MiR-125b Level In Pbmcs Of Pediatric Patients With Tuberculosis And Its Relationship With Biological Behaviors Of Macrophage Chltivation In Vitro

PART ONE:THE EXPRESSION OF MIR-125 B AND ITS TARGET GENE RAF1 IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF CHILDREN WITH PULMONARY TUBERCULOSIS AND NORMAL CHILDRENObjective To investigate the expression of mi R-125 b, target gene Raf1 and proto-oncogene serine/ threonine protein kinase(RAF1) in peripheral blood mononuclear cells(PBMCs) of pediatric patients with pulmonary tuberculosis(PTB).Methods PBMCs of patients with PTB and healthy children was collected and separated, real time fluorescence quantitative PCR was adopted to detect m RNA expression level of mi R-125 b and RAF1, and Western blot was used to detect the protein level of RAF1.Results 1.The expression of mi R-125 b in PBMCs in pediatric patients with PTB was down regulated,Compared with normal children.2.m RNA and protein level of RAF1 were up regulated,Compared with normal children.Conclusion The expression of mi R-125 b and the expression of its target gene- RAF1 were increased,in children with pulmonary tuberculosis in PBMCs; Mi R-125 b may be involved in the mechanism of immune response in children with tuberculosis by target-regulating the expression of RAF1.PART TWO:THE RELATIONSHIP BETWEEN MIR-125 B AND THE BIOLOGICAL BEHAVIORS OF MACROPHAGE CULTIVATION IN VITROObjective to detect whether mi R-125 b target-regulate the expression of RAF1 in THP-1 macrophages and to observe the regulation of mi R-125 b on macrophage cell apoptosis and cell activity.Methods THP-1 macrophages were transinfected into mi R-125 b mimic, negative control mimic(NC-mimic), mi R-125 b inhibitor and negative control inhibitor(NC-inhibitor) respectively by Lipofectamine TM RNAi MIX reagent,for 48 hours of cultivation.They were named by mi R-mimic group,NC-mimic group,mi R-inhibitor group and NC-inhibitor group.Western blot was used to detect the expression of RAF1 in THP-1macrophage, annexin Ⅴ-FITC/PI double staining combined with flow cytometry(FCM) was carried out to detect cell apoptosis, and CCK-8 assay was adopted to detect cell activity.Results 1.In mi R-mimic group of THP-1 macrophages, compared with the control group —NC-mimic group, which the expression of mi R-125 b was high,the RAF1 expression was decreased; in mi R-inhibitor group of THP-1 macrophages, compared with the control group—NC-inhibitor group, which the expression of mi R-125 b was inhibited, and RAF1 expression was increased.2.In THP-1 macrophages, group mi R-mimic, compared with the group NC-mimic,which the expression of mi R-125 b was high,promoted the cell apoptosis and reduced the cell viability; mi R-inhibitor,compared with the group NC-inhibitor, which the expression of mi R-125 b was inhibited,inhibited the cell apoptosis and increased cell viability.Conclusion mi R-125 b regulates target gene RAF1 in THP-1macrophages.The expression of mi R-125 b level in THP-1 macrophage was up regulated to promote THP-1 macrophages apoptosis and decrease cell activity,and was down regulated to inhibit THP-1 macrophages apoptosis and incresed cell activity.