| ObjectiveSkin usually damaged by stimulation of certain pathogenic factors such as scratches,burns,bites,sunburn,frostbite,knife,electric burns and others can be collectively called skin injury.The lightest trauma often limited to the skin epidermis,more severe skin damage had skin and subcutaneous tissue rupture and wound,the most most serious injury may have the muscle,nerve and bone fractured.The former can be healed by epithelial regeneration,but the latter two can not achieve complete repair by autotherapy because of the injuries of the skin appendages such as hair follicle,sweat glands and sebaceous glands.The reparative process often requires manual intervention.The traditional methods of treatment about skin injury include autologous skin transplantation,allogeneic skin transplantation,tissue engineering skin transplantation,wound protection agent and so on.With the continuous development of medicine,depending stem cell tissue repair technology which was regarded as one of the most promising therapeutic approaches emerged.Meanwihle,stem cells themselves have the property of multi differentiation potential and low immunity,these qualities make them the ideal seed cells for the acute and chronic trauma of the skin.Hair follicle stem cells(HFSCs)which were located in the bulge of hair follicles has participated in the periodic growth of the hair and hair follicle under physiological conditions.When the skin is hurted,the HFSCs break away from the rest to move to the center of the wound in a linear way and then proliferate and differentiate into epithelial cells,hair follicles,sebaceous glands and so on.How to promote the proliferation and differentiation of HFSCs effectively is of great significance for tissue repair of skin.Studies have shown that the Wnt/β-catenin signaling pathway not only promotes the formation of epidermis and hair follicles during embryonic development,but also maintains the self-renewal and differentiation of adult stem cells after birth.Traditional Chinese medicine has a long history in the treatment of skin trauma,and the system info of Traditional Chinese medicine becomes more mature in the Ming and Qing dynasties.There are many classic works on Department of Dermatology this time and formed different academic schools.“Tortoise shell powder" is a effective prescription to cure skin ulcer and widely used in the clinic.Principal drug in this prescriptiton is plastrum testudinis which flavours Kennedy sex cold and belongs to kidney,liver channel can nourish yin to suppress yang,tonify kidney and strengthen bone,replenish blood for nourishing heart.In our previous study,we found that plastrum testudinis can regulate the express level of Wnt in the rat bone marrow mesenchymal stem cells(MSC),can also induce the proliferation of epidermal stem cells(ESC)and prevent the apoptosis of epidermal stem cells caused by UV damage or serum damage.It is speculated that Plastrum Testudinis extracts can also promote the proliferation of HFSCs so as to achieve the purpose of promoting wound healing by activating the Wnt/β-catenin signalin pathway because of the homology between HFSCs and ESC.Methods1.Newly born Sprague-Dawley(SD)rats were choosen to separate hair follicle stem cells using two step enzyme digestion method.Immunofluorescence detection of the expression of CD34 and CD71 the cell surface markers of HFSCs.The cytoactive of HFSCs which were stimulated by Cholesterol Myristate(S8)and(+)Cholesten-3-one(S9)were detected by Cell Counting Kit-8 techniques.Immunofluorescence,Western-blot,real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)detected the express of β-catenin,GSK-3β,c-myc,Lefl,Tcf3,cyclin D1.2.Divide the male rats between 200～220 g into control group(normal saline group),model group(saline solution containing 10%DMSO),positive control group(Epidermal growth factor,EGF),the experimental group(treat with S8,S9 respectively).Detect the changes in skin wound repair by observing the rats wound healing score,wound healing rate,pathological HE staining.Effects of S8 and S9 on the expression of Wnt/β-catenin signaling pathway in rat skin wound healing were detected by immunofluorescence.3.The cultured HFSCs was suspended by PBS with a density of 106/mL.Establish skin defect rat model,the successful model of the rats were randomly divided to control group,model group and S9 group.The rats in control group were were injected with 20 u L PBS at the midpoint of upper edge and the right edge of the wound,the model group was injected with 20 u L HFSCs suspended with PBS in the same way,S9 group was injected with PBS suspensions of HFSCs pretreated with S9,normal diet feeding 4 d.The pathological changes of each group were detected by HE staining.The changes of collagen were detected by VG staining.Results1.HFSCs were isolated and cultured in vitro with two step enzymatic hydrolysis.These were adherent cell which form small,transparent soma,strong refraction.The cells are like typical paving stone closely spaced between them which were CD34 and CD71 positive.Both S8 and S9 could significantly promote the proliferation of HFSCs,and the optimum concentration was 30 μg/mL.Under the influence of S8 and S9,the expression of GSK-3β was decreased,the expression of β-catenin was increased,the expression of Lefl,c-myc and cyclinDl were increased,the expression of Tcf3 was increased under the action of S8 and decreased under the action of S9.The results indicated that S8 and S9 could activate Wnt/β-catenin signaling pathway in HFSCs.2.Establish skin defect rat model.According to different groups of different treatment making criterion to observe the wound healing.The results of semi quantitative statistics showed that:after 4 d and 7 d’s treating,the average score and healing rate of S8 group and S9 group were higher than that of control group and model group.The pathological results showed that in 1 d after operation,the inflammatory response of the wound was obvious in all groups.4 days after operation,the control group and the model group had obvious hyperemia and inflammatory cell infiltration,and some of them were covered with blood crusts,while the epidermis of S8 group,S9 group and positive control group were completely covered with no congestion,and the epidermis and dermis were relatively firm.On the 7 day after operation,the inflammatory reaction of SD rats in each group was completely subsided.In the control group and model group,the thickness of the skin was significantly increased,especially in the spinous layer of the skin,while S8 group and S9 group showed a slight thickening of the epidermis,confirmed that the repair was better than the control group and model group.The above results suggest that S8 and S9 in the early and mid-term of wound healing can reduce inflammation,inhibit wound bleeding,wound scar playing a positive role in promoting wound healing.Detecting the Wnt/β-catenin signaling pathway with immunofluorescence assay after S8,S9 were used in the SD rat whiskers proves that S8 and S9 could promote the wound healing by activating Wnt/β-catenin signaling pathway.3.HFSCs suspended in PBS were injection into whisker wound in rats.The results of HE staining showed that on the fourth day after operation,the congestion and inflammatory reaction were obvious the in control group,and the epidermis and dermis were not connected;there was a slight congestion in the skin area of the wound,no obvious inflammatory reaction,and the epidermis and dermis were connected firmly in the S9 group.VG staining showed that the collagen near the wound edge of normal skin counts,while wound center decrease.In control group,the epidermis and dermis were not well connected and there was almost few collagen fiber filling;a small amount of collagen fibers exist in the center of the wound;the collagen fiber content in S9 group was abundant.Conclusion1.The cells which were Isolated and cultured from epidermis were HFSCs.2.S8 and S9 can promote the proliferation of HFSCs by activating the Wnt/β-catenin signaling pathway.3.S8 and S9 can activate Wnt/β-catenin signaling pathway to promote the wound healing.4.Under the joint action of S9 and HFSCs,the wound healing of rats could be improved. |
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