Effect Of Mycobacterium Tuberculosis Eis Gene On THP-1 Cell Immune

Retention and resistance are two major problems at present of TB control,and the retention of Mycobacterium tuberculosis(Mycobacterium tuberculosis, MTB)is an important cause of chronic infection of tuberculosis. The retention of TB is an essential cause of a series of problems what is latent infection, recurrence, long-term treatment and resistance, etc. MTB keeps its stability and environmental non reactive, escape destruction from medcine and the immune system through a variety of ways. Wo could not get the most clear mechanism so far and that is why wo can not find the treatment method of infection. The study found that MTB can enter macrophages in order to adapt the macrophage intracellular environment to achieve retention effect. With the elucidation of complete genome sequence of MTB and the development of genetic techniques,it provides effective means to find MTB retention mechanism. MTB eis gene exists only in the pathogenicity of MTB, and it can inhibit the immune reaction who protects cell,and it also can increase retention of non pathogenic MTB in host cell.The 42 kd protein which is the product of EIS gene can affect the secretion of IL-10 and TNF-α,lead suppression of macrophage activation,and play a certain role in MTB retention. Our previous studies have confirmed that Eis protein can improve the M.S ’s survival rate in host macrophage, but the mechanism of MTB intracellular retention is not clear. Research shows that changes in secretion of local cytokine influence the MTB ’s retention in macrophages. Whether or not it is have the relationship between the the secretion of cytokinesbut and retention in macrophagein,but the report in the literature is very few so far.IL-10 is an efficient immunosuppressive cytokines, also known as cytokine synthesis inhibitory factor,which has extensive biological activities. It can change the immune response and the expression of MHC class II antigens, also can selectively inhibit certain aspects of the function of macrophages, and has the obvious effect on T cell and B cell function. The study was designed to explore the effect of EIS gene on the secretion of cytokines of mononuclear macrophage, screen differentially expressed cytokines and provide experimental basis for retention mechanism from the cellular immunity.Methods:1.The 1640 culture medium containing 10% fetal bovine serum was used to culture the THP-1 cell whose concentration was1×105 /ml.The THP-1cell was induced to differentiate into macrophages by PMA. MS-pmv261-eis and the control bacteria MS-pmv261 was cultured 7days and then was centrifuged. Flora was hard winded, shocked and mixed, and then counted with UV Spectrophotometry. Finally, according to the MOI=10:1 infected differentiated macrophages.2.Taked the cell supernatant, tested the level of IL-10 in THP-1 by ELISA. The THP-1 cells were inoculated to 6 pore plate, induced differentiation for 24 hours by PMA,washed by PBS and then infected byMS-pmv261-eis. The cell supernatant was collected after 18 hours, 1000 g centrifugal 15 min at room temperature.Taked 100 ul and tested IL-4, IL-6, IL-10,IL-12, INF-γby ELISA. Drew the standard curve according to the sample, calculated the concentration of cytokines.3.Total RNA was extracted from the collected cells, further detection of IL-10 gene expression by RT-PCR. Infected cells were collected after 18 h, then extracted total RNA. Specific IL-10 primer was designed according to the results of the above ELISA. Further confirmed the cytokine gene expression by RT-PCR. Primer sequences: the upstream primer: 5’-GGCTTCCTAACTGCTACA-3’; the downstream primer: 5’-CTCCTGACCTCAAGTGAT-3. PCR conditions: 95 ° Cinitial denaturation 3 min; 95 ° C denaturation 30 s, 60 oC annealing 30 s, 72 oC extension for 30 s, 35 cycles, in the end 72 oC extension of 10 min. Collected PCR products,1% agarose gel electrophoresis and analysis.4.Analysed data with the method of statistics by SPSS 15.0.Results:1.Thp cells were Successfully induced into macrophages by PMA. MS-pmv261-eis and MS-pmv261 MOI=10:1 had infected differentiated macrophages.2.ELISA results showed that: eis gene could cause IL-10 OD values increased significantly(P<0.05),and had no significant influence on the expression of cytokines in IL-4, IL-6, IL-12, INF-γ(P>0.05).3.Extracted total RNA and tested by RT-PCR, the results showed mRNA strip of IL-10 reinforced.Conclusions:1.ELISA results showed that: eis gene could cause IL-10 increased significantly(P<0.05),and had no significant influence on the expression of cytokines in IL-4, IL-6, IL-12, INF-γ(P>0.05).2.RT-PCR detection further showed that eis gene could cause IL-10 increased from the gene level.