The Effect Of Hair Follicle Neural Crest Stem Cells On Perineurial Cells And Its Mechanism

With the development of nerve regeneration medicine,many researchers have focused on more effective method for peripheral nerve injury repair.Apart from forming the bloodnerve barrier,perineurial cells contribute to peripheral nerve injury repair,such as forming the perineurial cell bridges,clearing cell debris,and reciprocal interactions with Schwann cells.During nerve regeneration,perineurial cells may be the first cell type to migrate to injury area,clear cell debris,and form cell bridges,which is earlier than the activation of Schwann cells.Schwann cells migration and axon regeneration occur adjacent to perineurial cells.Perineurial cell may play a key role in the treatment of long-nerve defects through multiple cell mobilization,which is worth studying.However,the ability of the proliferation and migration of perineurial cells is weak.Promoting the proliferation and migration of perineurial cells and accelerating the formation of perineurial cell bridges may be a new strategy to peripheral nerve injury repair.At present,there is no effective method to purify and culture the perineurial cells in vitro,which leads to hardly studying the mechanism of perineurial cells in peripheral nerve injury repair.Hair follicle neural crest stem cells（hfNCSCs）derived from neural crest,which were found above the bulge in the hair follicle.hfNCSCs resembled stem cells in the brain and expressed nestin and readily formed neurons,Schwann cells and many other cell types.hfNCSCs could promote peripheral nerve injury repair,whether it could be applied to activate perineurial cells is still unknown at present.A “limited digestion – differential adherence – drug treatment” method was designed to obtain high pure perineurial cells in this project,and hfNCSCs of rat whisker pad were cultured.The co-culture system of hfNCSCs and perineurial cells was established on transwell assay to explore the effects of hfNCSCs on the proliferation and migration of perineurial cells.Moreover,hfNCSCs exosomes（hfNCSCs-Exos）were obtained and identified,hfNCSCs-Exos were used to treat perineurial cells.hfNCSCs-Exos micro RNA was sequenced to analyse the biological function and pathways of its target genes.The possible mechanism of hfNCSCs on the proliferation and migration of perineurial cells was discussed to provide important theoretical basis and experimental basis for promoting the proliferation and migration of perineurial cells.Part Ⅰ Isolation,culture,purification and identification of perineurial cells and hfNCSCsObjective: To obtain perineurial cells with high purity and hfNCSCs,and provide a stable cell source for studying the mechanism of peripheral nerve injury repair.Methods: Isolation,culture,purification and identification of perineurial cells: Sciatic nerves were harvested from Sprague-Dawley rats（weighing 100 grams）.Epineurium and nerve fibers were stripped off to obtain perineurium.The perineurium was incubated in collagen I-coated 12-well plates without culture medium at 37 ℃,95% CO2,5% O2 for 1h,then perineurial cell medium was added.After 10 days of culture,the nerve fragments were removed.A “limited digestion – differential adherence – drug treatment” method was designed to purify the cells: The cells were treated with 0.25% trypsin-EDTA for 10 s to eliminate the Schwann cells.The cell suspension containing the remaining cells was transferred to a T75 flask at 37 ℃,95% CO2,5% O2 for 30 min to allow fibroblasts to adhere to the plate.The cell suspension was then put into a new T75 flask.The above steps were repeated three days later.After inoculation for 24 h,100μmol/L concentrations of cytarabine（Ara-c）was added for 24 hours to eliminate the remaining fibroblasts.Immunofluorescence staining was performed after 2 days.Antibodies to Claudin-1,S100 and vimentin were used to label perineurial cells,Schwann cells and fibroblasts,respectively.Isolation,culture and identification of hfNCSCs: The bulges in the hair follicle were harvested from Sprague-Dawley rats（males,weighing 50 grams）.The bulges were incubated in collagen I-coated 12-well plates without culture medium in an incubator（37℃,95% CO2,5% O2）for 30 min.Then hfNCSCs primary culture medium was added.Cells of passages 2-3 were used for immunofluorescence.Antibodies to nestin,S100 and beta III Tubulin were used to lable hfNCSCs,Schwann cells and neurons,respectively.Results: 1.For immunofluorescence analysis of perineurial cells,the percentage of cells without purification positive for Claudin-1 was 42.33%,vimentin was 50.33%,and S100 was 15%.During limited digestion and differential adherence,the percentage of cells positive for Claudin-1 was increased to 65%,the percentage of vimentin-positive cells was 37.66%,while no cells was labeled with S100.During 100 μmol/L drug treatment after limited digestion and differential adherence purification,the Claudin-1 positive rate was 97.66%,the vimentin positive rate was 5.66%,and S100 was negative.2.For immunofluorescence analysis of hfNCSCs,the positive rate of nestin was more than 96%,while the positve rate of S100 and beta III Tubulin were nearly zero.Conclusion: Via "limited digestion – differential adherence – drug treatment" method,the perineurial cells with high purity and good growth state are obtained.The hfNCSCs obtained are mostly undifferentiated cells.Part Ⅱ The effects of hfNCSCs on the proliferation and migration of perineurial cellsObjective: To explore the effects of hfNCSCs on the proliferation and migration of perineurial cells.Methods: hfNCSCs conditioned medium was obtained.Different concentrations of hfNCSCs conditioned medium diluted with DMEM-2% FBS at 1:1 or 1:2,DMEM-2% FBS（control）were added to culture the perineurial cells for 24 h,48h and 72 h.CCK8 kit and immunofluorescence staining were used to measure the OD value and the expression of PCNA.For the Transwell co-culture,perineurial cells were added to the upper insert.hfNCSCs were seeded in the bottom wells.DMEM-5% FBS was added to other bottom wells as the acellular group.After 6h,12 h and 18 h co-culture,the number of migrating perineurial cells in each group was counted.Results: Under microscope,the growth state of perineurial cells in hfNCSCs conditioned medium was better than those in control medium.For CCK8 detection,after 24 h and 48 h co-culture,there was no difference in OD value and cell viability of perineurial cells between the hfNCSCs conditioned medium and control medium（P > 0.05）.After co-culture for 72 h,perineurial cells with hfNCSCs conditioned medium proliferated more and were more viable than perineurial cells in control medium（P < 0.05）.There was no difference in OD value and cell viability of perineurial cells in the two concentrations of hfNCSCs conditioned medium（P > 0.05）.For immunofluorescence analysis,after 72 h co-culture,PCNA fluorescence in hfNCSCs conditioned medium was significantly higher than those in control medium（P < 0.05）.PCNA fluorescence in hfNCSCs conditioned medium（1:1）was significantly higher than those in hfNCSCs conditioned medium（1:2）（P < 0.05）.After 6h,12 h and 18 h co-culture,few perineurial cells migrated to the acellular wells,while the number of migrating perineurial cells to the hfNCSCs wells was significantly higher（P < 0.05）.Conclusion: The effects of hfNCSCs on the proliferation and migration of perineurial cells are induced by a paracrine mechanism.Part Ⅲ The effects of hfNCSCs-Exos on the proliferation and migration of perineurial cells and its mechanismObjective: To explore the effects of hfNCSCs-Exos on the proliferation and migration of perineurial cells and its possible mechanism.Methods: hfNCSCs-Exos were obtained and identified.Various concentrations（0,5,20,50μg/m L）of hfNCSCs-Exos were added for 3d and 7d to perineurial cells.CCK8 kit was used to measure the OD value and cell viability of perineurial cells.For the Transwell coculture,perineurial cells were added to the upper insert.Various concentrations（0,5,20,50μg/m L）of hfNCSCs-Exos were added in the bottom wells.After 6h,12 h and 18 h coculture,the number of migrating perineurial cells in each group was counted.Quality assessment and sequencing analysis were used to get top 10 expression of hfNCSCs-Exos micro RNA.GO and KEGG enrichment analysis were used to predict the relationship between micro RNA target genes and cell proliferation,cell migration and nerve regeneration.Results: hfNCSCs-Exos were cup-shaped construction under transmission electron microscope;The particle concentration distribution of hfNCSCs-Exos was unimodal,the peak of particle size was 114.4 nm,accounting for 98.7%.The particle concentration was 1.1E+10 Particles / m L.Western blotting results showed that hfNCSCs-Exos expressed CD9,CD63,CD81 and TSG101.After 3d co-culture,the OD value and cell viability of perineurial cells in various concentrations of hfNCSCs-Exos were significantly more than those in control（P < 0.05）.There was no difference in OD value and cell viability of perineurial cells in the different concentrations of hfNCSCs-Exos（P > 0.05）.After 7d co-culture,there was no difference in OD value and cell viability of perineurial cells between the low concentration of hfNCSCsExos and control（P > 0.05）.Compared with the control,the medium and high concentration of hfNCSCs-Exos（20μg/m L,50μg/m L）promoted the proliferation of perineurial cells more obviously（P < 0.05）.However,this two concentration of hfNCSCs-Exos had no difference（P < 0.05）.After 6h,12 h,18h of co-culture,the number of perineurial cells migrating to hfNCSCs-Exos wells was greater than these migrating to control wells（P<0.05）.With the increasing concentrations of hfNCSCs-Exos,the number of migrating perineurial cells was increasing at statistical significance（P < 0.05）.The quality assessment of hfNCSCs-Exos micro RNA met the experiment requirement.The sequencing analysis showed that top 10 expression of known hfNCSCs-Exos micro RNA were as following: rno-mi R-214-3p,rno-mi R-23a-3p,rno-let-7b-5p,rno-let-7c-5p,rnomi R-130a-3p,rno-mi R-423-5p,rno-mi R-24-3p,rno-mi R-21-5p,rno-mi R-25-3p,rno-let-7a-5p;Top 10 expression of novel hfNCSCs-Exos micro RNA were as following: 1₂148,KL567892₁₃4843,KL567892₁₃4844,13₂4036,7₁5469,3₈734,3₇984,5₁2158,5₁1883 and 14₂5744.For GO enrichment analysis,target genes participated in biological function such as cell growth,cell migration and nerve regeneration.For KEGG enrichment analysis,target genes were closely associated with synaptic vesicle cycle（nerve regeneration）,m TOR signaling pathway（cell proliferation）and Notch signaling pathway（cell migration）,indicating that the signaling pathways that hfNCSCs-Exos micro RNA target genes participated in may play a key role in the proliferation and migration of perineurial cells.Conclusion: hfNCSCs-Exos promote the proliferation and migration of perineurial cells.There is concentration dependence at a degree in the promotion of migration.hfNCSCs-Exos micro RNA target genes and associated signaling pathways may play a key role in this process.