Effect Of Electric Field On Microglia Polarization By MiRNA-155 And MiRNA124

Objective:By Treating microglia cells with different immunophenotypes using Tumor Treating Fields(TTFields)as a platform,we explored the role of mi RNAs in the effect of TTFields on microglia cell polarization(focusing on mi RNA-155-5p,mi RNA-124-3p)to provide relevant experimental and basic research support for further exploring the mechanism of action of TTFields in the treatment of intracranial tumors.Methods:(1)Construction of TTFields experimental equipment:(1)Formula application: The length and width of the petri dish and the thickness of the side wall of T25 cells were accurately measured with vernier calipers,and the dielectric constant of the dish was determined by searching physics books,and the field strength of the self-made model was simulated and calculated by relevant experts.The formula was as follows :E2=U/ε2(d1/ε1+d2/ε2),and the voltage required by the two side walls was specified when used.(2)The bottom edge of the T25 cell culture bottle is coated with biononconductive adhesive,and the distance is1 cm wide groove is reserved.The double-sided conductive copper foil tape is affixed close to both sides of the groove and the bottom edge to make the cell culture bottle required by the basic experimental equipment of TTFields.(3)Disinfect the incubator in advance,and put the positive and negative alligator clip connecting wires into the constant temperature incubator through the reserved holes in the incubator;(4)Connect the whole set of equipment,adjust the experimental parameters in advance,and directly switch on the power before the experiment begins.(2)Cell experiment:(1)Cell culture: Mouse BV-2 microglia were cultured(37℃,5%CO2).(2)Cell grouping: The experiment was divided into blank control group(group A1),lipopolysaccharide induction group(group B1)and interleukin-4 intervention group(group C1).At the same time,the three groups of cells treated in the same way were placed in the electric field,and the blank electric field group(A2),lipopolysaccharide electric field group(B2)and interleukin-4 electric field group(C2)were generated again.Group significance: A2,B2 and C2 were the formal experimental part,and group B1 and C1 were the mode group with microglial cell polarization of M1 and M2,respectively.Under the same conditions,6 groups of cell culture vials were inoculated with 2×105 BV-2 cells,and the same amount of DMEM complete medium(1ml)was added for culture.b.After 12 hours,the cells could be completely adherent to the wall.In this time,the concentration of lipopolysaccharide in LPS group(B1)with lipopolysaccharide as inducer should be0.2mg/ml,and the concentration of IL-4 in interleukin 4 group(C1)should be 0.2ug/ml for induction preservation.c.The same three groups of cells were treated in the same way and placed in the electric field,labeled as blank electric field group(A2),lipopolypaccharide electric field group(B2)and interleukin-4 electric field group(C2);d.After 48 h of electric field treatment,cells in blank control group(A1),lipopolysaccharide induction group(B1),interleukin-4 intervention group(C1),blank electric field group(A2),lipopolysaccharide electric field group(B2)and interleukin-4 electric field group(C2)were collected again,with a total of six groups.e.The six groups of cells were collected by cell precipitation and stored at-80℃.Immunofluorescence labeling,flow cytometry and RT-q PCR were performed,and the results were statistically analyzed.Results:Both immunofluorescence and flow cytometry showed that:(1)M0 cells of mouse BV-2 microglia promoted the simultaneous transformation into M1 and M2 after 48 h TTFields treatment.(2)Lipopolysaccharide-induced mouse BV-2 microglia M1 cells showed a trend of polarization toward M1 after 48 h TTFields treatment.(3)Interleukin-4 induced mouse BV-2 microglia M1 cells inhibited the tendency of M2-polarization after 48 h TTFields treatment.The results of RT-q PCR showed that:(1)TTFields treated BV-2 microglia(group A2)tended to promote the polarization of M1 and M2,while the contents of mi RNA55-5p and mi RNA124-3p increased.(2)BV-2 microglia treated with lipopolysaccharide and TTFields(group B2)tended to promote the polarization of M1,while the content of mi RNA155-5p was significantly higher than that of blank group(A1)and lipopolysaccharide induced group(B1).(3)BV-2 microglia treated with interleukin4 +TTFields tended to inhibit M2 polarization,while the content of mi RNA124-3p was significantly higher than that in blank group(A1)and interleukin-4 induced group(C1)Conclusion:(1)TTFields promoted the polarization trend toward M1 and M2 of mouse BV-2microglia M0 cells;(2)TTFields induced further polarization toward M1 cells of mouse BV-2 microglia.(3)TTFields further inhibited M2-oriented polarization of mouse BV-2 microglia M2 cells.(4)TTFields may induce microglia to polarize towards M1 type by stimulating the increase of mi RNA-155-5p content;(5)The reason why TTFields could further inhibit the polarization of mouse BV-2microglia M2 cells towards M2 may not be closely related to mi RNA-124-3p.