本文選題：鋅轉運體 + 生長激素�。� 參考：《山東大學》2014年碩士論文

【摘要】：研究背景： 鋅是一種對生命至關重要的微量元素,在許多生物化學反應中起著舉足輕重的作用。鋅是細胞增殖及分化所必需的,并參與蛋白質、核苷酸、糖以及脂類的代謝以及調節(jié)機體的免疫功能。哺乳動物中有兩個鋅轉運體蛋白家族直接參與細胞內鋅離子的穩(wěn)態(tài)平衡,即SLC39A和SLC30A兩大家族。SLC39A編碼ZIP蛋白,能夠增加細胞外鋅內流或促進細胞器內鋅釋放入胞漿,從而提高胞漿中鋅的含量。SLC30A編碼CDF蛋白,CDF蛋白也被稱為ZnT蛋白,通過促進胞漿內鋅外流和鋅在細胞器內區(qū)室化分布而降低胞漿中鋅的含量。 缺鋅普遍存在,在兒童中尤為常見。缺鋅時骨生長減慢可導致脊柱動物生長遲緩。生長激素是由垂體分泌的,能刺激骨的生長和兒童生長密切相關。鋅可以誘導人類生長激素的二聚化并能增強其生物學功能,因此鋅和生長激素存在一定的聯(lián)系。對人類鋅調節(jié)的基因進行全基因陣列分析發(fā)現(xiàn)ZIP2對缺鋅是最敏感的,近來有研究發(fā)現(xiàn)ZIP8在多器官形成及造血等方面有重要作用,ZIP8敲除后小鼠生長遲緩,多種組織內鋅水平降低。因此,我們探討身材矮小兒童的生長激素與鋅及鋅轉運體的關系；并構建了大鼠缺鋅模型,原代培養(yǎng)大鼠垂體細胞,在體內外研究缺鋅情況下大鼠垂體ZIP2和ZIP8的表達水平。研究目的：通過觀察矮小兒童生長激素激發(fā)試驗中鋅轉運體的表達情況,探討生長激素與鋅及鋅轉運體之間的關系；構建大鼠缺鋅模型,原代培養(yǎng)大鼠垂體細胞,體內外研究缺鋅對大鼠垂體中ZIP2及ZIP8表達的影響。 實驗方法： 一、生長激素激發(fā)試驗用來鑒定是否存在生長激素缺乏。分別于空腹及服用刺激生長激素分泌的藥物30分鐘、60分鐘及90分鐘后抽血檢測生長激素水平。臨床收集接受生長激素激發(fā)試驗的6名身材矮小兒童及15名正常對照兒童的新鮮抗凝外周血標本,并收集其血清檢測鋅含量。以密度梯度離心法分離樣本中的單個核細胞(PBMCs),分別提取總RNA及總蛋白。通過實時定量的方法檢測鋅轉運體ZIP1, ZIP2, ZIP6ZIP8及ZnT1mRNA的表達情況,通過Western-blot的方法檢測ZIP2和ZIP8的蛋白表達情況。 二、構建Wistar大鼠缺鋅模型,檢測缺鋅時大鼠垂體中ZIP2和ZIP8mRNA的表達情況。同時原代培養(yǎng)大鼠垂體細胞,用不同濃度TPEN缺鋅處理后觀察細胞形態(tài),并檢測ZIP2和ZIP8mRNA的表達情況。實驗結果： 一、在矮小兒童生長激素激發(fā)試驗中,生長激素濃度和ZIP1及ZIP2mRNA水平呈正相關(r=0.5133, P=0.0371; r=0.6719, P=0.0030);和ZIP8呈負相關(r=-0.5264, P=0.0285)。矮小兒童組ZIP2的表達水平高于對照組(P0.05),而ZIP6和ZIP8表達水平低于對照組(P0.05,P0.05)。 二、缺鋅組大鼠垂體中ZIP8的表達量低于對照組(P0.05),ZIP2的表達量也低于對照組,但無統(tǒng)計學意義(P0.05)。對培養(yǎng)的大鼠垂體原代細胞進行缺鋅處理后ZIP2和ZIP8表達上調,并且TPEN濃度越高,表達上調越明顯,特別ZIP2上調非常顯著(P0.05,P0.05)。結論： 生長激素濃度和ZIP1及ZIP2mRNA水平呈正相關；和ZIP8呈負相關。矮小兒童組ZIP2的表達水平高于對照組,而ZIP6和ZIP8表達水平低于對照組。提示生長激素與鋅及鋅轉運體之間存在一定的內在聯(lián)系。 體內缺鋅時大鼠垂體中ZIP8的表達降低,但是對培養(yǎng)的大鼠垂體原代細胞進行缺鋅處理后ZIP2和ZIP8的表達均明顯上調,提示在體內外垂體細胞對缺鋅環(huán)境中ZIP2和ZIP8的表達水平不同。
[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.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R725.8

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