The Influence Of An AG-type Oligodeoxynucleotide On HMGB1Secretion In RAW264.7Cell Stimulated By CPG Oligodeoxynucieotide

Objective:This study aims to find the effect of MS19（an AG-type single strandedoligodeoxynucleotide）on the expression and the intracellular localization of HMGB1inRAW264.7cells treated with CpG ODN, in order to elucidate the role of MS19in the acutelung injury murine model deriving from H1N1influenza viruses.Methods：1. The cells used in this experiment were divided into such groups as medium, CpGODN stimulation, MS19stimulation, and CpG ODN plus MS19stimulation. We observedthe growth state of RAW264.7cells from different murine groups by a microscoperegularly after stimulation of the cells.2. By real-time quantitative PCR, we detect the effect of MS19on the HMGB1mRNA expression in RAW264.7cells stimulated by CPG oligodeoxynucleotide.3. By immunofluorescence, we detect the effect of MS19on HMGB1localization inRAW264.7cell stimulated by CPG oligodeoxynucleotide.4. Western blot was used to detect MS19affect the amount of extracellular HMGB1inRAW264.7cell stimulated by CPG oligodeoxynucleotide.Results:2、1. We observed，by a microsope，tEach group of RAW264.7cells stimulated withCpG ODN, MS19and CpG ODN combined MS19growth state with no obvious abnormalities compared to the control group at6h/12h/18h, the cells were adherent growth,consistent density, strong refractive index, significant quantities increased cell in goodcondition, broadly consistent form and clearly Contour.2. After the real-time PCR results showed that CpG ODN stimulation, MS19andMS19combined CpD ODN stimulation to stimulate RAW264.7cells in different timeperiods, HMGB1mRNA levels were no significant differences in contrast.3．By immunofluorescence, we observe the effect of MS19on HMGB1proteinlocalization in RAW264.7cells stimulated with CpG ODN at18h. We found that greenfluorescence localization inside and outside the cell appeared both in cells stimulated withCpG ODN and control group, extracellular fluorescence intensity of cells stimulated byCpG ODN are stronger than control group. Compared with control group,greenfluorescence of the cells stimulated by MS19was mainly located in the cell and the cellmembrane. Compared CpG ODN group with CpG ODN combined MS19group,twogroups both inside and outside were visible green fluorescence，while CpG ODN combinedMS19group intracellular fluorescence-based positioning.4．To detect the intracellular protein amount of HMGB1with Western blot, we foundthat when the RAW264.7cells were stimulated for48hours，the amount of HMGB1incells compared with control group had no significant difference; and that when the RAW264.7cells were stimulated for72hours，the amount of HMGB1in different groups couldbe ordered，from most to least，as followed：MS19group，CpG ODN combined MS19group，the control and CpG ODN group.Conclusion:1. MS19has no significant effect on the growth of RAW264.7cells stimulated withCpG ODN.2. MS19has no significant effect on the HMGB1mRNA expression in RAW264.7cells.3. MS19can inhibit HMGB1to transfer from the inside of cells to the outsidestimulated by CPG oligodeoxynucleotide.