Study On Effect Mechanism Of Promoting Angiogenesis Of Shengji Yuhong Collagen Induce Adipose-derived Stem Cells To Initiate MiRNA-lncRNA Targeted Regulation Of P38 Pathway

Objective:To study the in vivo and in vitro mechanisms of the biological scaffold collagen combined with traditional Chinese medicine surgery for the treatment of chronic wound classic recipe "Shengji Yuhong Cream" to regulate the paracrine of human adipose-derived stem cells?hADSCs?and promote angiogenesis.Research content:Extraction,isolation,and cultivation of human-derived adipose stem cells;preparation and evaluation of Shengji Yuhong collagen.According to the results of microarray and bioinformatics analysis to screen the most different microRNA?miRNA?and long-chain non-coding RNA?lncRNA?and their relationship with the predicted target protein p38 when Shengji Yuhong collagen regulates the function of adipose-derived stem cells.With the model of lower limb ischemia in nude mice to verify the results in vitro experiments and some previous experiments.Methods:1.Human adipose stem cells were isolated by collagenase digestion centrifugation,induced adipogenic,osteogenic and chondrogenic differentiation,identified by staining,and identified by flow cytometry;2.Shengji Yuhong Cream extract was extracted by alcohol extraction,and its adhesion rate and proliferation were evaluated after binding with collagen protein;3.RT-qPCR verification the most significant difference expression of miRNA which was found in the Microarray.silenced the gene by lentivirus transfection technology,observe GFP expression under fluorescence microscope,detected miRNA and p38 expression levels,and exam the reverse effect of Shengji Yuhong collagen on miRNA and p38;4.With the same method of selecting miRNA to screen the most significant different lncRNA,detect the expression of IncRNA with miRNA-silenced hADSCs,and detect the effect on miRNA and p38 expression level after silencing the lncRNA by lentivirus transfection technology;5.Establish the lower limb ischemia model of nude mice,divide into groups for 28 days,with weeks as time nodes,recorded by ultrasonic Doppler,test the expression of target miRNA,target IncRNA,target gene and their expression in exosomes by RT-qPCR.Results:1.The adipose-derived stem cells have good multiple differentiation ability,which is defined as adipose stem cells supported by cell surface antigen;2.The adhesion rate ofShengji Yuhong collagen to hADSCs is more than 90%,which is suitable for hADSCs to be used as scaffolds for tissue engineering;3.Shengji Yuhong collagen may act on hADSCs through miR-21/lncRNA TCONS₀0178845/p38 signaling pathway plays an important role in promoting angiogenesis and healing;4.The feasibility of the signaling pathway obtained from cell experiments was verified by experiments in nude mice,and the cooperative use of hADSCs in promoting healing may be achieved by paracrine exosomes carrying relevant regulatory factors to the wound site.Conclusion:hADSCs can promote angiogenesis through paracrine exosomes carrying regulatory factors miR-21 to influence lncRNA TCONS 00178845 through miR-21/lncRNA TCONS 00178845/p38 signaling pathway,which can promote healing.