缺锌上调儿童PBMCs的ZIP2表达及ZIP2对淋巴细胞Jurkat，E6-1分泌IFN-γ的影响

发布时间：2018-02-28 06:31

本文关键词： 锌锌转运体 Jurkat E6-1 低锌基因表达　出处：《山东大学》2013年硕士论文　论文类型：学位论文

【摘要】：研究背景：锌是生物体所必须的微量元素之一,人体内300多种酶和转录因子的重要组成成分,起着稳定结构、催化和调节的作用。在世界范围内,因缺锌引起的疾病发生率估计超过20%,在发展中国家锌缺乏症可能影响到了二十多亿人的健康。锌缺乏症的发生主要是由于营养不良、衰老和天生的或是获得性锌吸收障碍所导致的,而缺锌主要会导致胸腺萎缩,免疫功能障碍,感染性疾病发病率增高并伴随着着生长迟缓和内分泌功能障碍。在临床上,补锌可能是辅助治疗由缺锌引起的疾病的有效的措施之一,尤其是易感人群中儿童和老人。在细胞免疫和体液免疫中,锌离子是必不可少的,缺锌会导致免疫功能紊乱,包括损伤细胞的固有免疫调节,锌在免疫系统的功能上广泛研究。体内锌平衡是通过胃肠道高度调节,但这种紧密的调节需要多种锌转运体相互协调作用。哺乳动物中,两大锌转运体基因家族参与锌在细胞内外的转运：ZIP和ZnT。由于两锌转运体基因家族在锌转运功能方面表现出相反的作用,因此锌转运体在维持细胞内外锌平衡中起到至关重要的作用。近年来研究发现,ZIP2蛋白可能与人的免疫力相关,尤其是在低锌的条件下。在ZIP2基因敲除鼠中,对野生型和基因敲除鼠进行基因表达分析,发现ZIP2在免疫系统的一些未成熟的树突状细胞中活跃表达；幼儿过敏性哮喘患者血清锌明显低于正常水平,且外周血单个核细胞(PBMCs)中ZIP2mRNA量显著高于正常人。提示ZIP2在免疫细胞中发挥一定的作用。因此,探讨在免疫细胞缺锌与ZIP2的表达水平的关系。研究目的：体内外探讨缺锌对儿童PBMCs中ZIP2表达的影响。将已构建成功的ZIP2表达载体转染人T淋巴细胞Jurkat, E6-1细胞株,研究ZIP2对Jurkat, E6-1细胞分泌细胞因子的影响。实验方法：一、临床收集20例缺锌患儿以及20例正常对照儿童和不同年龄段的正常人的抗凝外周血液样本、原子吸收分光光度计检测血清锌含量、密度梯度离心法分离样本的PBMCs,通过Real-time PCR方法检测PBMCSS锌转运体ZIP2、ZIP1、 ZIP6、ZnT1mRNA表达。体外培养正常儿童的外周血单个核细胞,TPEN处理,每周检测ZIP2的表达,至第七周,探讨PBMCs体外长时间缺锌条件下对ZIP2的影响；不同浓度的TPEN处理Jurkat, E6-1细胞株12h,选择一合适的TPEN浓度。二、将已构建完成的pEGFP-N1-ZIP2真核表达载体转染至人T淋巴细胞。转染36h后,TPEN处理12h转染48h后,Real-time PCR和Western-blot技术检测淋巴细胞中ZIP2mRNA和蛋白质的表达水平,然后采用Real-time PCR技术检测ZIP2过表达对其他锌转运体ZIP1、ZnT1、ZnT5、ZIP6、ZIP10及细胞因子INF-y和IL-6mRNA水平产生的影响；通过MTT比色法检测ZIP2对细胞增殖的影响。实验结果：一、低锌患儿组与正常对照儿童相比,Real-time PCR结果显示低锌患儿PBMCs中ZIP2的表达量明显高于正常对照组(P0.05)；低锌患儿中,ZnTl表达量上调,ZIPl的表达量下调,而ZIP6的表达没有显著差异。不同年龄的成年人与正常儿童比,青年组中ZIP2的表达最低。体外培养的正常儿童外周血细胞,结果显示,ZIP2的表达下降后上调的。二、ZIP2真核表达载体pEGFP-N1-ZIP2转染至Jurkat, E6-1细胞株后,结果显示ZIP2在Jurkat, E6-1细胞株内高表达,ZnT1mRNA表达量下调(P0.05)而其他锌转运体ZIP1、ZnT5、ZIP6、ZIP10mRNA表达量均没有显著改变(P0.05)；细胞因子INF-y的mRNA表达量上调(P0.05),TPEN处理后,INF-y的mRNA表达量下调(P0.05),而IL-6的表达量没有明显改变(P0.05),且对细胞的增殖没有显著影响(P0.05)。结论：本研究在体内外检测了缺锌时儿童PBMCs中ZIP2的表达,结果显示缺锌上调儿童的PBMCs中ZIP2的表达,缺锌处理细胞株Jurkat, E6-1,使ZIP2表达上调,推测在缺锌状态下ZIP2在维持锌的动态平衡和免疫调节过程中起一定作用。同时,将ZIP2真核表达载体pEGFP-N1-ZIP2成功转染至Jurkat, E6-1细胞株中,观察细胞株中细胞因子的分泌情况,结果发现ZIP2高表达可使细胞因子INF-y的表达上调,IL-6的表达没有显著改变,提示ZIP2可能在淋巴细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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R153.2

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