The Experimental Study Of Seed Cells For Repairing Muscular Lumen Defects Of Urinary Tract

Part1 Primary culture of beagle dogs'adipose-derived stem cells and differentiation induction into myoblastsObjective:To explore the method of culture of beagle dogs adipose- derived stem cells (ADSCs) and differentiation induction into myoblasts .Methods: Adipose tissues were obtained from beagle dogs, and were isolated by enzyme digestion and cultured into ADSCs . The expression of surface antigen CD90, CD105 and CD34 was detected by immunofluorescence and flow cytometry . ADSCs of the second passage with logarithmic growth were obtained, and culture media containing 5- azacytidine (5-aza) and basic culture media were employed for cells in induction group and control group, respectively . The induction lasted for 7 d, 14 d, 21 d and 28 d, respectively . Cell growth and cell morphology were observed by inverted phase contrast microscope, and immunofluorescence and flowcytometry were utilized to detect the expression of myoblast specific antigens desmin and myosin .Results: ADSCs were successfully isolated and cultured, and were identified to be stem cells . On the 21th day of induction, cells in induction group displayed"swirl"morpholgy, and in 4th week of induction , multinucleation was observed . It was revealed by immunofluorescence and flowcytometry that the highest expression rates of desmin and myosin were 59.4% and 56.1% , respectively on the 28th day of induction, while there was no expression before induction and in control group.Conclusion:ADSCs can be isolated and cultured from beagle dogs adipose tissues, and can further differentiate into myoblasts after induction by culture media with 5-aza.The expression of myoblast specific antigen is the highest on the 28th day of induction .Part2 Experimental study about the primary culture of beagle dogs'epidermal cells and identificationObjective: The feasibility of canine epidermal cells'primary culture, and provide a reliable seed cells for the next experiment.Methods: Oral epithelial tissues were obtained from male beagle dogs, and were isolated by enzyme digestion, using i3T3 as the cell layer and cultured into epidermal cells. Cell growth to about 80% of culture dish passaged line treatment; observed changes in cell morphology under inverted microscope daily; immunofluorescence and flowcytometry were utilized to detect the expression of marker proteins AE1/AE3 .Results: Epidermal cells were successfully isolated and cultured. Epidermal cells grow slowly. On the 21th day of primary culture, the cell can achieve about 80% of the 100mm culture dish. After passage growth is significantly faster,it needs 10 day to achieve the same area. It was revealed by immunofluorescence and flowcytometry that the result of epidermal cells'specific antigens- AE1/AE3 is satisfactory.Conclusion:this study further evidence of epidermal cells can be obtained by primary culture and are able to be used and reliable as seed cells for further experiments.