Zinc Deficiency and Thl Functions: Molecular Mechanisms

锌在人体免疫功能中起着重要作用。在缺锌人群中观察到胸腺素活性降低，IL- 2产生减少，NK细胞裂解活性降低，细胞溶解性T细胞减少，能量降低，CD4/CD8降低，CD4+ CD45RA+/CD4+ CD45RO+比值降低，这些异常可以通过补充锌来纠正。为了了解锌对IL-2产生的作用机制，我们提出利用ThO人恶性淋巴母细胞样细胞株HUT-78进行研究。人IL-2基因启动子包含一个真正的Rel/NF-kB因子的结合位点，NF-kB与DNA的结合被锌螯合剂特异性阻断，并通过添加锌来重建。NF-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，NF-kB的活化和向细胞核的易位以及NF-kB的基因表达会减少，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)通过凝胶移位试验和共聚焦成像对NF-kB的核结合；2)用含有NF-kB增强子元件的荧光素酶报告基因载体对细胞进行基因转染；3)测定细胞细胞质部分中磷酸化、未磷酸化和泛素化形式的IkB （NF-kB复合物的抑制分子）。我们将利用反义p105表达载体在HUT-78细胞中检测NF-kB对锌对IL-2和IL-2受体α转录激活的作用。我们还假设，在PHA刺激的缺锌人单核细胞中，NF-kB激活、IL-2产生和IL-2 mRNA会降低，这些异常将通过体内和体外补锌来纠正。在本研究中，我们将从养老院中选择适当数量的健康流动老年人受试者。我们以前的经验表明，大约30%的健康老年人缺锌。

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.