Over-expression Of ZIP2in PBMCs From Children With Zinc Deficiency And Effected Of ZIP2on The IFN-γ Secretion Of Jurkat, E6-1Cells

BackgroundZinc (Zn) is a ubiquitous trace element. Zinc is an important component of more than300kinds of enzymes and transcription factors. It plays an important role in structure stable, catalytic and regulatory function. Worldwide, the incidence of diseases caused by zinc deficiency is estimated to more than20%. Zinc deficiency may affect the health of more than two billion people in developing countries. Zinc deficiency occurs mainly due to malnutrition, aging, and inborn or acquired impairment of zinc uptake, and can lead to thymic atrophy, malfunction of immune cells, and subsequently, high incidence of infections, accompanied by growth retardation and endocrine dysfunction. In clinical practice, zinc supplementation may be one of the effective adjunctive treatments of diseases caused by zinc deficiency, especially vulnerable populations:children and the elderly.Zinc is essential in both cell-mediated and humoral immunity. Zinc deficiency results in a multitude of alterations in immune function, including impairment of cellular mediators of innate immunity. The immune system is regulated delicately by zinc homeostasis. The functions of zinc in the immune system have been studied extensively. Zinc homeostasis is highly regulated via the gastrointestinal tract. This tight regulation requires multiple transporters to work in coordination. Two families of mammalian zinc transporters exist:ZIP and ZnT. As the opposite zinc transporting function of the two zinc transport gene families, they play a crucial role in maintenance of both intracellular and extracellular zinc homeostasis.In recent years, studies have found that ZIP2protein may be related with the immunity, especially under conditions of low zinc. Compared with ZIP2knockout mice the ZIP2gene expressed actively in immature immune cells of the wild mice by gene expression analysis. ZIP2was up-regulation in peripheral blood mononuclear cells (PBMCs) of asthma infants whose serum zinc level was lower than normal infants. These studies suggest that ZIP2may play an important role in the immune cells. Therefore, our research was to explore the relationship between the expression levels of ZIP2and zinc deficiency in immune cells.ObjectiveInvestigate the impact of zinc deficiency on ZIP2expression in PBMCs of children in vivo and vitro.Transfect the constructed pEGFP-N1-ZIP2expression vector into the T lymphocyte cells Jurkat E6-1transiently to explore the effect of ZIP2on cytokines secreting.MethodsThe anticoagulant blood samples of20children with zinc deficiency and20normal children, severed as controls, as well as blood samples of different ages adults were collected. Serum zinc content was measured by atomic absorption spectrometry. PBMCs was separated by density gradient centrifugation. The mRNA expression of zinc transporters:ZIP2、ZIP1、ZIP6and ZnTlof PBMCs were measured via real-time PCR method. Pripheral blood mononuclear cells from normal children was cultured in vitro, treated with TPEN and the expression of ZIP2was detected every week till the seventh week to explore the effect of long time zinc deficiency on ZIP2expression in vitro.Different concentrations of TPEN processing Jurkat, E6-1cell lines12h, to select a suitable concentration of TPEN.Transfect the constructed pEGFP-Nl-ZIP2expression vector into the T lymphocyte cells Jurkat E6-1transiently, treated with TPEN for12h after transfection36h, mRNA level and protein level of ZIP2were measured by Real-time PCR and Western-blot after transfection48h. After pEGFP-N1-ZIP2vector was successfully transfected into cells, other zinc transporters such as ZIP1、ZnT1、ZnT5、ZIP6、ZIP10and cytokines:INF-γ和IL-6were examined using the real-time PCR; proliferation capacity of target cells was measured by MTT assay.Results:Real-time PCR results showed the expression of ZIP2mRNA was significantly up-regulation in the children with zinc deficiency, compared with the normal children (P<0.05). While ZnT1was up-regulation, ZIP1was down-regulation, ZIP6had no change. The result showed that the ZIP2expression of the infant and the old-people were significant higher than the youth in the PBMCs from different age groups. The data showed that ZIP2mRNA expression of the cultured PBMCs with the deprivation group appears as first decreased and then increased compared with the normal group.Real-time PCR showed that over-expression of ZIP2was found after transfection of pEGFP-N1-ZIP2for48h and the expression of ZnT1was down-regulated(P<0.05), while expression of other zinc transporters genes:ZIP1、ZIP6、ZIP10and ZnT5had no changes (P>0.05). The cytokine INF-γ(P<0.05) was up-regulation and IL-6had no change (P>0.05); but the expression of INF-γ was down-regulation (P<0.05) and IL-6expression had no difference when the transfected cells were treated by TPEN. The proliferation capacity of cells with transfection of pEGFP-N1-ZIP2had no significant difference compared with no transfection cells.(P>0.05)Conclusion:Our research detected the expression of ZIP2in PBMCs from children under the condition of zinc deficiency in vivo and vitro. The results showed that ZIP2mRNA expression was significantly up-regulated in children with zinc deficiency compared with the normal children,treated Jurkat, E6-1cell line with TPEN can up-regulate the expression of ZIP2. It suggested that ZIP2may play an essential role in maintaining zinc homeostasis while zinc deficiency.At the same time, Transfect the constructed pEGFP-N1-ZIP2expression vector into the T lymphocyte cells Jurkat E6-1transiently to observe the expression of cytokines. The result showed that over-expression of ZIP2could up-regulate the expression of INF-γ while there was no change of IL-6. It suggested that ZIP2may play an important role in the immune regulation.