The Experimental Study Of Human Adipose-derived Stem Cells Transplanted Into Nude Mice Subcutaneous Stratum

Objective:1.To isolate,culture and identificate the human adpose-derived stem cells(hADSCs) in vitro.2.To inject the marked hADSCs subcutaneously and observe the survival and transformation of the cells.Methods:Human adipose tissues was isolated and cultured by the method of collagenaseⅠdigestion and adhering to the culture flask .The morphological changes of the cultured cells were observed. The hADSCs were cultured under the conditions of adipogenic induction and osteogenic induction for 3 weeks. Oil red staining and calcium staining were used to observe the multi-differentiation ability of hADSCs. Freshly isolated cells, primary passage cells and the third passage cells were tested by the fluorescence-activated cell sorting(FACS). Surface marker of CD45,CD34 and CD133 were used. The hADSCs were treated by liposome-mediated pEGFP-N3 plasmid transfection and G418 screening. The green fluorescent protein(GFP)positive ratios were observed. After the liposome-mediated pEGFP-N3 plasmid transfection and G418 screening for 5 passages, the transfected hADSCs were transplanted into nude mice subcutaneously by means of injection. 4,8 and 12 weeks later, the gross observation and micro-observation were used for evaluating the cell-transplantation effects.Results:1.Use the method of collagenaseⅠdigestion , the lots of hADSCs can be isolated by collagenase/adipose ration of 3:1, collagenase concentration of 1 mg/ml and digestion time of 1 hour. In Vitro human ADSCs are in the shape of fibroblast-like cells.2.Under the condition of adipogenic induction or osteogenic induction, hADSCs can differentiate into adipocytes or osteoblasts.3.In the freshly isolated cells, a large number of cells are CD45~+CD34~- (37.62%) and CD45~-CD34~+(24.22%).In the primary passage, a large number of cells are CD34~+CD45-(58.76%) and no cells are CD45~+ or CD133~+.In the third passage, the CD34~+CD45~- cells is lowered to 30.47%; the number of CD45~+ cells is very low and no cells are CD133~+.4.After the transfection of pEGFP-N3 plamid for 24 hours, a large number of hADSCs express GFP and the positive cells'rate is (3.0±3.5)%. After screened by G418, the rate reach to (65.0±5.4)%.5.By means of injection, the pEGFP-N3 transfected hADSCs can be transplanted into nude mice subcutaneously. 8 weeks and 12 weeks later, green fluorescent positive areas can be seen by gross observation. Under the observation of microscope, the green fluorescent positive cells can be seen in the subdermis area and are corresponding to the adipocytes. There is no green fluorescent structure in the dermis.Conclusion:1.A large number of hADSCs can be isolated from adipose tissue,which have the multi-differentiating ability and have the same phenotype characters of stem cells, CD34~+CD45~-CD133~-.2.By liposome-mediation, the pEGFP-N3 plasmid can be transfected into hADSCs and the G418 screening is helpful to increase the positive rate.3.hADSCs can transform into adpocytes in nude mice after being injected subcutanteously and thus hADSCs may be used as the seed cells for adipose tissue engineering.