The Effects Of Chemokine SDF-1 On Adscs Differentiation Into VECS And SHF-DPC Development

The cashmere produced by Arbas goat is described as "soft gold",which has the very high economic value.During cashmere growth process,the anagen,catagen and telogen are caused by the periodical change of hair follicle.Previous research has shown that the blood vessel density and volume around the hair follicle in the anagen period was increased due to the up-regulated expression of various inducing factors.The formation of blood vessels is a dynamic process which is regulated by chemokines and internal factors.Stromal cell-derived factor-1(SDF-1)is an important chemokine.It has been demonstrated that adipose mesenchymal stem cells treated by SDF-1 could quickly repair damaged wounds,which indicated that SDF-1may play a key role in the formation of vascular network and tissue repair process.In this research,SDF-1 chemokine was added in the culture media for adipose derivedstem cells(ADSCs)differentiation into vessel endothelial cells(VECs),and its effect on differentiation and growth of blood vessels(angiogenesis)was observed,and the effect of induced vessel endothelial like cell(VECs-like)on secondary hair follicle-dermal papilla cell(SHF-DPC)growth was explore as well,so that to provide experiment basis for the understanding of villi growth mechanism in the future.1.Induced differentiation of adipose mesenchymal stem cells into vessel endothelial cellsADSCs were used as experimental materials,and 30 ng/ml,50 ng/ml,70 ng/ml SDF-1 were added in induced culture media for ADSCs differentiation into VECs,the induced VECs-like cells were detected at 72 h and 21 d,respectively.The results showed:(1)The induction process could trigger the cell death,and the proportion of death was positively correlated with the induction time.The cell morphology was transformed from spindle to long spindle,the color of cytoplasm was deepened,and the nuclear position was obvious,but there was no significant difference between groups.(2)VECs-like cells after inducted 72 h were marked by rabbit source von Willebrand factor(VWF),then were detected by flow cytometry.Each treatment of inducing was successful,but 50 ng/ml SDF-1 was the best condition.(3)The expression of CD34,VWF and CXCR4 in VECs-like cells after inducted 72 h were detected by RT-q PCR,and the most favorable expression of VWF was in 30 ng/ml SDF-1,which was 1.45 times of the control group(p<0.05).SDF-1 in 50 ng/ml and70ng/ml had significant effect on CXCR4 expression,which were 40.85 and 12.96 times of control group respectively(p<0.01).The expression of CD34 was thehighest in 50 ng/ml SDF-1,which was 1.741 times of the control group(p<0.05).(4)30 ng/ml and 50 ng/ml SDF-1 had a significant effect on the formation of specific tubular structure of VECs-like cells after inducted 21 d.At the same time,the cell morphological changes were significant and the intercellular lines were obvious.The apoptosis rates were about 50% compared with the cells before induction.(5)Filamentous connection between cells and a sample tube structure were observed in the VECs-like cells induced after 21 d.2.The functional detection of vessel endothelial like cells.VECs-like cells induced after 24 h and 72 h were detected by Propidium(PI)staining,CCK-8,Annexin V-FITC/PI of double staining,scratch method respectively,cell cycle,active,apoptosis,migration ability of VECs-like cells were observed.The results showed:(1)Cell cycle was blocked in S and G2 stage,and the highest block proportion was in 50 ng/ml SDF-1.(2)VECs-like cell activity was detected 4 days by microplate reader.During the observation,the cell activity was not high 3 days before,and was increased until 4 d.SDF-1 had no obvious effect on cell activity,and there was no significant difference between the experimental groups.(3)The survival rate of VECs-like cells was significantly increased after treatment with 50 ng/ml SDF-1,and the proportion of early apoptosis was significantly reduced.(4)VECs-like cells treated with scratch method was observed in 12 h,24 h,36 h and 48 h respectively,the rates of cell migration were significantly inhibited in 30 and 50 ng/ml SDF-1treatments after 36 h.3.The effects of induced vessel endothelial like cells on secondary papillary cells.First,VECs-like cells were induced,the correlation factors in VECs culture media after 24 h were detected by ELISA.The results showed that the content b FGF was the highest in ADSCs supernatant,followed by treatment group of 30 ng/ml SDF-1.There was no significant difference in the content of PDGF and IGF-1 in each group.The VEGF content of 50 ng/ml SDF-1 treatment group was the highest,with the lowest secretion in 0ng/ml SDF-1 treatment group.Second,the supernatant fluid cultured VECs-like cells for 24 h used to culture SHF-DPC,the influence of VECs-like cells to SHF-DPC on activity and proliferation was assessed.The results showed that the activity of SHF-DPC was no significant difference between the experimental groups.During continuous count of 7 d,SHF-DPC began exponential growth from 3 d,SHF-DPC treated with VECs-like cells supernatant fluid grew accelerated significantly than that of control from 4 d.It indicated that the supernatant fluid composition in experimental groups had promoting effect on proliferation.Third,VECs-like cells(lower chamber)after induced 72 h were cocultured with SHF-DPC(upper chamber)in Transwell for 24 h and 48 h,the protein expression of the Wnt/ beta-catenin in SHF-DPC was detected.The results showed that the promoting effect in 50 ng/ml SDF-1 was the most obvious.The results of this reserach indicated that SDF-1 factor was not significantly improve the efficiency of induction,but could promote the function of the VECs-like cells to a certain extent,improve anti-apoptosis ability of cells,and have positive regulatory role to protein expression and proliferation ability of SHF-DPC.