate of weight maintenance is99.8%,95.19%,90.63%,in the same observation time, the average weight of the control group277.05±6.33mg,230.55±9.28mg,202.37±7.73mg,the rate of weightmaintenance is69.29%,57.64%,50.59%, the difference of the experimentalgroup and control group was significant (P <0.01).4Observed the microvascular of graft: Elected four high-power field ineach period of peripheral and central areas, counting microvascular volume ofeach vision.Dyed brown single endothelial cells, endothelial cell clusters as ablood vessel counts, calculation of microvessels per square millimeter area ofcontent, find the mean. The central area density of blood vessels at7days,14days,28days in the experimental group was29.44±3.86,45.74±5.07,60.79±7.36, in the control group was16.83±3.13,32.01±5.23,44.04±6.10,the vascular density of the surrounding area at7days,14days,28days in the experimental group was65.10±4.18,89.55±6.37,73.30±6.33,inthe control group was47.24±4.49,71.27±3.02,53.54±3.80.SPSS13.0statistical software for statistical analysis. Compared the central area vasculardensity between the experimental and control group in the same time.7daysgroup (P＜0.01),14days group (P＜0.01),28days group (P＜0.01).Compared the around area vascular density between the experimentaland control group in the same time.7days group(P＜0.01),14days group(P＜0.01),28days group(P＜0.01).This result showed whatever central oraround area vascular density in the experimental group should be higher thanthe control group;Compared the experimental group vascular density betweenthe central and around area in the same time.7days group(P＜0.01),14daysgroup(P＜0.01),28days group(P＜0.01).Compared the control groupvascular density between the central and around area in the same time.7daysgroup(P＜0.01),14days group(P＜0.01),28days group(P＜0.01).Thisresult showed whatever experimental or control group vascular density in thearound area should be higher than the central area.Compared the experimentalgroup central area vascular density in there different time (P＜0.01).Compared the experimental group around area vascular density in there different time(P＜0.01).Compared the control group central area vasculardensity in there different time(P＜0.01).Compared the control group aroundarea vascular density in there different time(P＜0.01).This result showed thedifferences in experimental group and control group whatever around orcentral area the number of vascular are statistically significant in threeperiods.And we can see with the time increase, whatever the experimentalgroup or control group around area vascular density highest at14days,28days group is higher than the14days group, vascular density of the centralarea is increased gradually over time.5Immunohistochemical staining detected calibration by BrdU antibodyand nuclei were stained brown fat cells,this result to prove that transplantationof stem cells in the body has become a live and grow into mature fat cells.Conclusion: This study confirmed that human umbilical cordmesenchymal stem cells play an important role in the process of fat transplantas seed cells.1. The hUC-MSCs which subsequently exhibited the phenotypeof adipose cell were mixed with the granule adipose tissue implanted, part ofthe stem cells become the mature fat cells, the formation of receptors innormal, alive adipose tissue.2.The transplanted stem cells accelerate thetransplanted fat angiogenesis, and transplantation of adipose tissue survivalthough paracrine, thus improve the survival rate of transplanted fat.3.Humanumbilical cord mesenchymal stem cells help to form blood vessels and bloodvessel, to promote the growth of transplanted fat.4.Stem cells can generate anew generation of fat cells, the existing fat cells after apoptosis, the new fatcells will be replaced by a vividly,continuous,steady flow of vital force. |
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