矮小儿童PMBCs中锌转运体的表达及缺锌对垂体ZIP2和ZIP8表达的影响

发布时间：2018-05-08 16:36

本文选题：锌转运体 + 生长激素　；参考：《山东大学》2014年硕士论文

【摘要】：研究背景：锌是一种对生命至关重要的微量元素,在许多生物化学反应中起着举足轻重的作用。锌是细胞增殖及分化所必需的,并参与蛋白质、核苷酸、糖以及脂类的代谢以及调节机体的免疫功能。哺乳动物中有两个锌转运体蛋白家族直接参与细胞内锌离子的稳态平衡,即SLC39A和SLC30A两大家族。SLC39A编码ZIP蛋白,能够增加细胞外锌内流或促进细胞器内锌释放入胞浆,从而提高胞浆中锌的含量。SLC30A编码CDF蛋白,CDF蛋白也被称为ZnT蛋白,通过促进胞浆内锌外流和锌在细胞器内区室化分布而降低胞浆中锌的含量。缺锌普遍存在,在儿童中尤为常见。缺锌时骨生长减慢可导致脊柱动物生长迟缓。生长激素是由垂体分泌的,能刺激骨的生长和儿童生长密切相关。锌可以诱导人类生长激素的二聚化并能增强其生物学功能,因此锌和生长激素存在一定的联系。对人类锌调节的基因进行全基因阵列分析发现ZIP2对缺锌是最敏感的,近来有研究发现ZIP8在多器官形成及造血等方面有重要作用,ZIP8敲除后小鼠生长迟缓,多种组织内锌水平降低。因此,我们探讨身材矮小儿童的生长激素与锌及锌转运体的关系；并构建了大鼠缺锌模型,原代培养大鼠垂体细胞,在体内外研究缺锌情况下大鼠垂体ZIP2和ZIP8的表达水平。研究目的：通过观察矮小儿童生长激素激发试验中锌转运体的表达情况,探讨生长激素与锌及锌转运体之间的关系；构建大鼠缺锌模型,原代培养大鼠垂体细胞,体内外研究缺锌对大鼠垂体中ZIP2及ZIP8表达的影响。实验方法：一、生长激素激发试验用来鉴定是否存在生长激素缺乏。分别于空腹及服用刺激生长激素分泌的药物30分钟、60分钟及90分钟后抽血检测生长激素水平。临床收集接受生长激素激发试验的6名身材矮小儿童及15名正常对照儿童的新鲜抗凝外周血标本,并收集其血清检测锌含量。以密度梯度离心法分离样本中的单个核细胞(PBMCs),分别提取总RNA及总蛋白。通过实时定量的方法检测锌转运体ZIP1, ZIP2, ZIP6ZIP8及ZnT1mRNA的表达情况,通过Western-blot的方法检测ZIP2和ZIP8的蛋白表达情况。二、构建Wistar大鼠缺锌模型,检测缺锌时大鼠垂体中ZIP2和ZIP8mRNA的表达情况。同时原代培养大鼠垂体细胞,用不同浓度TPEN缺锌处理后观察细胞形态,并检测ZIP2和ZIP8mRNA的表达情况。实验结果：一、在矮小儿童生长激素激发试验中,生长激素浓度和ZIP1及ZIP2mRNA水平呈正相关(r=0.5133, P=0.0371; r=0.6719, P=0.0030);和ZIP8呈负相关(r=-0.5264, P=0.0285)。矮小儿童组ZIP2的表达水平高于对照组(P0.05),而ZIP6和ZIP8表达水平低于对照组(P0.05,P0.05)。二、缺锌组大鼠垂体中ZIP8的表达量低于对照组(P0.05),ZIP2的表达量也低于对照组,但无统计学意义(P0.05)。对培养的大鼠垂体原代细胞进行缺锌处理后ZIP2和ZIP8表达上调,并且TPEN浓度越高,表达上调越明显,特别ZIP2上调非常显著(P0.05,P0.05)。结论：生长激素浓度和ZIP1及ZIP2mRNA水平呈正相关；和ZIP8呈负相关。矮小儿童组ZIP2的表达水平高于对照组,而ZIP6和ZIP8表达水平低于对照组。提示生长激素与锌及锌转运体之间存在一定的内在联系。体内缺锌时大鼠垂体中ZIP8的表达降低,但是对培养的大鼠垂体原代细胞进行缺锌处理后ZIP2和ZIP8的表达均明显上调,提示在体内外垂体细胞对缺锌环境中ZIP2和ZIP8的表达水平不同。
[Abstract]:Research background:
Zinc is a vital trace element for life and plays an important role in many biochemical reactions. Zinc is essential for cell proliferation and differentiation, and is involved in the metabolism of proteins, nucleotides, sugar and lipids, and regulating the immune function of the body. In mammals, two zinc transporter proteins are directly involved in the thin mammals. The homeostasis of intracellular zinc ions, namely, SLC39A and SLC30A two large family.SLC39A encoding ZIP protein, can increase the extracellular zinc flow or promote the release of zinc into the cytoplasm in the organelle, thus increasing the content of zinc in the cytoplasm.SLC30A encoded CDF protein, and CDF protein, also known as ZnT protein, through the promotion of intracellular zinc Exodus and zinc in the intracellular compartment. It reduces the content of zinc in the cytoplasm.
Zinc deficiency is common in children and is particularly common in children. Slow bone growth can lead to growth retardation in the spine. Growth hormone is secreted by the pituitary gland, which can stimulate bone growth and children's growth. Zinc can induce human growth hormone dimerization and enhance its biological function, so zinc and growth hormone are present. A full gene array analysis of human zinc regulated genes found that ZIP2 is the most sensitive to zinc deficiency. Recent studies have found that ZIP8 plays an important role in the formation and hematopoiesis of multiple organs. The growth retardation of ZIP8 knockout mice and the decrease of zinc levels in a variety of tissues. Therefore, we explore growth hormone and zinc in short stature children. The relationship between the zinc transporter and the zinc deficiency model of rats, the primary culture of rat pituitary cells, and the expression level of ZIP2 and ZIP8 in the rat pituitary under the condition of zinc deficiency. A rat model of zinc deficiency was established. Primary cultured rat pituitary cells were used to study the effects of zinc deficiency on the expression of ZIP2 and ZIP8 in the pituitary gland in vivo and in vitro.
Experimental methods:
First, the growth hormone stimulation test was used to identify the presence of growth hormone deficiency. 30 minutes, 60 and 90 minutes of growth hormone levels were detected on the fasting and 60 and 90 minutes, respectively. Clinical collection of fresh anticoagulants of 6 stature children and 15 normal control children with growth hormone stimulation test The content of zinc in peripheral blood was collected and the content of zinc was collected. The total RNA and total protein were extracted by density gradient centrifugation (PBMCs). The expression of ZIP1, ZIP2, ZIP6ZIP8 and ZnT1mRNA in zinc transporter was detected by real-time quantitative method. The protein expression of ZIP2 and ZIP8 was detected through Western-blot method. Situation.
Two, the zinc deficiency model of Wistar rats was constructed to detect the expression of ZIP2 and ZIP8mRNA in the pituitary of rats with zinc deficiency. At the same time, the rat pituitary cells were cultured in the primary culture. The morphology of the cells was observed with different concentrations of TPEN zinc deficiency treatment, and the expression of ZIP2 and ZIP8mRNA was detected. The experimental results were as follows:
In the growth hormone stimulation test of small children, the growth hormone concentration was positively correlated with the level of ZIP1 and ZIP2mRNA (r=0.5133, P=0.0371; r=0.6719, P=0.0030), and negative correlation with ZIP8 (r=-0.5264, P=0.0285). The expression level of ZIP2 in the small children group was higher than that of the control group (P0.05), and the level of ZIP6 and expression was lower than that of the control group.
Two, the expression of ZIP8 in the pituitary of rats with zinc deficiency was lower than that of the control group (P0.05), and the expression of ZIP2 was also lower than that of the control group, but there was no statistical significance (P0.05). The expression of ZIP2 and ZIP8 in the cultured rat pituitary cells was up to up after zinc deficiency treatment, and the higher the concentration of TPEN, the more obvious the expression was up, especially the up regulation of ZIP2 (P0.05, P0.05). Conclusion:
There was a positive correlation between the level of growth hormone and the level of ZIP1 and ZIP2mRNA, and negative correlation with ZIP8. The expression level of ZIP2 in the small children group was higher than that of the control group, while the expression level of ZIP6 and ZIP8 was lower than that of the control group.
The expression of ZIP8 in the rat pituitary decreased when zinc deficiency was in vivo, but the expression of ZIP2 and ZIP8 in the cultured rat pituitary cells were obviously up regulated after zinc deficiency treatment, suggesting that the expression level of ZIP2 and ZIP8 was different in the pituitary cells in vivo and in the zinc deficiency environment.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R725.8

【共引文献】