The Mechanism Of Activation Hair Follicle Stem Cells By Dermal Papilla Cells Of Arbas Cashmere Goat

Hair follicle(HF)is a tiny organ that can dynamically change in mammals.After HF matures,the dynamic change is cycle from the anagen phase to the telogen phase.A variety of cells and signaling pathways are involved in this cycle change,and the two most important cells are hair follicle stem cells(HFSCs)and dermal papilla cells(DPCs).HFSCs can be activated during the growth phase of HF to participate in the HF formation,so HFSCs are essential for mammalian hair regeneration.In the HF regeneration,DPCs mainly regulate the state transition of HFSCs from quiescence to activation,which is a key switch in the HF cycle.Therefore,exploring the interaction mechanism between DPCs and HFSCs is the core of understanding HF regeneration.In order to understand the mechanism of interaction between DPCs and HFSCs,the primary hair follicles stem cells(PHFSCs),secondary hair follicles stem cell(SHFSCs),primary dermal papilla cells(PDPCs)and secondary dermal papilla cells(SDPCs)of Arbas cashmere goats were used as the research objects in this study.First,PHFSCs and SHFSCs were isolated and identified from Arbas cashmere goats,and the multipotency of PHFSCs and SHFSCs were analysed in this study.PHFSCs were co-cultured with PDPCs by transwell system.Similarly,SDPCs and SHFSCs were co-cultured by transwell system.These co-culture models were observed the biological functions effects of the two types of DPCs on two types of HFSCs,respectively.Here,we explored the changes of related signaling pathways of DPCs and HFSCs,so as to understand the molecular mechanism of the effect of DPCs on the HFSCs biological function.1.Isolation and identification of PHFSCs and SHFSCsPHFSCs and SHFCs were obtained from primary hair follicles and secondary hair follicles of Arbas Cashmere goats,respectively.The cell lines were obtained by these methods that type IV collagen purification and passage of trypsin.The surface markers of HFSCs: K14,K19,K15,CD34 and LGR5 were identified by cell immunofluorescence,real-time quantitative PCR and Western Blot.The proliferation and morphology of PHFSCs and SHFSCs were evaluated by CCK8 and Giemsa staining.The results showed that the growth curves of two types of HFSCs were S-shaped.The morphology of the two types of HFSCs were no significant difference,they were cobblestone-like morphology,which was typical for skin stem cells.HFSC related pluripotency genes SOX2,OCT4 and SOX9 were identified.Their ability to differentiate into adipogenic,neurogenic,and hepatogenic cells was identified by induced differentiation.2.The effect of biological function of HFSCs co-cultured with DPCsTranswell system was used to co-culture PDPCs and PHFSCs,SDPCs and SHFSCs,respectively.Proliferation of PHFSCs and SHFSCs were detected by Edu assay,flow cytometry and Western Blot.The result showed that PDPCs and SDPCs could promote proliferation of PHFSCs and SHFSCs,respectively.Futhermore,the expression of genes related to HF differentiation was detected by q RT-PCR,and we found that PDPCs and SDPCs induced the differentiation of PHFSCs and SHFSCs into HF,respectively.PDPCs and SDPCs could promote the migration ability of PHFSCs and SHFSCs by transwell migration,respectively.At the same time,the ALP activity of PDPCs and SDPCs,the genes related to inducibility,and the Wnt signaling pathway were detected in the co-culture system.It was found that the activity ALP of PDPCs and SDPCs was increased,and the inducibility was enhanced.3.Transcriptome reveals the mechanism of biological function changes of HFSCs activated by DPCsTranscriptome sequencing of PHFSCs co-cultured with PDPCs and SHFSCs co-cultured with SDPCs showed that there were 424 differential genes in the PHFSCs group and 318 differential genes in the SHFSCs group.Seven genes were randomly selected from each group of differential genes,and their expressions were analyzed by q RT-PCR.The results showed that expression levels of these genes were consistent with the trend of transcriptome datas.Therefore,the transcriptome datas were credible.The differential genes were further analyzed by GO function enrichment and KEGG pathway enrichment,and it was found that most of the differential genes of PHFSCs groups were enriched in gene functions such as extracellular matrix organization,extracellular matrix and nucleic acid binding.The differential genes of SHFSCs groups were enriched in gene functions such as heme binding,tetrapyrrole binding and oxidoreductase activity.KEGG analysis revealed similar pathways that the differential genes of PHFSCs and SHFSCs groups could be enriched to,including: proteoglycan in cancer,ECM-receptor interaction,focal adhesion,protein digestion and absorption and P13K-AKT signaling pathway.These pathways constitute a regulatory network involved in the proliferation、differentiation and migration of PHFSCs and SHFSCs.Screening of 11 hub genes associated with proliferation,differentiation and migration in the PHFSCs and SHFSCs groups by PPI networks.We also combined the transcriptome data from the skin of cashmere goats during different stage and selected and verified 4 hub genes(ITGA5,PLOD3,CDSN and PTGS2),which were associated with the transformation from telogen to anagen phase of HF.In conclusion,Arbas cashmere goat PHFSCs and SHFSCs had pluripotent differentiation characteristics.When PHFSCs and SHFSCs co-cultured with PDPCs and SDPCs respectively,they could be induced to proliferate,differentiate and migrate.The expression of ITGA5 and PLOD3 in co-cultured PHFSCs may be activated via ECM-receptor interaction signaling and focal adhesion pathway and effect PHFSCs proliferation and differentiation.The expression of CDSN and PTGS2 were effected in co-cultured SHFSCs and activate SHFSCs proliferation and differentiation.These findings provided an important experimental basis for future functional verification of cells and research on the growth mechanism of HF in vivo.