The Study On The Effect And Mechanism Of The PBM-activited Ca²⁺/NFAT On The Proliferation And Differentiation Of ESCs And Wound Healing

Photobiomodulation（PBM）is a method to regulate the activity of cell through non-Photothermolysis using mainly the Non-ionization light in range of the visible and infrared spectrum（Laser,Optical Diode etc.）.It could alleviate the pain and inflammation significantly and furthermore promote the wound recovery.It is so far one of the common therapies which are non-surgical in Plastic Surgery and Dermatology.Recently,the research of the biological effect of PBM draws great attention of the researchers among the world,however,the mechanism of PBM in wound recovery has not been investigated sufficiently.Skin is the largest organ and shield in human body.Its swift and effective recovery after the damage is significant for maintaining the balance of the internal environment of the body.The differentiation and proliferation of epidermal stem cells（ESCs）are furthermore the basis of the recovery of wound.According to the latest research,the cytokines of the cell in wound area are integrated by transcription factor with the information transferred by growth factors and initiate the transcription,which could promote the recovery of wound.It was observed clinically that the Photodynamic therapy possessed a significant effect on wound healing.It was shown in the experiment that the spike of[Ca²⁺]in epidermis of wounded skin could prompt the differentiation of epidermal cells and accelerate the wound healing.Thus,it is reasonable to infer:If the increase of[Ca²⁺]in the skin cells of wound margin could be regulated precisely,then the expression of its downstream transcription factor could be controlled and thus PBM could benefit for the wound healing.A sufficient comprehension of the regulation mechanism of[Ca²⁺]and transcription factor in wound recovery could provide a new idea for clinical wound treatment.According to previous studies about wound recovery,Notch,Jun B,AP-1 and multiple transcription factors were involved in it.But the effect of the Nuclear factor of Activated T cells（NFAT）on recovery is still unknown.NFAT is a sort of transcription factor which is involved in the regulation of the cell evolution,growth and differentiation.Especially,it plays an important role in the proliferation and differentiation of human epidermal cells.It was reported by previous works that the NFAT family had 5 subtypes,those were NFAT1（NFATc2）,NFAT2（NFATc1）,NFAT3（NFATc4）,NFAT4（NFATc3）and NFAT5.Subtype1-4 of which were mainly controlled by[Ca²⁺]in cell and NFAT5 was controlled by the osmotic pressure.This work is concentrated on the effect and mechanism of NFAT and[Ca²⁺]on the proliferation,differentiation and the wound healing in ESCs.It was observed in some researches that the re-epithelization of wound was accompanied by the increase of[Ca²⁺]concentration and considered that the[Ca²⁺]was one of the earliest signals of the start of re-epithelization.In non-excitatory cells,the store-operated calcium-entry（SOCE）is an important pathway for extracellular[Ca²⁺]of mediated cells to enter the cell,which consists of the ORAI 1 protein in cell membrane and the stromal interaction molecule 1（STIM1）in endoplasmic reticulum.When the Ca²⁺in endoplasmic reticulum is exhausted,cytoplasmic structural domain STIM-CT can control the ORAI pathway open directly and let Ca²⁺flows in.It infers that it is possible to prompt the expression of NFAT by regulating SOCE pathway to increase the intracellular[Ca²⁺].Hence,this work,using light to control SOCE pathway(LOVsoc plasmid controls the influx of[Ca²⁺]),through transfecting the LOVsoc plasmid to ESCs and the transplanted transfection skin,investigates the pathophysiologic mechanisms of differentiation and re-epithelization of ESCs cells from in Vivo and in Vitro aspects after PBM released[Ca²⁺]activates NFAT.This work consists of 3 sections:Section 1.The evaluation study of the effect of clinical Photodynamic therapyUse PBM treat the refractory wounds of 5 patient by the clinically common Photodynamic therapy and observe their healing to clarify that PBM has promotion on wound healing.Methylene blue（MB）is used as photosensitizer and red Optical Diode as light source with wavelength 635nm,energy 120 J/cm²,power 100m W/cm².Irradiate the wound at 5cm from the wound vertically once a week till it heals.Section 2.The investigation of regulation mechanism through in Vitro cell experiment2.1 The extraction and detection of primary epidermal stem cellsIsolate and cultivate hp-ESCs from human preputial samples.According to the detection with ICC,in 90%of the primary ESCs,CK19,Integrinβ1（stem cell markers）had positive signals.As the PCR result showed,NFAT1,NFAT2,NFAT4 subtypes of m RNA in human primary ESCs all expressed,but NFAT3 did not.The same conclusion was made from the detection with ICC of the expression of NFAT subtypes in human primary ESCs.2.2 The clarification of the influence of NFATs on the proliferation and differentiation of hp-ESCs via AM404 restraining the expression of NFAT in hp-ESCs.（1）Use CCK8 to investigate the influence on cell activity after different duration of treatment of AM404（15um）.It was shown that the activity of hp-ESCs was reduced to 50%after 3-day-treatment of AM404.（2）Use Western Blot and ICC to detect hp-ESCs after 3-day-treatement of AM404（AM404 group）.It showed that the expression of NFAT1,NFAT2,NFAT4 subtypes was restrained.（3）In Ed U cell proliferation experiment,more ICC positive cells in AM404 group were detected,that is that the proliferation of hp-ESCs rises after the expression of NFAT was restrained.（4）Western Blot and ICC showed that the expression of CK10 protein,which is the index of cell differentiation,in AM404 group was lowered.（5）Detect the migration of cells by Transwell experiment.It showed that the transmembrane hp-ESCs in AM404 group were far less than that in CTL group.2.3 Cultivation of hp-ESCs,clarification of the change of[Ca²⁺]after transfected by LOVsoc plasmid and regulated by PBM.（1）Transfect LOVsoc plasmid to hp-ESCs,use CCK8 to investigate the influence on the activity of cell with different duration of PBM,select the best irradiation duration 30min.（2）Transfect LOVsoc plasmid to hp-ESCs given the best irradiation duration to treat the cells,observe the relative[Ca²⁺]fluorescence intensity,using FV3000 Calcium Imaging.A physiological calcium oscillation wave occurred in experiment group.（3）Use PBM to regulate hp-ESCs and detect the differentiation index CK10 of hp-ESCs by ICC,clarify the promotion of PBM on the differentiation of hp-ESCs.（4）Use PBM to regulate hp-ESCs.Via ICC,the entry of NFAT1,NFAT2,NFAT4 in nucleus were clearly detected in hp-ESCs and that the expression of the differentiation index CK10 rose was detected by Western Blot.Section 3.The investigation of mechanisms by in Vivo experiment（1）In wound healing model on mice,using qPCR,it was observed that the NFAT m RNA expressed during the normal recovery of the skin tissue of wound margin on mice.（2）Transfect LOVsoc plasmid.It was observed that the recovery rate of the wound increased significantly after PBM treatment.Clarify that PBM could activate NFAT and prompt the healing of wound via[Ca²⁺].In sum up.PBM can prompt the differentiation of hp-ESCs and healing of the wound by regulating[Ca²⁺]/NFAT pathway.