本文關(guān)鍵詞： 鋅 鋅轉(zhuǎn)運體 Jurkat E6-1 低鋅 基因表達(dá)　出處：《山東大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景： 鋅是生物體所必須的微量元素之一,人體內(nèi)300多種酶和轉(zhuǎn)錄因子的重要組成成分,起著穩(wěn)定結(jié)構(gòu)、催化和調(diào)節(jié)的作用。在世界范圍內(nèi),因缺鋅引起的疾病發(fā)生率估計超過20%,在發(fā)展中國家鋅缺乏癥可能影響到了二十多億人的健康。鋅缺乏癥的發(fā)生主要是由于營養(yǎng)不良、衰老和天生的或是獲得性鋅吸收障礙所導(dǎo)致的,而缺鋅主要會導(dǎo)致胸腺萎縮,免疫功能障礙,感染性疾病發(fā)病率增高并伴隨著著生長遲緩和內(nèi)分泌功能障礙。在臨床上,補鋅可能是輔助治療由缺鋅引起的疾病的有效的措施之一,尤其是易感人群中兒童和老人。 在細(xì)胞免疫和體液免疫中,鋅離子是必不可少的,缺鋅會導(dǎo)致免疫功能紊亂,包括損傷細(xì)胞的固有免疫調(diào)節(jié),鋅在免疫系統(tǒng)的功能上廣泛研究。體內(nèi)鋅平衡是通過胃腸道高度調(diào)節(jié),但這種緊密的調(diào)節(jié)需要多種鋅轉(zhuǎn)運體相互協(xié)調(diào)作用。哺乳動物中,兩大鋅轉(zhuǎn)運體基因家族參與鋅在細(xì)胞內(nèi)外的轉(zhuǎn)運：ZIP和ZnT。由于兩鋅轉(zhuǎn)運體基因家族在鋅轉(zhuǎn)運功能方面表現(xiàn)出相反的作用,因此鋅轉(zhuǎn)運體在維持細(xì)胞內(nèi)外鋅平衡中起到至關(guān)重要的作用。 近年來研究發(fā)現(xiàn),ZIP2蛋白可能與人的免疫力相關(guān),尤其是在低鋅的條件下。在ZIP2基因敲除鼠中,對野生型和基因敲除鼠進行基因表達(dá)分析,發(fā)現(xiàn)ZIP2在免疫系統(tǒng)的一些未成熟的樹突狀細(xì)胞中活躍表達(dá)；幼兒過敏性哮喘患者血清鋅明顯低于正常水平,且外周血單個核細(xì)胞(PBMCs)中ZIP2mRNA量顯著高于正常人。提示ZIP2在免疫細(xì)胞中發(fā)揮一定的作用。因此,探討在免疫細(xì)胞缺鋅與ZIP2的表達(dá)水平的關(guān)系。 研究目的： 體內(nèi)外探討缺鋅對兒童PBMCs中ZIP2表達(dá)的影響。將已構(gòu)建成功的ZIP2表達(dá)載體轉(zhuǎn)染人T淋巴細(xì)胞Jurkat, E6-1細(xì)胞株,研究ZIP2對Jurkat, E6-1細(xì)胞分泌細(xì)胞因子的影響。 實驗方法： 一、臨床收集20例缺鋅患兒以及20例正常對照兒童和不同年齡段的正常人的抗凝外周血液樣本、原子吸收分光光度計檢測血清鋅含量、密度梯度離心法分離樣本的PBMCs,通過Real-time PCR方法檢測PBMCSS鋅轉(zhuǎn)運體ZIP2、ZIP1、 ZIP6、ZnT1mRNA表達(dá)。體外培養(yǎng)正常兒童的外周血單個核細(xì)胞,TPEN處理,每周檢測ZIP2的表達(dá),至第七周,探討PBMCs體外長時間缺鋅條件下對ZIP2的影響；不同濃度的TPEN處理Jurkat, E6-1細(xì)胞株12h,選擇一合適的TPEN濃度。 二、將已構(gòu)建完成的pEGFP-N1-ZIP2真核表達(dá)載體轉(zhuǎn)染至人T淋巴細(xì)胞。轉(zhuǎn)染36h后,TPEN處理12h轉(zhuǎn)染48h后,Real-time PCR和Western-blot技術(shù)檢測淋巴細(xì)胞中ZIP2mRNA和蛋白質(zhì)的表達(dá)水平,然后采用Real-time PCR技術(shù)檢測ZIP2過表達(dá)對其他鋅轉(zhuǎn)運體ZIP1、ZnT1、ZnT5、ZIP6、ZIP10及細(xì)胞因子INF-y和IL-6mRNA水平產(chǎn)生的影響；通過MTT比色法檢測ZIP2對細(xì)胞增殖的影響。 實驗結(jié)果： 一、低鋅患兒組與正常對照兒童相比,Real-time PCR結(jié)果顯示低鋅患兒PBMCs中ZIP2的表達(dá)量明顯高于正常對照組(P0.05)；低鋅患兒中,ZnTl表達(dá)量上調(diào),ZIPl的表達(dá)量下調(diào),而ZIP6的表達(dá)沒有顯著差異。不同年齡的成年人與正常兒童比,青年組中ZIP2的表達(dá)最低。體外培養(yǎng)的正常兒童外周血細(xì)胞,結(jié)果顯示,ZIP2的表達(dá)下降后上調(diào)的。 二、ZIP2真核表達(dá)載體pEGFP-N1-ZIP2轉(zhuǎn)染至Jurkat, E6-1細(xì)胞株后,結(jié)果顯示ZIP2在Jurkat, E6-1細(xì)胞株內(nèi)高表達(dá),ZnT1mRNA表達(dá)量下調(diào)(P0.05)而其他鋅轉(zhuǎn)運體ZIP1、ZnT5、ZIP6、ZIP10mRNA表達(dá)量均沒有顯著改變(P0.05)；細(xì)胞因子INF-y的mRNA表達(dá)量上調(diào)(P0.05),TPEN處理后,INF-y的mRNA表達(dá)量下調(diào)(P0.05),而IL-6的表達(dá)量沒有明顯改變(P0.05),且對細(xì)胞的增殖沒有顯著影響(P0.05)。 結(jié)論： 本研究在體內(nèi)外檢測了缺鋅時兒童PBMCs中ZIP2的表達(dá),結(jié)果顯示缺鋅上調(diào)兒童的PBMCs中ZIP2的表達(dá),缺鋅處理細(xì)胞株Jurkat, E6-1,使ZIP2表達(dá)上調(diào),推測在缺鋅狀態(tài)下ZIP2在維持鋅的動態(tài)平衡和免疫調(diào)節(jié)過程中起一定作用。 同時,將ZIP2真核表達(dá)載體pEGFP-N1-ZIP2成功轉(zhuǎn)染至Jurkat, E6-1細(xì)胞株中,觀察細(xì)胞株中細(xì)胞因子的分泌情況,結(jié)果發(fā)現(xiàn)ZIP2高表達(dá)可使細(xì)胞因子INF-y的表達(dá)上調(diào),IL-6的表達(dá)沒有顯著改變,提示ZIP2可能在淋巴細(xì)胞中扮演重要的角色。
[Abstract]:Research background:
Zinc is one of trace elements necessary for organism, an important component of the human body 300 kinds of enzymes and transcription factors, plays a stable structure, catalytic and regulatory role. In the world, because the incidence is estimated at more than 20% diseases caused by zinc deficiency, zinc deficiency in developing countries may affect the health of about two billion people. Zinc deficiency is mainly due to the occurrence of malnutrition, aging and inborn or acquired disorders caused by the absorption of zinc, and zinc deficiency will lead to atrophy of thymus gland, immune dysfunction, infectious disease incidence and with growth retardation and endocrine dysfunction. In clinic, zinc may be one of effective adjuvant therapy the measures caused by zinc deficiency disease, especially susceptible to children and elderly people.
In the process of cellular immunity and humoral immunity, the zinc ion is essential, zinc deficiency can lead to immune dysfunction, including innate immune cell damage regulation, extensive research of zinc in the function of the immune system. Zinc balance is to adjust the height of the gastrointestinal tract, but this tight regulation requires a variety of zinc transporter interaction in mammals. Two, zinc transporter gene family involved in the transport of zinc in and out of cells: ZIP and ZnT. as the two zinc transporter gene family showed opposite effects on zinc transport function, therefore zinc transporters play a crucial role in the maintenance of intracellular zinc homeostasis.
In recent years, the study found that ZIP2 protein may be related to the immune system, especially in low zinc conditions. In ZIP2 knockout mice, knockout mice by gene expression analysis of the wild type and gene expression of ZIP2 was found to be active in some immature dendritic cells of the immune system in children with allergic asthma; the serum zinc was significantly lower than the normal level, and peripheral blood mononuclear cells (PBMCs) in ZIP2mRNA was significantly higher than that in normal people. Suggesting that ZIP2 play a role in immune cells. Therefore, to explore the relationship between the expression level of immune cells in zinc deficiency and ZIP2.
The purpose of the study is:
The effect of zinc deficiency on the expression of ZIP2 in children's PBMCs was explored in vivo and in vitro. The successful ZIP2 expression vector was transfected into human T lymphocyte Jurkat and E6-1 cell line to study the effect of ZIP2 on cytokines secreted by Jurkat and E6-1 cells.
Experimental methods:
A normal person, anticoagulation collect 20 clinical cases with zinc deficiency and 20 cases of normal children of different ages and peripheral blood samples, atomic absorption spectrophotometer serum zinc content detection, separation of samples using PBMCs density gradient centrifugation method for the detection of PCR PBMCSS by Real-time ZIP2 ZIP1, ZIP6 zinc transporter, ZnT1mRNA, expression in vitro. Children of normal peripheral blood mononuclear cells, TPEN treatment, ZIP2 expression detected every week, to seventh weeks, to investigate the in vitro PBMCs long time zinc deficiency conditions influence on ZIP2; TPEN treatment of different concentrations of Jurkat, E6-1 cell line 12h, selecting a suitable concentration of TPEN.
Two, the constructed pEGFP-N1-ZIP2 eukaryotic expression vector was transfected into the T cells. After transfection of 36h, TPEN 12h 48h after transfection, the expression level of ZIP2mRNA protein and detection of lymphocyte Real-time PCR and Western-blot technology, and then use Real-time PCR technology to detect the ZIP2 expression of other zinc transporters ZIP1, ZnT1, ZnT5. ZIP6, ZIP10 and INF-y and IL-6mRNA levels of cytokines produced by detecting ZIP2; MTT colorimetric assay for cell proliferation.
Experimental results:
A low zinc group compared with the normal children, Real-time PCR results showed that the expression level of ZIP2 PBMCs in low zinc were significantly higher than the normal control group (P0.05); low zinc in children with ZnTl expression, the expression of ZIPl was down regulated, while the expression of ZIP6 had no significant differences between different age and normal adults. Children than in young group, ZIP2 was the lowest. In vitro peripheral blood cells of normal children showed decreased expression of ZIP2 up-regulated.
Two, ZIP2 eukaryotic expression vector pEGFP-N1-ZIP2 was transfected into Jurkat cell line, E6-1, showed that ZIP2 in Jurkat, E6-1 cells with high expression, down-regulation of ZnT1mRNA expression (P0.05) and other zinc transporters ZIP1, ZnT5, ZIP6, ZIP10mRNA expression showed no significant change (P0.05); cytokine INF-y mRNA expression the amount of increase (P0.05), after TPEN treatment, INF-y mRNA expression (P0.05), and the expression of IL-6 did not change significantly (P0.05), and had no significant effect on cell proliferation (P0.05).
Conclusion:
This study examined the expression of ZIP2 in children with PBMCs zinc deficiency in vivo, the results showed that the expression of ZIP2 increased in PBMCs children zinc deficiency, zinc deficiency treatment of Jurkat cells, E6-1, up-regulated the expression of ZIP2, presumably in zinc deficiency state of ZIP2 plays a certain role in homeostasis and immune regulation in the process of maintenance of zinc.
At the same time, the ZIP2 eukaryotic expression vector pEGFP-N1-ZIP2 was successfully transfected into Jurkat E6-1 cells, secretion of cytokines were observed in cell lines. The results showed that high expression of ZIP2 can increase the expression of INF-y cytokines, no significant change of the expression of IL-6, suggesting that ZIP2 may play an important role in lymphocyte.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R153.2

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