Study On The Mechanism Of Adipose Derived Stem Cells Promoting The Proliferation Of Dermal Papilla Cells In Hypoxic Microenvironment

Hair follicles are skin appendages that control hair growth and can be self-renewing through periodic structural changes.Mature hair follicles have complex structure,including keratinocytes,melanocytes and DPCs?dermal papilla cells?.DPCs are highly specialized from fibroblasts originating from the dermal interstitial cells and play a key role in the cycle of hair follicles.Even in vitro,DPCs still have the function of inducing hair follicle formation and promoting hair follicle growth.ADSCs?adipose derived stem cells,ADSCs?surrounded the outer layer of DPCs can optimize the microenvironment to promote hair folliclesgrowth.ADSCsoriginatedfrommesodermhave multi-directional differentiation potential.Due to their abundant sources and low immunogenicity,ADSCs have broad application prospects in cell therapy and tissue engineering in recent years.Oxygen is an important component of the stem cell microenvironment.The survival,maintenance and behavior of stem cells are inseparable from the regulation of oxygen.Hypoxic microenvironment is essential for promoting the proliferation of ADSCs,maintaining their normal morphology and multi-directional differentiation potential.Therefore,the cultivation of ADSCs in hypoxic microenvironment may promote the growth of hair follicles.However,it is still unclear which molecular mechanisms are involved in this process.Therefore,it is of great theoretical significance to study the mechanism of ADSCs promoting the growth of hair follicles in hypoxic microenvironment.In this research,ADSCs and DPCs of Arbas Cashmere Goats were used as the research objects.The hypoxic microenvironment of ADSCs was established to study the changes of ADSCs function in hypoxic.Further,DPCs were cultured in ADSCs conditioned medium,the changes of DPCs growth and the expression mechanism of related growth factors were detected,so that to provide experimental basis for understanding hair follicle growth and its molecular mechanism.1.Establishment of hypoxic microenvironment and its effects on the function of adipose derived stem cellsIn order to establish the hypoxic microenvironment of ADSCs,this study firstly examined the effects of different concentrations of hypoxic mimics DFO?desferrioxamine,DFO?and CoCl₂?cobalt chloride,CoCL₂?on the proliferation of ADSCs cultured for 24 h,48 h and 72 h respectively.The results showed that the proliferation of ADSCs cultured in 25?M DFO and 150?M CoCl₂ for 48 h was the strongest.At the same time,the cells were cultured in physical hypoxia.Compared with ADSCs cultured in normoxia,it was found that the proliferation activity of ADSCs cultured in both the physical hypoxia and the hypoxia mimic was increased.The detection of HIF-1?in ADSCs cultured in hypoxia for 24h,48 h and 72 h showed that HIF-1?was expressed,and the expression was higher at 48 h.In addition,the expression of ADSCs markers CD44 and CD90 were positive,and the hematopoietic stem cell markers CD34 and CD45 were negative after hypoxia culture.The expression of pluripotency genes NANOG,OCT4 and SOX2 were not only continuously expressed,but also increased after hypoxia culture.At the same time,in the physical hypoxia group,the expression of proliferating gene TERT increased and promoted cell proliferation.The expression of tumor suppressor gene p53decreased,and the proportion of apoptotic gene Bcl-2/BAX increased and inhibited cell apoptosis.Taking the established physical hypoxic model as the experimental object,the changes of cell function of ADSCs were examined,including cell migration ability,angiogenic ability and paracrine level.The results showed that cell migration ability was stronger in hypoxic environment.Under the action of matrigel,ADSCs could form vascular-like structure in vitro,and both the number and length of blood vessels per unit area were significantly higher in hypoxic culture than that in normoxic culture.And the results of paracrine levels of ADSCs showed that the Hypo-cm?hypoxic conditioned medium,Hypo-cm?contained more growth factors,including VEGF,bFGF and PDGF.2.Effects of conditioned medium of adipose derived stem cells on the growth of dermal papilla cellsPHF-DPCs?primary hair follicle drmal papilla cells,PHF-DPCs?and SHF-DPCs?secondary hair follicle dermal papilla cells,SHF-DPCs?were coultured in Hypo-cm and Nor-cm?Normoxic conditioned medium,Nor-cm?.The results of detecting the growth of DPCs found that conditioned medium,especially Hypo-cm,could significantly increase the proliferation of DPCs.In addition to the higher rate of EdU positive cells,the cells of expressing proliferation gene Ki67 were also significantly higher than the control group.Cell cycle results showed that DPCs cultured in conditioned medium were significantly increased the proportion of cells entering S phase and the expression of CDK2.In addition,the proportion of apoptosis cells was decreased,which also proved that conditioned medium could promote the proliferation of PHF-DPCs and SHF-DPCs.The results of ALP detection showed that the ALP staining and activity of DPCs cultured in conditioned medium were significantly higher than those of the control group.The results of other DPCs specific marker genes SOX2,?-SMA,NCAM and Version expressions showed that the DPCs specific marker genes could be improved by culturing DPCs in conditioned medium,especially in Hypo-cm.It was also proved that conditioned medium could promote the growth of PHF-DPCs and SHF-DPCs and enhance their hair follicle induction ability.3.The mechanism of ERK1/2 signaling pathway and HIF-1?promoting DPCs growth regulated by ADSCsThe results of detection that regulatory network involved in the secretion of hair follicle growth factors VEGF,bFGF and PDGF in ADSCs showed,the hypoxic microenvironment activated the ERK1/2signaling pathway of ADSCs.Whereas,when treated with ERK inhibitor PD98059,the increased expressions of growth factors in the hypoxic environment were reduced,which indicated that ERK1/2 induced the expressions of VEGF,bFGF and PDGF.At the same time,HIF-1?plays an important role in the hypoxic microenvironment.On the one hand,HIF-1?increased the expressions of VEGF,bFGF,PDGF,and inhibited by the HIF-1?inhibitor YC-1.On the other hand,HIF-1?inhibited the expressions of BMP2,BMP4 and BMP7,in which BMP7 was significantly decreased after overexpression of HIF-1?,and recovered after addition of YC-1.Further HIF-1?expression vector PcDNA3.1-HIF-1?and BMP7 promoter luciferase reporter vector pGL4.1-BMP7 were constructed.The results of detection showed that HIF-1?significantly inhibited the activity of BMP7 promoter,thereby negatively regulated the expression of BMP7.In summary,this study revealed that hypoxic microenvironment could enhance the proliferation and the cell function of ADSCs.The ADSCs conditioned medium,especially Hypo-cm,could promote the growth of DPCs and increased the expressions of DPCs specific marker genes.ERK1/2 signaling pathway and HIF-1?promoted the growth of DPCs regulated by ADSCs in hypoxic microenvironment.The above results have important theoretical and practical significance for understanding the development of hair follicles and their regeneration mechanism.