The HMME-PDT Promotes The Differentiation Of Adipose-Derived Mesenchymal Stem Cells Into Fibroblasts And Its Underlying Mechanism

BackgroundThe integrity of skin structure and function plays an important role in maintaining the homeostasis of the body.When the skin is damaged,the wound tissue can repair itself with a variety of cells and regulators factors.Wound healing is a dynamic process which includes stages of inflammation,proliferation and remodeling.Any problem in any stage will lead to delayed wound healing.When the wound healing is delayed for more than three months,it is called chronic wound.Chronic wounds can be induced by a variety of factors,including in-fection,poor blood flow,diabetes,etc.These factors can reduce the migration of inflammato-ry cells in the wound,reduce the polarization of macrophages,and reduce the secretion of cy-tokines,thus making fibroblasts,endothelial cells,epithelial cells and other repair cells unable to fully activate and play the original wound repair function,leading to the prolongation of the inflammatory period of the wound.Due to the complexity of the mechanisms related to the occurrence and healing of chronic wounds,traditional treatments often fail to achieve good results.In recent years,regenerative medicine has attracted extensive attention because it can create lost or dysfunctional tissues and organs with normal structures and functions.Mesen-chymal stem cells(MSCs)participate in the process of tissue regeneration due to their ability to self renew,differentiate,secrete regulatory factors and so on,having great potential in the treatment of chronic wound healing.Therefore,researches on stem cells in the treatment of chronic wounds have gradually been attracted people’s attention.Photodynamic therapy(PDT)is a combination of photosensitizers and light to make pho-tosensitizers react with oxygen under the excitation of optimum wavelength light for disease diagnosis and treatment.High-dose PDT can produce high-dose of reactive oxygen species(ROS),which can destroy DNA and protein of cells,and cause cell death,while low-dose PDT can produce ROS that can be controlled.ROS,as a signal regulator,activates intracellu-lar signaling pathways to regulate cell survival,proliferation,secretion,differentiation and other functions.Hemoporfin,a derivative of Hematoporphyrin monomethyl ether(HMME),is the second generation of porphyrin monomer photosensitizer.Compared with other com-monly used hematoporphyrin derivative photosensitizers,HMME has the advantages of short incubation time,short half-life,low toxicity and stable structure.At present,HMME-PDT has good clinical efficacy in the treatment of angioproliferative diseasesUsing stem cell tissue engineering to repair wounds has become a hot topic in basic re-search and clinical practice.Among them,mesenchymal stem cells have the potential to dif-ferentiate into various tissues of mesoderm,and can improve the damage of almost all major tissues and organs,including skin defects.Adipose mesenchymal stem cells are easy to obtain and abundant,and have become important candidate cells in tissue engineering.Therefore,this topic will explore the possible mechanism of photodynamic promotion of chronic wound healing by studying the effects of PDT on the proliferation,regulation and other functions and directional differentiation of adipose mesenchymal stem cells.Methods1.Adipose mesenchymal stem cells(ADSCs)were isolated from human adipocyte and cultured as usual and its activity was detected by CCK-8 assay.The proper photodynamic therapy parameters combined the concentration of photosensitizer(HMME)and light fluences on cells were also selected by CCK-8 assay.2.ADSCs were divided into four groups:control group(blank control),HMME group(only incubated with HMME),Irridation group(only irrideated by red light)and PDT group(HMME combined with irradiation).The effect of PDT on cell apoptosis was detected by flow cytometry,and cell proliferation was detected by Ed U staining.The effect of PDT on cell migration was detected by cell scratch assay,and cell regulation was detected by Transwell chamber.3.ADSCs were treated in different conditions as mentioned above,then feed in the spe-cial differentiation medium to induces cell differentiation into fibroblasts.Recording the dif-ferentiated days,cell phenotype,cell differentiation rates by flow cytometry.Masson staining was used to detect the collagen deposition.Immunofluorescence and RT-PCR were used to detect the extracellular matrix secretion.4.Western Blot was used to detect the changes of related signal molecules in MAPK pathway before and after PDT treatment,so as to clarify the possible molecular mechanism of PDT inducing the directional differentiation of ADSCs.Results1.When ADSCs were treated by low concentration HMME(2μg/ml)with low fluences light illumination(3 J/cm~2),the activity of cells is the best and the proliferation rate was sig-nificantly increased compared with other groups.2.PDT could accelerate the migration of ADSCs and enhance the ability of ADSCs to regulate the migration of Ha Ca T cells,compared with the control group,3.PDT could significantly accelerate the differentiation of ADSCs into fibroblasts and increase the secretion of extracellular matrix,compared with the control group,4.After PDT treatment,the phosphorylation level of ERK in MAPK pathway was signif-icantly increased,while the phosphorylation level of p38 and JNK had no significant change.After inhibition of ERK pathway with inhibitor,the extracellular matrix secretion function in PDT+inhibitor group were significantly decreased compared with PDT group.Conclusion1.Low dose HMME-PDT can promote the cell activity and proliferation rate of ADSCs,promote cell migration,and enhance the regulatory function of the cells.2.Low dose HMME-PDT can promote the differentiation of ADSCs into fibroblasts,re-duce the differentiation time,enhance the secretion function of cells.3.PDT regulates the differentiation function of ADSCs by activating MAPK/ERK path-way.