| Hair follicle is a miniature organ composed of outer root sheath,inner root sheath and hair shaft,which is always in a periodic cycle of growth,regression and resting period.Intracutaneous adipose tissue is an important macroscopic environment of hair follicle,it is found that the subcutaneous adipose tissue is closely related to the formation and function of hair follicle.In fact,many studies have shown that the regeneration cycle of hair follicles is induced by stem cells,and is closely related to adipose tissue and adipose cell lineage cells.The mechanism of paracrine is considered to be the key to promotion of the regeneration of hair follicle tissue by adipose mesenchymal stem cells(ADSCs).It is believed that the growth factors,cytokines and chemokines secreted by stem cells,affect the survival,proliferation,migration and gene expression of cells in tissue specific.Among many factors affecting the ADSCs function,the oxygen concentration in the surrounding environment,especially the hypoxic environment,plays an important role.Hypoxiapromotes the proliferation and self-renewal ability of ADSCs,and facilitates its regenerative potential.At the same time,the paracrine of ADSCs can be enhanced by enhancing the secretion of some growth factors.In this study,cashmere goat ADSCs were selected as the research object,and Deferoxamine(DFO)was used as analogue of hypoxia.At first,ADSCs were cultured with DFO,and the effects of hypoxia on the growth,proliferation and paracrine of ADSCs were detected.Then the hair follicle stem cells(g HFSCs)were cultured by conditioned medium to detect the change of growth and colonization ability of g HFSCs,and the effect of the paracrine function of ADSCs on g HFSCs was observed.Finally,the signal pathways related to the growth of g HFSCs were analyzed to explore the role of ADSCs on the development of hair follicles,and to further understand the characteristics of hair follicle development and molecular regulatory mechanisms of cashmere goats,so that to provide experimental basis for cashmere goat villus growth mechanism.1.Effects of DFO on growth,proliferation and paracrine of ADSCsIn this study DFO treatment concentration and processing time were filtered through the method of CCK-8 activity detection,ADSCs were cultured with DFO solution concentrated in 25,50,100,200?mol/L for 24,48,72 h respectively.The results showed that the cell activity was the highest in the treatment of DFO concentrated in 25?mol/L for 48 h.Then the ADSCs treated with 25?mol/L DFO for48 h were detected by the method of cell count,CCK-8 activity detection,cell cycle and apoptosis detection.The results showed that compared with the cells in thecontrol group,the cells treated with 25?mol/L DFO for 48 h in G0/G1 phase were found to have an evident decline(p<0.05),and that in S-phase was not significant different(p>0.05),and that in M-phase were increased significantly(P<0.05).The cell cycle was stopped in M-phase by this treatment,and the number of apoptotic cells decreased significantly(P<0.01);The change in contents of the factors related to hair follicle,IGF-1,VEGF,b FGF and PDGF,in g ADSCs was detected by ELISA quantitative detection before and after the DFO treatment.The results showed that the content of IGF-1 and PDGF was increased in the supernatant of g ADSCs treated with 25?mol/LDFO for 48 h compared with that of g ADSCs untreated and normally cultured,but the difference was not significant(P>0.05).The content of VEGF and b FGF was increased,and the difference was extremely significant(P<0.01).The results indicated that the hypoxia stimulant,DFO,could significantly enhance the growth and proliferation of ADSCs,and significantly promote its paracrine function.2.Effects of ADSCs conditioned medium on the activity,cell cycle,apoptosis and related gene expression of g HFSCsIn this study,g HFSCs were cultured in conditioned media cultured ADSCs for48 h with DFO or not,then were detected by the method of cell count,CCK-8 activity detection,cell cycle and apoptosis detection.The results showed that the g HFSCs cultured in conditioned medium cultured ADSCs for 48 h with DFO were the most active.Compared with the control group cells cultured not in the conditioned medium,the number of g HFSCs cultured in conditioned medium cultured ADSCs withoutDFO in G0/G1-phase were decreased and in M-phase were increased,but the difference was not significant(p>0.05),and in the S-phase was increased significantly(P <0.05).The number of g HFSCs cultured in conditioned medium cultured ADSCs with DFO in G0/G1-phase was significantly reduced(P <0.05),and in S-phase and M-phase cells was significantly increased(P <0.05).It was indicated that the cells were blocked in S and M-phase by this conditioned medium treatment,and the number of apoptotic cells decreased significantly(p <0.01).A real-time quantitative detection was used to detect the change of HIF-1,VEFG and ?-catenin in g HFSCs before and after the conditioned medium treatment.The results showed that the transcription levels of HIF-1,VEGF and ?-catenin in the cells treated by conditioned medium were all increased compared with that in the control cells,and the difference was very significant(P<0.01).The results indicated that g ADSCs played an important role in the development of hair follicle,and this effect could be enhanced by hypoxia.3.The molecular mechanism of the indirect promotion of g ADSCs cultured by hypoxia simulant DFO on the growth of g HFSCsIn this study,a real-time quantification PCR and Western blot detection were performed to detect the changes of content of HIF-1,VEGF and ?-catenin in g ADSCs treated with DFO for 48 h.The results showed that the transcription levels of HIF-1,VEGF and ?-catenin gene in the cells treated by DFO hypoxia simulant were higher than those in normal cultured cells without DFO treatment,and the difference was extremely significant(P<0.01).The results of Western blot test showed that thecontent of HIF-1,VEFG and ?-catenin protein increased.The signal pathway of ERK and Akt in g HFSCs cultured by the culture medium were detected by western blot detection as well,the results showed that ADSCs could also activate the proliferation-related signal Akt and ERK in hair follicles.The results of this research indicated that the proliferation activity of g ADSCs was significantly increased after hypoxia treatment,the paracrine effect was more prominent.The conditioned medium could stimulate hair growth,which could be enhanced by hypoxia.ADSCs could also promote hair follicle regeneration by activating proliferation-related signals Akt and ERK in g HFSCs. |
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