發(fā)布時(shí)間：2018-05-08 16:36

  本文選題：鋅轉(zhuǎn)運(yùn)體 + 生長(zhǎng)激素　； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：研究背景： 鋅是一種對(duì)生命至關(guān)重要的微量元素,在許多生物化學(xué)反應(yīng)中起著舉足輕重的作用。鋅是細(xì)胞增殖及分化所必需的,并參與蛋白質(zhì)、核苷酸、糖以及脂類的代謝以及調(diào)節(jié)機(jī)體的免疫功能。哺乳動(dòng)物中有兩個(gè)鋅轉(zhuǎn)運(yùn)體蛋白家族直接參與細(xì)胞內(nèi)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(xì)胞外鋅內(nèi)流或促進(jìn)細(xì)胞器內(nèi)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(guò)促進(jìn)胞漿內(nèi)鋅外流和鋅在細(xì)胞器內(nèi)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(jiàn)。缺鋅時(shí)骨生長(zhǎng)減慢可導(dǎo)致脊柱動(dòng)物生長(zhǎng)遲緩。生長(zhǎng)激素是由垂體分泌的,能刺激骨的生長(zhǎng)和兒童生長(zhǎng)密切相關(guān)。鋅可以誘導(dǎo)人類生長(zhǎng)激素的二聚化并能增強(qiáng)其生物學(xué)功能,因此鋅和生長(zhǎng)激素存在一定的聯(lián)系。對(duì)人類鋅調(diào)節(jié)的基因進(jìn)行全基因陣列分析發(fā)現(xiàn)ZIP2對(duì)缺鋅是最敏感的,近來(lái)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(zhǎng)遲緩,多種組織內(nèi)鋅水平降低。因此,我們探討身材矮小兒童的生長(zhǎng)激素與鋅及鋅轉(zhuǎn)運(yùn)體的關(guān)系；并構(gòu)建了大鼠缺鋅模型,原代培養(yǎng)大鼠垂體細(xì)胞,在體內(nèi)外研究缺鋅情況下大鼠垂體ZIP2和ZIP8的表達(dá)水平。研究目的：通過(guò)觀察矮小兒童生長(zhǎng)激素激發(fā)試驗(yàn)中鋅轉(zhuǎn)運(yùn)體的表達(dá)情況,探討生長(zhǎng)激素與鋅及鋅轉(zhuǎn)運(yùn)體之間的關(guān)系；構(gòu)建大鼠缺鋅模型,原代培養(yǎng)大鼠垂體細(xì)胞,體內(nèi)外研究缺鋅對(duì)大鼠垂體中ZIP2及ZIP8表達(dá)的影響。 實(shí)驗(yàn)方法： 一、生長(zhǎng)激素激發(fā)試驗(yàn)用來(lái)鑒定是否存在生長(zhǎng)激素缺乏。分別于空腹及服用刺激生長(zhǎng)激素分泌的藥物30分鐘、60分鐘及90分鐘后抽血檢測(cè)生長(zhǎng)激素水平。臨床收集接受生長(zhǎng)激素激發(fā)試驗(yàn)的6名身材矮小兒童及15名正常對(duì)照兒童的新鮮抗凝外周血標(biāo)本,并收集其血清檢測(cè)鋅含量。以密度梯度離心法分離樣本中的單個(gè)核細(xì)胞(PBMCs),分別提取總RNA及總蛋白。通過(guò)實(shí)時(shí)定量的方法檢測(cè)鋅轉(zhuǎn)運(yùn)體ZIP1, ZIP2, ZIP6ZIP8及ZnT1mRNA的表達(dá)情況,通過(guò)Western-blot的方法檢測(cè)ZIP2和ZIP8的蛋白表達(dá)情況。 二、構(gòu)建Wistar大鼠缺鋅模型,檢測(cè)缺鋅時(shí)大鼠垂體中ZIP2和ZIP8mRNA的表達(dá)情況。同時(shí)原代培養(yǎng)大鼠垂體細(xì)胞,用不同濃度TPEN缺鋅處理后觀察細(xì)胞形態(tài),并檢測(cè)ZIP2和ZIP8mRNA的表達(dá)情況。實(shí)驗(yàn)結(jié)果： 一、在矮小兒童生長(zhǎng)激素激發(fā)試驗(yàn)中,生長(zhǎng)激素濃度和ZIP1及ZIP2mRNA水平呈正相關(guān)(r=0.5133, P=0.0371; r=0.6719, P=0.0030);和ZIP8呈負(fù)相關(guān)(r=-0.5264, P=0.0285)。矮小兒童組ZIP2的表達(dá)水平高于對(duì)照組(P0.05),而ZIP6和ZIP8表達(dá)水平低于對(duì)照組(P0.05,P0.05)。 二、缺鋅組大鼠垂體中ZIP8的表達(dá)量低于對(duì)照組(P0.05),ZIP2的表達(dá)量也低于對(duì)照組,但無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。對(duì)培養(yǎng)的大鼠垂體原代細(xì)胞進(jìn)行缺鋅處理后ZIP2和ZIP8表達(dá)上調(diào),并且TPEN濃度越高,表達(dá)上調(diào)越明顯,特別ZIP2上調(diào)非常顯著(P0.05,P0.05)。結(jié)論： 生長(zhǎng)激素濃度和ZIP1及ZIP2mRNA水平呈正相關(guān)；和ZIP8呈負(fù)相關(guān)。矮小兒童組ZIP2的表達(dá)水平高于對(duì)照組,而ZIP6和ZIP8表達(dá)水平低于對(duì)照組。提示生長(zhǎng)激素與鋅及鋅轉(zhuǎn)運(yùn)體之間存在一定的內(nèi)在聯(lián)系。 體內(nèi)缺鋅時(shí)大鼠垂體中ZIP8的表達(dá)降低,但是對(duì)培養(yǎng)的大鼠垂體原代細(xì)胞進(jìn)行缺鋅處理后ZIP2和ZIP8的表達(dá)均明顯上調(diào),提示在體內(nèi)外垂體細(xì)胞對(duì)缺鋅環(huán)境中ZIP2和ZIP8的表達(dá)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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.8

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