Effects Of CD9 On Keratinocytes Migration In Electric Field And Its Mechanisms

Background: Wound healing is a complex and orderly physiological process,including the migration and proliferation of many types of cells,of which keratinocytes migration is the key to whether the wound can heal completely.The wound bioelectric field is generated immediately after the wound surface is formed.The wound edge is the positive electrode and the center of the wound surface is the negative electrode.Its intensity increases slowly and then gradually decreases.It continues until the re-epithelialization is completed and disappears.The electric field plays a key role in wound healing.In vivo studies have shown that the electric field can promote the directional migration of epidermal cells,but the mechanism is not yet clear.CD9 is a membrane protein that belongs to the family of four transmembrane proteins.It binds to other transmembrane molecules or proteins and affects cell migration,differentiation,and signal transduction.Our previous research found that CD9 was highly expressed in normal skin,and the expression was significantly reduced in the early and mid-term wound healing,which promoted the migration of keratinocytes,and returned to normal levels in the later period of wound healing.Combining the corresponding relationship between the wound bioelectric field and CD9 in wound healing,we speculate that the electric field may regulate cell migration by regulating the expression of CD9 in keratinocytes.AMPK is an AMP-dependent protein kinase,a key molecule that regulates bioenergy metabolism,and is also closely related to cell migration.Studies havefound that inhibiting the activity of AMPK can improve the motor ability of keratinocytes,and AMPK is an upstream signaling molecule of the four-transmembrane protein family.We suspect that AMPK may be involved in the regulation of CD9 by the electric field.This study proposed the scientific hypothesis that bioelectric field regulates CD9 expression through AMPK to promote epidermal cell migration.The exogenous electric field simulates the wound electric field to detect the effect of CD9 on epidermal cell migration under electric field conditions,and explores the regulation of electric field on epidermal cell AMPK and the electric field Under the conditions,AMPK regulates CD9,clarifies the mechanism of electric field regulating epidermal cell migration,and provides new theoretical basis for clinical wound repair.Objective: Explore the role of CD9 in the electric field to promote the migration of keratinocytes,and clarify the mechanism of EF regulation of keratinocytes migration.Methods: HaCaT cell line and mouse keratinocytes(MKS)were used in the experiment.Firstly,the effect of EF(200mV/mm)on the migration of keratinocytes was observed,and the movement track was analyzed statistically.The expression of CD9,AMPK and p-AMPK were observed after the cells were treated with EF.Using recombinant adenovirus(Ad-CD9-GFP)to establish CD9 high expression cell model and observe the effect of CD9 on cell movement.After using AMPK activator AICAR and AMPK inhibitor Compound C,we observed the effect of EF on the expression of CD9 by AMPK signaling pathway.The living cell workstation was used to observe the speed,direction and track of cell movement in a continuous period of time;Western blot was used to detect the protein expression of CD9,AMPK and p-ampk in keratinocytes;immunofluorescence staining was used to detect the expression anddistribution of CD9 in cells.Results: The biological intensity electric field promoted the migration of keratinocytes from the wound edge to the wound center,increased the movement speed,and decreased the expression of CD9.The expression of CD9 decreased gradually with the increase of treatment time and EF intensity.The cells with high expression of CD9 showed slow moving speed and scattered moving direction,but the overall migration direction remained unchanged.EF treatment can reduce the activity of AMPK,reduce the decrease range of CD9 in EF after activating AMPK,and increase the decrease range of CD9 in EF after inhibiting its activity.Conclusion: By regulating the AMPK signal pathway,EF can affect the expression of CD9 and promote the migration of keratinocytes.This study enriches the theoretical system of wound healing molecular mechanism and provides a new idea for the clinical treatment of wound healing.