Zinc Deficiency and Thl Functions: Molecular Mechanisms

锌在人体免疫功能中起着重要作用。缺锌者胸腺细胞活性降低，IL-2产生减少，NK细胞杀伤活性降低，细胞溶解T细胞减少，无能，CD_4/CD_8降低，CD_4~+CD45RA~+/CD_4~+CD45RO~+比值降低，这些异常可通过补锌纠正。为了了解锌对IL-2产生的作用机制，我们建议利用人恶性淋巴母细胞系HUT-78进行研究。人IL-2基因启动子含有一个真正的REL/NF-kB因子的结合部位，而NF-kB与DNA的结合被锌螯合剂特异性地阻断，并通过添加锌来重组。核因子-kB还与IL-2受体α的调控基因结合。我们推测，在缺锌的HUT-78细胞中，核因子-kB的活化和移位以及核因子-kB的基因表达将减少，从而导致IL-2和IL-2受体α的基因表达减少，IL-2、sIL-2受体α和总sIL-2受体的产生减少。不同浓度的锌对HUT-78细胞中NF-kB活化的影响将通过以下方法确定：1)凝胶位移分析和共聚焦成像检测核内NF-kB的结合；2)含NF-kB增强子元件的荧光素酶报告基因载体的细胞内基因转染；3)胞浆部分中磷酸化、非磷酸化和泛素化形式的IKB(核因子-kB复合体的抑制分子)的检测。利用反义p105表达载体在HUT-78细胞中检测核因子-kB在锌对IL-2和IL-2受体α转录激活中的作用。我们还假设，在PHA刺激的缺锌人单核细胞中，核因子-kB的激活、IL-2的产生和IL-2mRNA的表达将减少，这些异常将通过体内和体外补锌得到纠正。在这项研究中，我们将从养老院中选择适当数量的健康非卧床老年受试者。我们以往的经验表明，这些健康的老年人中约有30%是缺锌的。

Zinc plays an important role in immune functions in humans. Decreased thymulin activity, decreased production of IL- 2, decreased NK cell lytic activity, decreased cytolytic T cells, anergy, decreased CD4/CD8 and decreased CD4+ CD45RA+/CD4+ CD45RO+ ratios, have been observed in zinc deficient humans and these abnormalities are corrected by zinc supplementation. In order to understand the mechanism of zinc action on IL-2 production, we propose to utilize HUT-78, a ThO human malignant lymphoblastoid cell line for our studies. The human IL-2 gene promoter contains one binding site for genuine Rel/NF-kB factors and binding of NF- kB to DNA is specifically blocked by a zinc chelator and is reconstituted by addition of zinc. NF-kB also binds to the regulatory gene of IL-2 receptor alpha. We hypothesize that in zinc deficient HUT-78 cells, the activation and translocation of NF-kB to nucleus and the gene expression of NF-kB will be decreased and this will lead to decreased gene expression of IL-2 and IL-2 receptor alpha and decreased production of IL-2, sIL-2 receptor alpha and total sIL-2 receptors. The effect of different concentrations of zinc on NF-kB activation in HUT-78 cells will be determined by i) nuclear binding of NF-kB by gel shift assay and confocal imaging ii) gene transfection of cells with luciferase reporter gene vector containing NF-kB enhancer element and iii) assays of phosphorylated, unphosphorylated and ubiquitinated forms of IkB (the inhibitory molecule of the NF-kB complex) in cellular cytosolic fraction. The functional role of NF-kB on transcriptional activation of IL-2 and IL-2 receptor alpha as affected by zinc will be determined by using antisense p105 expression vector in HUT-78 cells. We are also hypothesizing that NF-kB activation, IL-2 production and IL-2 mRNA will be decreased in PHA stimulated zinc deficient human mononuclear cells and that these abnormalities will be corrected by in vivo and in vitro zinc supplementation. For this study we will select appropriate number of healthy ambulatory elderly subjects from a nursing home. Our previous experience indicates that approximately 30 percent of these healthy elderly are zinc deficient.