Effect And Function Of Sp110 Gene Silencing On Macrophages Against Mycobacterium Tuberculosis

Object: A specific si RNA was used to construct a cell model with reduced Sp110 expression,and on this basis,the influence of Sp110 on macrophages after Mycobacterium tuberculosis infection was studied in order to lay the foundation for promoting the transgene synthesis of anti-tuberculosis genes and discovering new methods of treating tuberculosis.Methods: Macrophages were transfected with Lipofectamin TM3000 Reagent wrapped si RNA.Real-time quantitative PCR was used to detect the m RNA expression level of Sp110 gene in RAW264.7 cells,Total protein was extracted from the cells.Expression of SP110 protein in macrophages was detected by Western Blot method.After successfully constructing macrophages with reduced expression of Sp110,macrophages were infected with BCG and H37 Ra respectively,and the cells were washed 6 hours after infection to remove extracellular Mtb and recorded as 0 h at this time.The acid-fast staining was used to analyze the phagocytosis of Mtb by macrophages,and the cell growth curve was drawn in four time periods of 0 h,24 h,48 h,and 72 h to understand the proliferation.Flow cytometry fluorescent antibody specific binding method was used to analyze cell death.Results:(1)After si RNA transfecting 24 hours,the cell morphology of the negative control group did not change significantly compared with the blank control.The cell body was round and highly refractive.It had few intracellular particles.Cell body of si RNA group was irregular;(2)After transfection of si RNA for 24 hours,the m RNA expression of Sp110 gene in the si RNA group was significantly reduced compared with the blank control group(P < 0.01).Compared with the negative control group,the m RNA expression of the si RNA group was significantly reduced.(P < 0.01).There was no difference between the blank control and the negative control(P > 0.05);(3)After transfection of si RNA for 48 hours,the relative expression of SP110 protein in the si RNA group was significantly reduced compared with the blank control group(P < 0.001).Compared with the negative control group,the expression of SP110 protein in the si RNA group was significantly reduced(P < 0.001).There was no difference in protein expression between the blank control group and the negative control group(P > 0.05);(4)The acid-fast staining results showed that about 30% of the cells engulfed at least one acid-fast bacilli.This shows that RAW264.7 cells can successfully engulf Mtb after Mycobacterium tuberculosis infection;(5)Without Mycobacterium tuberculosis infection,cells grow rapidly between 0 h and 24 h.Cells grew fastest between 24 h and 48 h,and slowed down between 48 h and 72 h.The decrease of Sp110 expression will not affect the proliferation of cells.When cells were infected with BCG,the cells proliferation in the control group showed an upward trend.The decrease of Sp110 expression will slow down the proliferation of cells.When H37 Ra was infected with cells,the cells proliferation in the control group showed an upward trend.Low expression of Sp110 would slow down the proliferation of cells,but there was no obvious difference in the effect of BCG and H37 Ra infection on the proliferation of cells;(6)After BCG infection,the apoptosis rate of the control group was 0.05%,and the apoptosis rate of the si RNA group was 4.7%.There is a statistical difference between the two groups(P < 0.001);After H37 Ra infection,the apoptosis rate of the control group was 1.13%,and the apoptosis rate of cells in si RNA group was 7.7%.There is a statistical difference between the two groups(P < 0.001).Conclusion:1.The low expression of Sp110 can reduce the proliferation of macrophages after infection,but BCG and H37 Ra have no difference in slowing down the proliferation of macrophages.2.The low expression of Sp110 can increase the apoptosis of macrophages.Mycobacterium tuberculosis with different virulence induced different apoptosis rates.