| Objective(s):Type ? collagen(Col I)in vitro of adipose stem cells(ADSCs)and endothelial progenitor cells(EPCs)regulatory mechanism of proliferation and differentiation,to provide good environment for growth of seed cells proliferation,provide simple method for stem cells in vitro,as fat grafts early vascularization and provide theoretical basis to improve the survival rate.Methods:(1)Rat fibroblasts,ADSCs and EPCs were cultured and identified in vitro,and a large number of fibroblasts were amplified and harvested.Collagen was extracted by acid extraction method and purified,and collagen was identified by Western Blot.(2)The optimal concentration for ADSCs proliferation by CCK-8 method was 0.1mg/mL,and the growth curve was plotted.0.1mg/mL of type ? collagen was used to detect the proliferation of ADSCs in different generations.0.1mg/mL type I collagen was coated in the lower compartment of Transwell(collagen group),and the lower compartment of the control group was not treated.The number of cells inoculated in the upper compartment of the collagen group and the control group was 3×104/well,and the number of migrated cells was counted after staining at 24h,48h and 72h,respectively.The expression of VEGFA in ADSCs in collagen group and control group was detected by RT-qPCR.Cells in the collagen group and the control group were treated with adipogenic differentiation,osteogenic differentiation and chondrogenic differentiation,respectively.Cells in each group were stained on the 3th,10th and 5th day,respectively,and their morphology was observed.Cells in the other part of the group were collected for RNA extraction,and RT-qPCR was used to detect the expression levels of early adipogenic,osteogenic and chondrogenic genes in each group.(3)The optimal concentration for EPCs proliferation by CCK-8 method was 5?g/cm2,and the growth curve was drawn.The proliferation ability of EPCs in different generations was detected by 5?g/cm2 type ? collagen.Type ? collagen with a concentration of 5?g/cm2 was coated in the lower compartment of Transwell(collagen group).The lower compartment of the control group was not treated.The number of cells inoculated in the upper compartment of the collagen group and the control group was 3×104/well,and the number of migrating cells was counted after staining at 24h and 48h,respectively.RT-qPCR was used to detect the expression of VEGFA,VEGFR1,VWF,CD133 and CD31 in EPCs of collagen group and control group,respectively.Rats in the collagen group and the control group were inoculated with EPCs on the matrix glue,respectively.Each tube was observed under a microscope at 2h and 4h,respectively.Five fields were randomly selected to take pictures,and the total length of the tubes and the number of branch nodes were counted with Image J.The EPCs samples were collected by Trizol method and sent to the company for genome sequencing,and the sequencing results were verified by RT-qPCR.The total RNA of EPCs in collagen treatment group and control group was extracted by Trizol method,and EPCs gene expression in collagen treatment group and control group was detected by real-time quantitative PCR(RT-qPCR).Results:(1)Type ? collagen was extracted and purified from fibroblasts.(2)0.1mg/mL type ? collagen had the strongest proliferation ability on ADSCs(p<0.05),and the proliferation ability of type ? collagen at 0.1mg/ml was stronger than that in the control group(p<0.05).(3)The migration ability of type ? collagen to ADSCs was significantly stronger than that of the control group(p<0.05).(4)The expression levels of VEGFA,PPARG,Runx2 and COMP in Type ?collagen group were significantly higher than those in the control group(p<0.05).(5)5?g/cm2 Type ? collagen had the strongest proliferation ability on EPCs(p<0.05),and the proliferation ability of different generations of ADSCs was stronger than that of the control group(p<0.05).(6)The migration ability of Type ? collagen to EPCs was significantly worse than that of the control group(p<0.05).(7)The expressions of CD31,VEGFR1,VEGFA and VWF of EPCs in Type ?collagen group were significantly lower than those in control group,and the expression of CD133 was significantly higher than that in control group(P<0.05).(8)Type ? collagen can up-regulate cell cycle,adhesion molecule and metabolic process.(9)Type ? collagen can activate PI3K/Akt signaling pathway,and the amount of PI3K and Akt gene expression in Type ? collagen group is significantly higher than that in control group(P<0.05).Conclusion(s):(1)Type ? collagen can be extracted from fibroblasts,providing a new method for the extraction of Type ? collagen.(2)Type ? collagen can induce the proliferation and chemotaxis of ADSCs,providing a good microenvironment for fat regeneration after fat transplantation.(3)Type ? collagen promotes EPCs proliferation by up-regulating cell cycle and metabolic process.(4)Type ? collagen promotes the angiogenesis of EPCs by up-regulating the PI3K/Akt signaling pathway and adhesion molecules,providing a theoretical basis for the early vascularization of fat transplantation.(5)Type ? collagen can maintain the multidirectional differentiation potential of stem cells,providing a new method for the application of fat transplantation combined with stem cells. |
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