| Objective: To explore the rabbit adipose tissue isolation, culture and identification of stem cell methods; observation of long-term culture in vitro of New Zealand white rabbits adipose-derived mesenchymal stem cells (Adipose-Derived Stem Cells, ADSCs) of the basic biological characteristics and orientation induced differentiation of cartilage capacity. Methods: The adipose tissue , 5 ~ 10g, is taken from the neck of male New Zealand white rabbit Under sterile conditions, I collagenase digestion, 1500r/min centrifugal 10min. when the cells were covered 80% of the bottom of the bottle,they were digested by 2.5 g / L trypsin and subcultured by 1:3 ratio. The third generation cells were added cartilage induction media , deteced of their related phenotype by immunofluorescence and RT-PCR after 14d , the cartilage differentiation of adipose-derived mesenchymal stem cells were detected by alcian blue staining and type II collagen immunohistochemical. Results: Rabbit adipose-derived mesenchymal stem cells morphology:the rabbit adipose-derived stem cells in primary and passaged were long spindle-shaped or polygonal adherent growth, active growth and differentiation. the growth curve of rabbit adipose-derived mesenchymal stem cell:the cell growth curve is "S" shape. the cells isolated and cultured New Zealand white rabbits adipose tissue were detected by flow cytometry, CD44, CD49d expression was positive, CD45 expression was negative. Induction of cartilage induced medium after 72h, the cells grow slowly, the shapes of cells were changed from long spindle gradually to round, and the cartilage nodules were formed gradually, the cytoplasm appeared blue particles, the surface of cells had more pseudopodias, with a normal organelle structure. The celles induced after 2 weeks, there are expressions of specific cartilaginous matrix of typeⅡcollagen and Aggrecan. The detected results of typeⅡcollagen, Aggrecan and Sox-9 mRNA expression, alcian blue staining and type II collagen immunohistochemistry by RT-PCR were positive. Conclusion: New Zealand white rabbits adipose adipose tissue can be isolated and cultured adipose-derived stem cells in vitro with growth and stability,the characteristics of rapid proliferation,biological characteristics and bone marrow-derived mesenchymal stem cells. |
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