新生SD大鼠毛囊干细胞的分离、培养及鉴定

Objectives: To investigate an efficient access to separate, purify, culture and identify the hair follicle stem cells (HFSCs) of newborn SD rat in vitro, and observe the biological characteristics of HFSCs. Methods: To cut off 1-week-old rat whisker hair follicle tissue under microscope, separate the hair follicle bulge under microscope, then use a two-step digestion of Dispase andⅠcollagenase to harvest single cell suspension, purify and culture by rapid adhering on collagen type Ⅳ. Determine growth curve and colony-forming rate of different generation of HFSCs. To identify the surface signs of these cells by immunofluorescence staining. Results: The HFSCs which were isolated, cultured, and purified were like a typical "slabstone", had strong adhesion ability and colony formation ability. Cultured cells were in exponential phase at 5~6d, reach breeding peak at 8d, then entering into the platform period. Proliferation of P2, P3, P5 HFSCs decreased gradually with the passage (P< 0.05); P2, P3, P5 HFSCs all had high clone formation rate, also decreased gradually with the passage (P<0.05). Hair follicle stem cell marker CD34, K15, and β1 integrin expression were positive by immunofluorescence staining. Conclusion: We successfully obtained rHFSCs of a high purity by using microscope isolated combined a two-step enzymatic digestion and collagen Ⅳ differential adhesion purification. Cultured HFSCs in vitro had a good vitality, high proliferative capacity and stem-cell like biology characteristics, which provided a reliable method for the study of stem cell therapy and may be a new seed cell for tissue engineering. ?????