| ObjectivePressure ulcers are a global healthcare problem greatly affecting the quality of patients life and are a major cost to the healthcare system,with high incidence and difficult controlling.Skin wound repair depends on the proliferation,differentiation and migration of multiple skin stem cells,among which the hair follicle stem cells do play very important roles.Understanding the regulation of hair follicle stem cells fate,either proliferation or differiation,and how they respond to physiological changes or certain drugs can not only provide us new ideas for skin wound treatment,but also give implications for new drug screens and novel treatment designs.The two most important pathways controlling hair follicle stem cells are Wnt and BMP signaling,which regulate the proliferation or differiation of hair follicle stem cells as a counterbalance,thus may be the potential key of skin wound treatment.Plastrum Testudinis was one of the top-grade materia medica listed in Sheng Nong’s herbal classic,with great effects of nourishing yin for suppressing hyperactive yang,tonifying kidney,and nourishing blood.Moreover,PlastrumTstudinis extraeted with ethyl acetate(PTE)and its effective constituents have been reported to have good anti-apoptosis effects and can promote the proliferation and osteogenesis differentiation of mesenchymal stem cells.Yet,their effects on hair follicle stem cells are rare.Our study tried to explore the effects of PTE and its effective constituents on hair follicle stem cells in vitro and the related signaling pathways,and their effects on treatment of stage I pressur ulcer in vivo,thus laying foundations for clinical application or new drugs development.Methods1.Rat hair follicle stem cells were derived from newborn SD rats by microdissection combined with two-step enzyme digestion,cultured in Keratinocyte serum-free medium,and indentified by specific markers.2.The effects of PTE and its effective constituents on hair follicle stem cells were assayed by Cell Counting Kit-8 and flow cytometry.Cell growth curves were drawn and the appropriate intervention time and drug concentrations were selected.3.The expression of β-catenin and BMP4 were detected by Western-blot and the mRNA of downstream genes were detected by PCR.4.To establish the IR cycle model in SD rats,with the evaluation of Stage I pressure ulcer.5.The effects of PTE and its effective constituents on treatment of Stage I pressure ulcer were observed and related mechanism was preliminary explored.Results1.The hair follicle stem cells in vitro cultivation system was established successfully.Several kinds of dyeing with hair follicle stem cells specific markers showed that the purification of hair follicle stem cells are more than 90%,which guarantees the use for subsequent experimental study;2.Cholesterol Myristate,(+)-Cholesten-3-one and PTE in proper concentration can promote the proliferation of hair follicle stem cells,with G1 phase shorten,or S phase prolonged.3.Cholesterol Myristate,(+)-Cholesten-3-one and low concentrations of PTE can up regulate the expression of β-catenin while down regulating the expression of BMP4;high concentrations of PTE regulate the two proteins in a reverse one4.The IR cycle model was successfully established in SD rats,with the evaluation of Stage I pressure ulcer.5.Cholesterol Myristate,(+)-Cholesten-3-one and PTE helps the process of wound repair by regulating Wnt/β-catenin and BMP signaling.Conclusions1.By adopting microdissection combined with two-step enzyme digestion,we can quickly isolate the hair follicle stem cells in good viavility and high purity.2.Cholesterol Myristate,(+)-Cholesten-3-One and PTE in proper concentration can promote the proliferation of hair follicle stem cells,which is related with the Wnt/β-catenin and BMP signaling.3.Cholesterol Myristate,(+)-Cholesten-3-one and PTE helps the process of wound repair by regulating Wnt/β-catenin and BMP signaling,but their target cells and the detailed mechanism still need further research. |
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