The Experimental Study To Construct Tissue Engineering Adipose With Human Umbilical Cord Mesenchymal Stem Cells

Objective 1.To explore the conditions of isolation,purification,and culture of Umbilical Cord mesenchymal stem cells(hUCMSCs) in vitro,and study its biological characteristics;2.To study the potential ability of hUCMSCs to differentiate into adipogenic cells after they were frozen and thawed;3.To observe the attachment,shape,function and activity of hUCMSCs cultured on silk fibroin porous scaffolds in vitro,and to construct the tissue engineering adipose With hUCMSCs and silk fibroin porous scaffoldsMethods 1.After stripping off arteries and veins,there maining parts of umbilical cord were cut into small sections and plated in 100mm culture dish. Adherent cells coming out of fragments.The morphology of hUCMSCs were observed by inverted phase contrast microscope,the growth curves of them were drawn and the surface antigens of hUCMSCs were detected by flow cytometry.To induce the cells to differentiate into adipose and bone,and the potentiality of its osteogenic and adipogenic differentiation were detected by chemistry staining RT-PCR,the stem cell marker gene was measured by RT-PCR;2.The one-passage hUCMSCs were preserved in liquid nitrogen by reducing the temperature gradually. Five months later,the cells were thawed and their surface antigens were detected.The 12-passage hUCBMSCs were treated with differentiation medium,and Oil Red-O analysis was empolyed to detect the adipogenic differentiation after 21 days post-culture,RT-PCR analysis was used to detect peroxisome proliferator-activated receptorγ-2 and lipoproteinesterase on the 7 days and 14 days post-inducing respectively;3.The silk fibroin porous scaffolds were subsequently seeded with hUCMSCs and cultured in vitro.The growth and function of hUCMSCs were observed and measured with fluorescence inverted microscopy,scanning electronic microscopy and MTT method,the cell-scafold complexe was induced under the induction of adipose medium for 6 weeks,and then transplanted this complexe into rats,The constructed adipose was observed with scanning electron microscope and Oil Red-O in 4th and 8th week after transplantation respectively.Results 1.4-6 days after primary culture,adherent cells came out of fragments and these cells could be maintained for 10 passages without obvious changes in morphology or growth pattern.Flow cytometry analysis revealed that CD13,CD44 were expressed on the cell surface.CD14,CD34 and HLA-DR were negative or weakly expressed.The induction in vitro demonstrated the cells exhibited multi-potential differenti -ation into osteogenic and adipogenic cells,RT-PCR results showed that these cells expressed oct-4;2.The living cells of hUCMSCs were86%in the 5th month after frozen.FACS revealed that these cells expressed common markers of MSCs,such as CD13,CD44 and CD71,but did not express such markers as CD14,CD34 and HLA-DR.Adipogenic differentiation was seen as Oil red-O-positive cells and was also analyzed by RT-PCR,in which the adipocyte marker LPL and PPARγ-2 was expressed in the adipogenic formula-treated cells;3. hUCMSCs were fixed on silk fibroin porous scaffolds lor2 days later,and muitiplicative growth was observed on the 5th to 7th day,After 10 days,The microholes of the scaffolds were overlayed by hUCMSCs.Scanning electronic microscopy and fluorescence inverted microscopy showed that the cells adhered to scafold well and there were a lot of extra cellular matrix surrounding cells,hUCMSCs could differentiate into adipogenic cell under the induction of adipose medium after 4 and 6 weeks,New mature adipose was observed in the scaffold in 4th and 8th week after transplantation,and the histological examination revealed the proliferated adipocytes and extracelluar matrixes.No new mature adipose was observed in the control group.Conclusion 1.hUCMSCs could be cultured and proliferated in vitro and also have the similar biological characteristics with bone marrow -mesenchymal stem cells;2,hUCMSCs could be obtained by means of the cultivation of small tissue sections,and preserved by cryopre -servation.The hUCMSCs frozen and thawed were cultured to 12-passage still have adipogenic differentiation potential ability when frozen for 5 month,so hUCMSCs could be regarded as seed cells for adipose tissue engineering;3.Silk fibroin porous scaffolds are ideal for attachment,growth, function main- tenance and activity of hUCMSCs,and the scaffolds could be used as natural scaffolds for hUCMSCs in 3D culture,The scaffold is an ideal biological and biodegradable scaffold,hUCMSCs seeded in this scaffolds could construct tissue engineering adipose.