Research On Changes Of PBMCs TLRs Expression And Cellular Immunity Function Of HFMD Children Infected By EV71

Objective: To explore the changes of cellular immunity,mRNA expression level of peripheral blood mononuclear cell’s Toll-like receptor,among children with hand foot and mouth disease caused by enterovirus 71.Methods: 67 children diagnosed with hand foot and mouth disease caused by EV71 in Anhui Provincial Children’s Hospital were enrolled in this study.The children were divided into 2 groups: severe group(37 cases)and mild group(30 cases).At the same time,25 healthy subjects for routine physical examination were also included as control group.Collected subjects’ peripheral blood to detect the lymphocyte subtypes of CD3~+,CD4~+,CD8~+T lymphocyte,B lymphocyte and NK cell with flow cytometry method.At the same time collected subjects’ serum to detect cytokine levels such as IL-2R,IL-6,IL-8,IL-10,TNF-α.Extracted PBMCs by density gradient centrifugation method then separated RNA to amplify PBMCs’ TLR3 mRNA,TLR7 mRNA,β-actin mRNA with RT-PCR and Southern Blot technology.Using formula: TLR3level=TLR3/β-actin,TLR7 level=TLR7/β-actin to estimate TLR3 mRNA and TLR7 mRNA expression levels.Results: Children with hand foot and mouth disease caused by enterovirus 71 had lower ratios of CD3~+,CD4~+,CD8~+T cell,NK cell both in severe group and mild group than healthy group(P<0.05),the severe group had much lower ratios of CD3~+,CD4~+T cell than mild group(P<0.05),however ratios of CD8~+T and NK cell had no significant differences between these two groups(P>0.05);B cell ratio of two disease groups were higher than control group(P<0.05),but severe group had much higher B cell ratio than control group(P<0.05).Levels of serum IL-2R,IL-6,IL-8,IL-10 and TNF-αin two disease group children were higher than healthy children,the differences were statistical(P<0.05),IL-2R and TNF-α levels in severe children were significantly higher than mild children(P<0.05),but there were no significant differences of IL-6,IL-8,IL-10 levels between severe group and mild group(P>0.05).TLR3 mRNA and TLR7 mRNA expression levels in EV71 infected children were higher than healthy children(P<0.05),but there were no significant differences between two disease groups(P>0.05).Conclusions:1)Children with hand foot and mouth disease caused by enterovirus 71 had lower ratios of CD3~+,CD4~+,CD8~+T cell and NK cell than healthy children,ratios of CD3~+,CD4~+T cell kept continuous declination as HFMD aggravated however HFMD children had higher ratio of B cell and kept increasing as HFMD aggravated.It is suggested that apoptosis of lymphocytes may be involved in disease aggravation.2)EV71 infected HFMD children’ immune function was abnormal,serum cytokine levels such as IL-2R,IL-6,IL-8,IL-10,TNF-αincreased obviously,suggesting that abnormal secretion of cytokines may be involved in disease aggravation.3)The expression levels of PBMCs’ TLR3 mRNA and TLR7 mRNA increased obviously,suggesting that activation of TLRs may participate in the pathogenesis of EV71.