Investigation On The Effects And Mechanisms Of Direct Current Electric Field And Ultraviolet On Adipose-derived Stem Cells

Objective: Adipose-derived stem cells（ADSCs）are a population of mesenchymal stem cells,with self-renewal properties and multi-lineage differentiation capacity.ADSCs are ideal seed cells for tissue engineering because of their abundant sources,few limitations of ethical issues and the ability of transdermal differentiation.Therefore,the possibility and conditions for ADSCs to differentiate into cells other than the known differentiation lineage are areas worthy of continuous attention of researchers.Electrical signal exists in organism broadly and plays an indispensable role in transmitting information and function implementation.As a form of electrical signal in vivo,endogenous direct current electric field is of vital importance for the process of embryo development and wound healing.The completion of these processes is closely related to the proliferation,migration and differentiation of stem cells.However,there are few studies on the response of ADSCs in direct current electric field.Ultraviolet phototherapy has a long history and wide application in dermatology,especially in the treatment of vitiligo.Ultraviolet can change the cytokines secreted by epithelial cells,induce cell DNA damage and apoptosis,and promote the proliferation,migration and activity of melanocytes.Ultraviolet can also promote the differentiation of melanocyte precursor cells into melanocytes.However,whether ADSCs can differentiate into melanocytes and whether ultraviolet can start or promote this process remains to be revealed.Therefore,in order to explore the effects of direct current electric field and ultraviolet on ADSCs,and whether it can be used as guidance for seed cells of tissue engineering and regenerative medicine,we carried out the following research.Methods: 1.In this study,the ADSCs were extracted from the discarded adipose tissue by collagenase digestion,and the expression of CD34,CD44,CD45,CD73,CD90,and CD105 were detected by flow cytometry.In addition,adipogenic and osteogenic differentiation medium culturing and oil red O and alizarin red S staining were used to confirm the multilineage differentiation ability of ADSCs.Under the guidance of other papers,we constructed a device to apply direct current electric field stimulation to cells,select appropriate stimulation parameters,and treated ADSCs.The control group and the direct current electric stimulation group samples were collected and the total RNA of were extracted.RNA integrity and gDNA contamination were measured using electrophoresis with agarose.Then the RNA library was established for sequencing.The sequencing data was analyzed to get the expression profile of circular RNA,long non-coding RNA,microRNA and mRNA,from which the volcano map was indicated;the dysregulated RNA were screened to indicate the heatmap.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to enrich and analyze differentially expressed mRNA and the related genes of other groups of RNA.TargetScan and miRanda were used to predict the interaction among mi RNA-mRNA,miRNA-circRNA and miRNA-lncRNA.Then the network was constructed by the predicted results using Cytoscape software.According to the predicted network,as well as considering the genes related to cell proliferation,migration and differentiation,several RNAs were selected for validation.Primer Premier software was used to help design the RNA primers,BLAST was used to analyze the specificity of the primers,and Quantitative Real-time PCR was used to verify the expression of the corresponding RNAs.3.PCR array was used to detect the changes of RNA expression.Protein interaction network was constructed using the expression profile.MTS method was used to detect the proliferation of ADSCs after intermittent ultraviolet irradiation in order to guide the selection of long-term irradiation dose.Quantitative Realtime PCR and immunofluorescence were used to detect the expression of melanocyte related markers in ADSCs after ultraviolet treatment for 7 or 14 days.Under the condition of melanocyte induction medium for inducing pluripotent stem cells（iPSCs）,the ADSCs were irradiated with ultraviolet light,and the expression of melanocyte related markers was examined by Quantitative Real-time PCR after 21 or 42 days.Results: 1.The expression of CD34 and CD45 of the cells was negative,CD44,CD73,CD90,and CD105 were positive.The cells had the potential of adipogenic and osteogenic differentiation.2.A total of 8944 circRNAs,18564 lncRNAs,678 miRNAs and 17252 mRNAs were detected by sequencing.Compared with the control group,the ADSCs stimulated by 300 mV/mm direct current electric field for 6 hours showed 66 differentially expressed circRNAs,164 differentially expressed lncRNAs,1310 differentially expressed mRNAs（fold change > 2,p < 0.05）,and 26 differentially expressed miRNAs（fold change > 1.5,p < 0.05）.The enrichment analysis of differentially expressed mRNA and other groups of RNA related target genes showed that direct current electric field may cause stress of ADSCs,and MAPK signaling pathway played an important role in the reaction of cells in direct current electric field.2.After matching the differentially expressed RNA and the prediction of the regulatory axis,ceRNA networks containing circRNA and lncRNA were separately mapped,including 7,21,148 circRNAs,miRNAs,mRNAs and 1,1,36 lncRNA,miRNA,mRNAs.On the basis of these RNAs and considering their related functions,we verified 22 RNAs,such as hsa_circ₀070764,which showed the changing expression in the same direction with the sequencing results.3.The results of PCR array and protein interaction analysis showed that VEGFA and other 9 up-regulated genes were the key factors.The dose with no obvious effect on cell proliferation after 72 h irradiation of UVA was 2J,and the dose of UVB with the lowest effect was 0.01 J.After 7-14 days of ultraviolet irradiation but without other intervention,the expression of TRP-1 and other melanocyte related markers showed no significant difference with the control group.The ADSCs were induced for 3-6 weeks by the medium which can induce iPSCs into melanocytes,and treated with ultraviolet radiation at the same time.It was found that UVA could accelerate the expression of KIT,and UVB could reduce the expression of KIT.Conclusion: 1.The cells in this study are ADSCs,which have the potential of adipoenic differentiation and osteogenic differentiation.After ADSCs were exposed to direct current electric field,the expression profiles of circRNA,lncRNA,miRNA and mRNA were changed.MAPK signaling pathway may play an important role in responses of ADSCs.2.Using the differential expression profile,we constructed the ceRNA network to predict the regulatory relationships of RNAs in direct current electric field.Some of the network related RNAs were selected to verify its expression in the control group and the experimental group.It was found that the up-regulation or down-regulation results were consistent with the sequencing results.3.The expression of VEGFA and other genes in ADSCs was up-regulated after UV irradiation,which might be related to the regulation of cell proliferation,apoptosis and other activities.Ultraviolet irradiation for 7-14 days could not induce differentiation of ADSCs into melanocytes.In the process of inducing ADSCs to differentiate into melanocytes through the medium containing cytokines and other components,UVA irradiation can accelerate the expression of KIT,thus accelerating the differentiation process.