| Objective: 1. To investigate the method of isolation, purification and culture human umbilical cord mesenchymal stem cells(h UCMSCs) in vitro, and study its biological characteristics; 2.To observe the attachment, purification, function and differentiation of h UCMSCs cultures on silk fibroin porous scaffolds, and to explore the possibility of construct the tissue engineering adipose with h UCMSCs and silk fibroin porous scaffolds.Methods: 1.The h UCMSCs were obtained from the full-term umbilical cords by utilizing tissue block method, the acquired cells were cultured and propagated in vitro. We observed the characteristics of the cells by inverted phase contrast microscope, the cells growth curve were measured by MTT method, and check the surface antigens by flow cytometry. The 3rd passage cells were induced to differentiate into adipose cells by means of an induction differentiation medium, the Oil Red-O analysis check the adipogenic differentiation after 21 days post-culture. 2. The h UCMSCs were seeded in silk fibroin porous scaffolds to construct cells-scafford composites in vitro. The adhesion ratio of silk fibroin porous scaffolds composite wih h UCMSCs were detected; the cells growth curve in scaffolds were measured by MTT; and we observed the compound situation of cells by the fluorescence inverted microscopy; the cells-scafford composites were induced under the induction of adiose medium for 2 weeks, and RT-PCR analysis was used to examined the expression of Oct-4, a P2, LPL on the 14 d post-inducing respectively. The induced cells-scafford composites were implanted into the subcutaneous tissue of SD mouse. ALL of implant were performed examination of HE staining 4th, 8th and 12 th week after transplantation respectively.Results: 1. After about 2 weeks culture, a few adherent fibroblast-likes cells could be observed around the fragments, cells fused more than 80%-90% after the 3 weeks. These cells could be maintained for more than 10 passages without obvious changes in morphology and growth pattern. The growth curve showed that proliferation ability of the 3rd and 10 th passage cells was powerfol. Flow cytometry shows that 90% and 97% of the 3rd passage cells expressed CD 44 and CD 105,while only 2.6% and 1.2% of the cells expressed CD14 and CD 34. The induction in vitro demonstrated the cells could be differentiate into adipogenic cells. 2. The h UCMSCs had a good quality of growth, multiplication and differentiation in the silk fibroin porous scaffolds. The adhesion ratio of the h UCMSCs were over 80% in the silk fibroin porous scaffolds at 12 h. The growth curve showed no statistical difference between experience group and control group. The cells extened and proliferation in the silk fibroin porous scaffolds were observed by fluorescence inverted microscopy. The RT-PCR verified the expression of a P2 and LPL on the 14 d post-inducing, and the expression of Oct-4 reduced. New adipose was observed in the scaffold in 4th, 8th and 12 th week after implantion, and the scaffold material partly absorbed. No mature adipose was observed in the control group.Conclusion: 1. The h UCMSCs could be culture and proliferated in vitro, and the characteristics of the cells were similar to the mesenchymal stem cells; 2. The h UCMSCs could be accorded as a kind of seed cells for adipose tissue engineering; 3. The silk fibroin porous scaffolds is an ideal biological scaffold for adipose tissue engineering, and hadn’t a negative impact on the growth, proliferation and differentiation of h UCMSCs. The induced cells-scafford composites could construct tissue engineering adipose. |
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