| Objective: Grasp isolation,cultivation,amplification and discuss part of biological characteristics of human adipose-derived stem cells.Study the effects of fibrin glue on proliferation of ADSCs.To build a new injectable biological scaffold material,establish the experimental basis for clinical repair soft tissue defect.Method: 1.ADSCs were isolated from liposuction's fat particles by collagen digestion.Observe the transform of ADSCs morphological in vitro under inverted microscope.Cell proliferative ability was assayed by cell growth curve using MTT.P3 cells separately adding DMEM contain insulin,IBMX,dexamethasone,indometacin and DMEM contain dexamethasone,phosphoglyceride,vitamin C induce to adipogenesis and osteogenesis.Oil red O staining,Alizarion red staining test,observe the transform of ADSCs under inverted microscope.2.Cultivate ADSCs in vitro,take P3 cells inoculated into 96-well plates.With different concentrations(fibrin and thrombin proportion: 4:1,2:1,8,3,5,2)fibrin glue soakage solution,MTT method to detect ADSCs proliferation in different concentrations of fibrin glue.3.the experiment was divided into 5 groups: Group A: pure fibrin glue group;Group B: pure fat tissue after purification;Group C: pure ADSCs group,which concentration is 3×106/ml;Group D: cells-fat mixture: ADSCs concentration is 3×106/ml and purification fat tissue 1:1 mixed into cells-fat composites;Group E: cells-scaffold mixture: ADSCs with 3×106/ml and the concentration of fibrin glue in 1:1 volume mixing.Each injected 0.2ml into nude mice(10,4w male)subcutaneously,the formation of 5 points,the nude mice were observed after implantation of vital signs and local inflammation reaction after 8 weeks.The new organization will be evaluated from general observation,volume,quality,HE-staining.Results: 1.ADSCs were successfully isolated and cultured from liposuction's adipose tissue.They appeared the morphology like fibroblast.Primary cells adherence after 3-4 hours like-class circle,extend after 2-4days typical like fibroblast.ADSCs enter the logarithmic growth phase in 5 days,single cells fusion rate achieved about 90 percent which cells arranged tightly into a spiral.After passage ADSCs adherence in 2-4 hours,extend rapidly achieved 80 percent in 2-4 days.P3-P8 cells proliferate rapidly,a little less later.Oil red O staining showed many lipid drops among ADSCs,Alizarion red staining showed that many white nodules among cells.2.Different concentrations of fibrin glue preparation soakage solucion were no difference betwee ADSCs growth curve.3.the experiment of 10 nude mice implanted in local stable vital signs after 8 weeks,the vital signs were stable,each mouse injection site showed no obvious inflammation,group B ? D ? E have new organization,group A and C were not formed.General observation,Group B?D showed obvious yellow fat particles,group E also has yellow fat particle but smaller than other groups.HE-staining showed that four group B?D?E have visible fat cells,especially group D contain more collagen and fat particles.Volume:group D>group B>group E;Quality:group D>group B>group E.Conclusion: A simple method of isolation and cultivation of ADSCs was successfully established in vitro.ADSCs could proliferate rapidly,readily available and multilineage differentiation.ADSCs can be an ideal seed cells for tissue engineering.Different concentrations of fibrin glue were not significantly affect ADSCs proliferation,fibrin glue is expected to become one source of stem cells to grow fat scaffold material.ADSCs can adherence,growth on fibrin glue,fibrin glue can be used as the carrier of ADSCs and the scaffold material in tissue engineering. |
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