Netrin-1 Improves Function Of Adipose-derived Stem Cells In Angiogenesis

Part one Extraction,cultivation and identification of human adipose derived stem cellsObjective: To extract human adipose derived stem cells(ADSCs)?As well as to identify the cells morphology,cell surface markers and multi-differentiation ability of ADSCs.Methods:1.ADSCs were extracted from abandoned adipose tissue in plastic liposuction surgery.The fat tissues using 0.1% type I collagenase digestion,then filtration and centrifugation.Meanwhile,the phenotype characteristics of the cells were observed.2.The expression of ADSCs exterior antigen CD29,CD44,CD73,CD90,CD105,CD117,CD166,CD31,CD34,CD45,CD106 were checked by flow cytometry.3.ADSCs were induced adipogenic differentiation,osteogenic differentiation and cartilage differentiation by using the special culture medium.The induced cells were separately assayed by oil red O,alizarin red and alcian blue staining to identify their multi-differentiation potential.Results:1.ADSCs were successfully isolated and cultured.The obtained large numbers of adherent cells with fusiform vortex like growth,morphology similar to fibroblast-like cells.2.Flow cytometry results showed that the ADSCs surface marker of CD29,CD44,CD73,CD90,CD105,CD117,CD166 were positive(>95%),while CD14,CD31,CD34,CD45(<5%)were negative.3.In adipogenic differentiation,oil red O staining showed that the cells have a large number of orange red fat droplets.In osteogenic differentiation,alizarin red staining presented orange typical red calcium nodules.In chondrogenic differentiation,alcian blue staining appeared blue bodies.Conclusion: Successfully isolation,cultivation and identification of ADSCs,as well as conform it has multi-differentiation ability.Part Two Netrin-1 improves bioactivity of adipose derived stem cellsObjective : This study was performed to examine the effects and mechanism of over-expression Netrin-1 on ADSCs proliferation,migration,invasion and angiogenesis.Methods:1.Cell transfection: over-expression-Netrin-1 plasmid was transferred into ADSCs by Lipo2000,the transfection efficiency of it was checked under fluorescence microscope.And the expression level of m RNA and protein in adipose derived stem cells after transfected Netrin-1 were detected by real-time quantitative PCR(q RT-PCR)and Western blot,separately.2.The proliferation of ADSCs was measured using Cell Counting Kit-8(CCK-8).3.Migration and invasion ability of ADSCs after transfected Netrin-1 were detected by transwell model.And the matrix metalloproteinase 2(MMP-2)and matrix metalloproteinase 9(MMP-9)were analysised at the transcript level.4.Metrics of angiogenesis included measurement the tube formation of ADSCs with or without co-cultured with Human Umbilical Vein Endothelial Cell(HUVEC).Various methods were adopted to detect vessel length,complicacy and the extent of maturity.As well as the level of m RNA and protein expression in Vascular endothelial growth factor(VEGF)and Placenta growth factor(Pl GF).Results:1.Over-expression of Netrin-1 in ADSCs,the cells conveyed high green fluorescent.And the level of m RNA(5.03±1.99)and protein(3.04±0.07)in transfected ADSCs expression were enhanced.2.Netrin-1 promoted proliferation of ADSCs.The proliferation of Netrin-1 group was significantly enhanced on the second day(1.39±0.06 vs 1.13±0.02),the third day(2.07±0.09 vs 1.34±0.50)and the fourth day(2.67±0.06 vs 1.39±0.07),presenting an increasing trend.3.Netrin-1 significantly increased ADSCs migration(121.3±3.22 vs 51.67±1.53)and invasion(76±1.73 vs 20±2)mainly through enhanced the m RNA expression of MMP-2(1.317±0.10)and MMP-9(3.53±0.87).4.In the tube formation assay,ADSCs alone culture group(18.33±1.53 vs 6 ±2)and co-cultured with HUVEC group(31.33 ± 4.16 vs 16 ± 3),Netrin-1 markedly increased the number of tubules,the length and complexity of the branchs.In the over-expression Netrin-1 group,the vascular endothelial growth factor m RNA(3.6 ± 0.39)and protein(3.54± 0.10)levels were significantly increased.At the same time,m RNA(3.1±0.50)and protein(2.36 ±0.03)level of placental growth factor also were improved(P<0.05).Discussion: This study demonstrated that over expression of Netrin-1 in adipose derived stem cells significantly enhanced the cells biological functions of proliferation,migration,invasion and angiogenesis.