摘要
研究背景:
2型糖尿病是遗传因素和环境因素长期作用的结果,胰岛素抵抗和胰岛素分泌不足(包括两者的相互作用)是目前公认的2型糖尿病发病机制的两个基本环节和特征。当前这方面的研究主要集中于高血糖、高血脂对胰岛功能的影响,而高浓度氨基酸对胰岛功能的影响则研究很少。目前亮氨酸对胰岛功能的影响尤其是对葡萄糖刺激的胰岛素释放的作用,这方面的研究很多,但是存在着很大的争议。Yang等发现亮氨酸可以促进葡萄糖刺激的胰岛素释放,然而Anello等则在他们的实验中发现长期亮氨酸以剂量依赖方式抑制葡萄糖刺激的胰岛素释放。并且,到目前为止,亮氨酸影响胰岛素释放和胰岛素含量的具体机制也尚未阐明。
PDX-1(Pancreas/Duodenum Homeobox-1),即胰腺十二指肠同源异型盒因子-1,是胰腺β细胞胰岛素基因的转录调控因子,此外,PDX-1还能转录胰岛素和调控胰岛素相关基因如葡萄糖转运蛋白2(GLUT-2)和葡萄糖激酶(GCK)等的表达,对胰岛细胞内分泌功能起重要调节作用。而葡萄糖刺激β细胞释放胰岛素,首先必须经细胞膜上GLUT-2蛋白转运进入细胞内,在葡萄糖激酶作用下磷酸化,增加ATP/ADP比值,使胰岛素颗粒释放。大量实验研究显示PDX-1与胰岛素分泌具有密切的关系,那么他们是否在亮氨酸影响胰岛功能的过程中发挥作用是我们研究的方向。因此,我们的实验旨在研究长期高浓度亮氨酸培养对葡萄糖刺激的胰岛素释放和胰岛素含量的影响,并且进一步研究其相关机制。
目的:
1.观察长期亮氨酸培养对大鼠胰岛β细胞株(DN-PDX-1# 28和PDX-1# 6)的胰岛素释放功能和胰岛素含量的影响。
2.观察长期亮氨酸对DN-PDX-1# 28和PDX-1# 6细胞中PDX-1、GCK/GLUT2mRNA表达的影响,探讨可能的机制。
3.观察长期亮氨酸是否通过(PDX-1)-(GCK/GLUT2)通路影响高糖刺激的胰岛素释放以及胰岛素含量。
研究方法:
1.DN-PDX-1# 28和PDX-1# 6胰岛细胞株的培养和分组:RPMI1640培养PDX-1#6细胞株与PDX-1不表达的PDX-1#28细胞,加以强力霉素(Doxycyline),使PDX-1过表达以及不表达。DN-PDX-1#28 and PDX-1#6细胞在RPMI1640培养基中培养,此外,还需添加100μg/ml潮霉素,100μg/ml G418和500 ng/ml的强力霉素。DN-PDX-1#28和PDX-1#6细胞分别在40mM亮氨酸或者500 ng/ml的强力霉素中培养。处理时间:48小时。
2.葡萄糖刺激的胰岛素释放功能以及胰岛素含量的测定与评价:细胞种于24孔板中,分组处理后,在葡萄糖浓度为3.3 mM、27.8 mM的KRB缓冲液分别孵育20min,收集上清,放射免疫方法测定分泌的胰岛素水平分别为基础胰岛素分泌(BIS)和葡萄糖刺激的胰岛素分泌(GSIS)。
3.PDX-1、GCK、GLUT2的mRNA检测:在DN-PDX-1#28和PDX-1#6细胞上,Real-time PCR检测PDX-1、GCK、GLUT2mRNA的表达。
结果:
1、胰岛素释放和胰岛素含量
①在DN-PDX-1# 28细胞中,与正常对照组相比,单纯亮氨酸组和单纯强力霉素组分别显著降低高糖刺激的胰岛素分泌36%和84%(P＜0.05),分别降低胰岛素含量23%和62%(P＜0.05)。与单纯亮氨酸组相比,强力霉素与亮氨酸共同培养组显著降低高糖刺激的胰岛素分泌67%(P＜0.05),降低胰岛素含量44%(P＜0.05)。
②在PDX-1# 6细胞中,与正常对照组相比,单纯亮氨酸组显著降低高糖刺激的胰岛素分泌26%(P＜0.05),降低胰岛素含量25%(P＜0.05);单纯强力霉素组显著增加高糖刺激的胰岛素分泌20%(P＜0.05),增加胰岛素含量21%(P＜0.05)。然而,与单纯亮氨酸组相比,强力霉素与亮氨酸共同培养组显著增加高糖刺激的胰岛素分泌36%(P＜0.05),增加胰岛素含量21%(P＜0.05)。
2、PDX-1、GCK、GLUT2mRNA表达
①在DN-PDX-1#28细胞中,与对照组相比,单纯亮氨酸组显著降低PDX-1、GCKmRNA和GLUT2 mRNA水平(P＜0.05);单纯强力霉素组也显著降低PDX-1、GCK mRNA和GLUT2 mRNA水平(P＜0.05);与单纯亮氨酸组相比,强力霉素与亮氨酸共同培养组显著降低PDX-1、GCK mRNA和GLUT2 mRNA水平(P＜0.05)。
②在PDX-1# 6细胞中,与对照组相比,单纯亮氨酸组显著降低PDX-1、GCKmRNA和GLUT2 mRNA水平(P＜0.05);单纯强力霉素组也显著增强PDX-1、GCK mRNA和GLUT2 mRNA水平(P＜0.05);与单纯亮氨酸组相比,强力霉素与亮氨酸共同培养组显著增强PDX-1、GCK mRNA和GLUT2 mRNA水平(P＜0.05)。
结论:
长期40mM浓度亮氨酸在转录水平通过降调β细胞PDX-1以及其下游因子GCK与GLUT2 mRNA表达,最终抑制高糖刺激的胰岛素释放以及胰岛素含量。
研究背景:
在甲状腺疾病的发生发展过程中,生长因子的作用日益受到重视,其中以血管内皮生长因子(VEGF)和胰岛素样生长因子-1(IGF-1)的作用较为突出。生长因子以内分泌、旁分泌和自分泌等方式,广泛而精细的地调节甲状腺细胞的生长、分化和功能,参与甲状腺疾病的发生发展过程。
VEGF是最强的可溶性血管形成因子之一,能增加微血管的通透性,促进血浆纤维蛋白外渗,为血管形成过程中多种细胞迁移提供一个纤维网络;并能直接刺激内皮细胞增殖,促进内皮细胞移动,利于血管形成。根据编码VEGF mRNA的剪接方式不同,即亚单位所含氨基酸数量的不同,至少分为5种亚型,即VEGF_(206)、VEGF_(189)、VEGF_(165) VEGF_(145)及VEGF_(121)。在大鼠中VEGF的亚型也有类似的编码,但比人类的少一个氨基酸。IGF-1是一种多功能的生长因子,具有广泛的生物的生物学作用,能促进细胞的增殖和抑制细胞的凋亡,对血管内皮细胞、平滑肌细胞等多种细胞的增殖、分化、代谢有重要的促进作用。另外,在内分泌肿瘤细胞中,IGF-1可以上调VEGF的mRNA和蛋白表达,从而参与新生血管形成。我们前期研究结果显示,GD患者血清和甲状腺组织中的VEGF与GD患者甲状腺内血管形成有关,IGF-I还与GD患者甲状腺体积有关。其机制研究多限于GD患者甲状腺自身免疫性抗体模拟TSH作用于TSH受体而引起,但临床中也存在大量非自身免疫性甲状腺肿大合并高代谢患者,其发病机制尚不明确。因此探讨甲状腺血管增生性肿大的形成机制,研发有效的治疗方法是十分必要的。
甲状腺的毒性症状表现在甲状腺激素(T3)引起的高代谢,嘌呤核苷酸循环增强,致ATP分解代谢增强,经过一系列代谢后生成小分子物质腺苷,并有研究报道甲状腺功能亢进患者血清腺苷浓度明显高与正常人群,而内源性腺苷的受体则为腺苷受体A2aR。腺苷受体属于G蛋白偶联受体家族,其中腺苷受体A2aR已经被发现在心肌细胞、免疫炎症细胞以及中枢神经细胞中有表达,尤其在中枢神经系统有大量表达。该受体在某些疾病中已经是潜在的调节新生血管形成的治疗靶点。先前的研究发现在不同的细胞中,腺苷受体A2aR对VEGF的影响是不尽相同的。早在1992年Ledent等第一次报道甲状腺肿大合并功能亢进的动物模型:腺苷受体A2转基因小鼠。这是腺苷受体在甲状腺的相关研究中第一次被关注。2003年和2004年国外研究发现:A2aR转基因模型同样可以引起高功能的甲状腺肿大,并且在这种转基因模型的甲状腺组织中,微血管主要位于增生区域,该区域VEGF蛋白的表达明显高于对照,并且IGF-1的mRNA表达也增高。故我们推测甲状腺肿大患者甲状腺局部腺苷水平的变化或者说其受体A2aR活性变化可能与甲状腺血管增生性肿大的发生发展有关。
基于上述研究现状,为了更深入的认识A2aR在甲状腺中的表达及其对VEGF、IGF-1的影响,以便为发现治疗甲状腺血管增生性肿大的分子靶点提供实验依据,本研究确定研究目的与方法如下:
目的:
1、确定A2aR在大鼠甲状腺细胞中的基因和蛋白表达:
2、通过A2aR激动剂或TSH刺激甲状腺细胞,观察A2aR和TSHR基因表达的变化,初步探讨A2aR和TSHR的关系;
3、通过A2aR激动剂、A2aR阻断剂、TSH或IGF-1刺激甲状腺细胞,观察VEGF基因和细胞内外蛋白表达的变化;以及通过A2aR激动剂、A2aR阻断剂刺激甲状腺细胞,观察IGF-1基因和细胞内外蛋白表达的变化,探讨A2aR对VEGF和IGF-1影响。
研究方法:
1、甲状腺细胞(Fisher rat thyroid cell line-5,FRTL-5)培养:Coon's改良的Ham's F12培养液中加入六种激素-促甲状腺激素10 mU/ml、胰岛素10μg/ml、转铁蛋白5μg/ml、氢化考的松5 ng/ml、生长抑素10 ng/ml、甘氨酸-组氨酸-亮氨酸10 ng/ml及5%胎牛血清,即配成甲状腺细胞培养液,称为6H(hormome)。待甲状腺细胞在培养皿中长至80%～85%时,PBS洗两遍,然后以4H培养基(6H培养基去除促甲状腺激素和胰岛素,改为其余四种激素加0.2%胎牛血清)为对照加入不同的处理(A2aR激动剂CGS21680、A2aR阻断剂ZM241385、TSH或IGF-1)作用48小时。另外,原代培养的大鼠神经元细胞作为A2aRmRNA表达的阳性对照。
2、采用RT-PCR检测A2aR、TSHR、IGF-1和VEGF各亚型mRNA水平变化。
3、采用免疫荧光反应检测A2aR在甲状腺细胞中表达的位置。
4、采用免疫沉淀-Western blot检测A2aR和IGF-1蛋白水平。
5、采用酶联免疫吸附剂测定法检测甲状腺细胞内外VEGF蛋白水平。
结果:
1、A2aR在甲状腺细胞中的表达:在甲状腺细胞和阳性对照神经元细胞中都能扩增出A2aRmRNA140bp大小的片段。与正常对照组相比,1.0μM CGS21680能上调甲状腺细胞A2aR的mRNA水平(P＜0.05),而10mU/ml TSH下调其mRNA水平(P＜0.05);采用了免疫共沉淀-Western blots的方法进行半定量实验结果显示在甲状腺细胞中CGS21680可以轻度调节A2aR蛋白水平(P＞0.05),采用了免疫荧光的方法进行定位实验结果显示A2aR蛋白表达在甲状腺细胞的细胞膜中。
2、TSHR在甲状腺细胞中的mRNA的表达:与正常对照组相比,10 mU/mlTSH下调30.5%(P＜0.05),而1.0μM CGS21680上调1.35倍(P＜0.05)甲状腺细胞TSHR的mRNA水平,并且都呈剂量依赖性。
3、VEGF各亚型在甲状腺细胞中的转录表达:正常培养甲状腺细胞中可以扩增出4个VEGF亚型:VEGF_(188),VEGF_(164),VEGF_(144)和VEGF_(120),其中,VEGF_(120)和VEGF_(164)是主要成分。
CGS21680对VEGF亚型mRNA的影响:与正常对照组相比,CGS21680明显上调VEGF_(188)和VEGF_(164)的表达(P＜0.05);TSH对VEGF亚型mRNA的影响:与正常对照组相比,TSH明显下调VEGF_(188)和VEGF_(164)的表达(P＜0.05);IGF-1对VEGF亚型mRNA的影响:与正常对照组相比,IGF-1明显上调VEGF_(164)和VEGF_(120)的表达(P＜0.05)。
4、VEGF蛋白在甲状腺细胞中的表达:
①首先对细胞内外的VEGF蛋白量进行相关分析:VEGF蛋白量细胞外量是细胞内量的22.3倍,相关分析显示两者之间呈正相关关系,相关系数为0.978。
②A2aR对VEGF蛋白表达的影响:CGS21680刺激甲状腺细胞后,细胞内外VEGF蛋白含量都明显增加,且随着CGS21680刺激浓度的增高;相反,ZM241385刺激甲状腺细胞后,细胞内外VEGF蛋白含量都明显下调,且随着ZM241385刺激浓度的增高,VEGF表达明显下调;ZM241385可以抑制CGS21680引起的VEGF上调表达。以上说明VEGF的蛋白表达与A2aR的活性变化呈剂量依赖性;
③TSH和IGF-1对VEGF蛋白表达的影响:加入10 mU/ml TSH后,与对照组相比,细胞外VEGF含量下降16.9%(P＜0.05);而加入50ng/ml IGF-1后,细胞外VEGF含量升高39.0%(P＜0.05)。
5、TSH和A2aR对IGF-1的影响:加入10 mU/ml TSH后,IGF-1mRNA水平下调。而A2aR激动剂CGS21680刺激甲状腺细胞后,与对照组相比,IGF-1 mRNA和蛋白水平上调,阻断剂ZM241385可以抑制IGF-1蛋白水平。
结论:
1、腺苷受体A2aR在甲状腺细胞FRTL-5中存在mRNA和蛋白的表达,并且该受体蛋白表达在细胞膜中。
2、腺苷受体A2aR的激动剂CGS21680上调甲状腺细胞A2aR、TSHR、VEGF亚型(主要是VEGF_(164))和IGF-1mRNA水平的表达;相反TSH受体的配体TSH下调以上三个基因的mRNA水平表达。本研究结果表明,同为G蛋白偶联受体的A2aR和TSHR之间可能存在某种协同关联。
3、A2aR调节甲状腺细胞VEGF和IGF-1的mRNA和蛋白水平的表达;IGF-1上调甲状腺细胞VEGF的mRNA(主要是VEGF_(164))和蛋白水平的表达,这提示在甲状腺细胞内A2aR上调VEGF表达可能与IGF-1参与有关。本研究结果表明A2aR可能通过上调VEGF和IGF-1而参与甲状腺血管增生性肿大的形成。
Part 1: PDX-1 INVOLVED IN INSULIN SECRETION UNDER LEUCINE EXPOSURE
Background:
Type 2 diabetes mellitus is a kind of heterogeneous syndrome of polygenic origin and involves both defective insulin secretion and peripheral insulin resistance.β-cell dysfunction is a sine qua non for the development of the disease.Some metabolic factors such as hyperglycemia and hyperlipidemia contribute to the progressive deterioration of glucose homeostasis characteristic of this disease.Recently,studies have attached great importance for the effect of leucine on glucose-stimulated insulin secretion(GSIS) and intracellular insulin content in pancreaticβ-cells.However,up to now,the results from different research groups have been quite controversial.Yang and his colleagues demonstrated that leucine was able to enhance GSIS in pancreaticβ-cells.However,Anello et al.reported that chronic leucine exposure impaired GSIS in a dose-dependent manner.Moreover,the mechanism of leucine affecting insulin secretion and content has not been elucidated yet.
Pancreatic/duodenal homeobox-1(PDX-1),a major transactivator of tightly regulating insulin and glucose transporter-2(GLUT2) at the transcriptional level,is an early and critical event in facilitating development and maintaining the function of pancreaticβ-cells.Its activation leads to insulin transcription potentiation that subsequently results in insulin synthesis increase.
Glucokinase(GCK),an enzyme phosphorylating glucose to glucose-6-phosphate, acts as a glucose sensor and regulates insulin secretion.GLUT2 is an important component for insulin secretion as well.Tiedge and Lenzen reported in their studies that the concordant regulation of GCK and GLUT2 genes might represent the basis regulation of GSIS.In 2006,Yang et al.firstly reported that leucine culture altered GCK expression in INS-1 cells,rat islets and human islets,moreover,glucokinase contributed tight control of insulin secretion.
Numerous studies in vitro and in vivo have demonstrated that chronic exposure to glucose or fatty acid is able to suppress PDX-1 expression,leading to decreased insulin secretion.The role of PDX-1 in pancreaticβ-cell insulin secretion derives from its effect on transactivating the expression of insulin and otherβ-cell-specific genes,such as GCK and GLUT2.
Objectives: Effect of leucine on GSIS in pancreaticβ-celts is quite controversial,and mechanism involved in the effect has not been elucidated yet.Consequently,we aimed to investigate effect of leucine on GSIS and its mechanism focusing on contribution of PDX-1,GCK and GLUT2.
Methods:
Rat insulinomaβ-cells(DN-PDX-1~# 28 and PDX-1~# 6) were cultured with or without 40 mM leucine or 500ng/ml doxycycline for 48h.
Results:
To further prove that the PDX-1 plays an important role in impaired insulin secretion and content induced by chronic leucine,DN-PDX-1#28 and PDX-1#6 cells were treated with or without 500 ng/ml doxycycline in the presence or absence of 40 mM leucine for 48 hrs.
1.glucose-induced insulin secretion and intracellular insulin content
In DN-PDX-1#28 cells,in comparison with control,leucine alone treatment and doxycycline alone treatment diminished high glucose-induced insulin secretion by 36%and 84%(P＜0.05),decreased intracellular insulin content by 23%and 62%(P＜0.05),respectively.In comparison with leucine treatment alone group,doxycycline plus leucine treatment decreased high glucose-induced insulin secretion by 67%(P＜ 0.05) and intracellular insulin content by 44%(P＜0.05).
In PDX-1#6 cells,compared with control,leucine alone treatment diminished high glucose-induced insulin secretion by 26%(P＜0.05) and insulin content by 25%(P＜0.05),doxycycline alone treatment increased high glucose-induced insulin secretion by 20%(P＜0.05) and insulin content by 21%(P＜0.05).However,relative to leucine alone treatment,doxycycline plus leucine treatment significantly increased GSIS by 36%(P＜0.05) and insulin content by 21%(P＜0.05).
2.The mRNA expression of PDX-1,GCK and GLUT2
In DN-PDX-1#28 cells,in contrast to control,40 mM leucine significantly diminished the mRNA level of PDX-1 and its downstream targets,GCK and GLUT2 (P＜0.05),doxycycline alone treatment decreased the mRNA level of PDX-1,GCK and GLUT2(P＜0.05).In comparison with leucine treatment alone group, doxycycline plus leucine treatment decreased PDX-1,GCK and GLUT2 mRNA level (P＜0.05).
In PDX-1#6 cells,compared with control,leucine alone treatment diminished PDX-1,GCK and GLUT2 mRNA level(P＜0.05),doxycycline alone treatment increased the mRNA level of PDX-1,GCK and GLUT2(P＜0.05).In comparison with leucine treatment alone group,doxycycline plus leucine treatment enhanced PDX-1,GCK and GLUT2 mRNA level(P＜0.05).
Conclusions:
The study indicated that chronic leucine might result in a decrease in PDX-1,in turn to depress GCK and GLUT2 resulting in decreased GSIS at high glucose and insulin content.
Part 2: PRESENCE OF ADENOSINE A2A RECEPTOR IN THYROCYTES AND ITS EFFECT ON VASCULAR ENDOTHELIAL GROWTH FACTOR
Background:
During the thyroid disease,the actions of growth factors,especially vascular endothelial growth factor(VEGF) and insulin like growth factor-1(IGF-1),are attached great importance to.It is thought growth factors can regulate the growth, differentiation and function of the thyroid cells through endocrine,autocrine, paracrine ways,and take part in the process of thyroid disease.
VEGF is one of the most important soluble angiogenic factors.It can increase the microvascular permeability and facilitate to the exudation of plasma fibrin which offers fibre network for the movement of many cells.It also can stimulate the proliferation of endothelial cells and make it easy for endothelial cells to move.All are favorable for vascular formation.Currently,at least five different mRNA species encoding VEGF have been characterized.These variants result from alternative splicing of the VEGF transcript and encode human isoforms containing VEGF protein of 121,145,165,189,and 206 amino acids.In the rat,a similar profile of VEGF splice variants has been described,each encoding one fewer amino acid than the human species.IGF-1 has the functions of accelerating proliferation,differention and metabolism of many cells,such as endothelial cells and smooth muscle cells. Furthermore,IGF-1 is also involved in angiogenesis via increasing VEGF gene transcript and protein secretion in thyroid carcinomas.
Our previous research showed that in patients with untreated GD,there was a significant increase in serum VEGF and IGF-1 levels,and localized VEGF and IGF-1 expression in thyroid tissues was higher in those patients compared with healthy subjects.The autoantibody(immunoglobulin G,IgG) against thyrotropin(TSH) receptor(TSHR),named thyrotropin receptor antibody(TRAb),is believed to act via the TSHR and mimic the role of TSH to bring biological effect through signaling cascades in thyroid and thus alter thyroid growth and function.In clinic,there are abundant patients with no TRAb developing goiter and hyperthyroidial toxic symptoms.Therefore,it is very important to study the mechanism of thyroid angiogenic goiter formation to find effective therapeutic targets for this disease.
Hyperthyroidism raises activity of the purine nucleotide cycle and thyroid hormones,especially T3,stimulate the synthesis and consumption of ATP simultaneously.Endogenous adenosine,ligand of adenosine A2a receptor(A2aR),is an intermediate product of adenosine nucleotide metabolism,which is released from metabolically active cells by facilitated diffusion or is generated extracellularly by degradation of released ATP.Adenosine receptors,members of the G protein-coupled receptor super-family,represent a potential therapeutic target for regulation of neovascularization.The adenosine A2aR,one of adenosine receptors,is found on cardiac myocytes,immune and inflammatory cells and,very abundantly,on some cells in the central nervous system.Previous studies have also implicated adenosine A2aR in the regulation of VEGF expression in various cell types.
The interest in targeting adenosine receptor in the realm of thyroid diseases first arose based on the mouse transgenic models that developed thyroid diseases.Ledent et al.reported thyroid expression of an A2 adenosine receptor induced thyroid hyperplasia and hyperthyroidism.The over-expression of adenosine A2aR(Tg-A2aR model) in the transgenic mice developed severe hyper-functioning and a large goiter. In goitrous Tg-A2aR mouse thyroid tissue,microvessels were mostly located in hyperplastic zones,VEGF proteins were more strongly expressed than in controls, and IGF-1 mRNA was observed to be up-regulated.However,less is known regarding A2aR activation in thyroid under various relative physiological microenvironments.
Objectives:
1.The mRNA and protein expresson of A2aR in thyroid cells;
2.The mRNA expresson of A2aR and TSHR influenced by A2aR agonist or TSH;
3.The effect of A2aR,TSH or IGF-1 on mRNA and protein expression levels of VEGF,and the effect of A2aR on mRNA and protein expression levels of IGF-1.
Methods:
1.Cell culture:FRTL-5 thyroid cells(ATCC catalog number CRTL-1486) were grown in 6H media consisting of Coon's modified Ham's F12 medium supplemented with 5%fetal bovine serum and a mixture of six hormones:bovine thyrotropin, insulin,hydrocortisol,transferrin,glycyl-L-histidyl-L-lysine acetate,and somatostatin. Fresh medium was added to all cells every 2 or 3 days,and cell passaging was done every 7-10 days.After cells were grown to 90%confluence in 6H media and washed twice with prewarmed phosphate-buffered solution(PBS),the cells continued incubation with 4H media containing0.2%FBS,and no thyrotropin,no insulin.In the individual experiments,cells maintained in 4H media were treated for 48 hours with or without A2aR selective agonist CGS21680 or antagonist ZM241385 or TSH, IGF-1.In addition,neuron cells in primary cultured Wister rat forebrain were cultured as previously described and used as positive control in the PCR assays for A2aR described here.
2.The mRNA levels of A2aR,TSHR and IGF-1 were measured using Real-Time PCR and Reverse-Transcription(RT)-PCR;RT-PCR was performed to detect VEGF isoforms mRNA levels.
3.Immunofluorescence was used to observe distribution of A2aR protein.
4.Immunoprecipitation(IP)-western blotting was used to semi-quantify A2aR and IGF-1 protein expression.
5.ELISA was used to quantify intra-and extra-cellular VEGF protein contents.
Results:
1.A2aR expression in FRTL-5
After PCR amplification,a distinct band of 140 bp for A2aR was observed in the 6H and 4H cells.The real-time PCR results showed that in contrast to the control,A2aR mRNA expressions were enhanced by 25.3%(P＜0.05) at 1.0μM CGS2168,while diminished by 34.6%(P＜0.05) at 10mU/ml TSH.
Immunoprecipitation-Western blot results showed that the A2aR protein level was increased by its selective agonist CGS21680 by about 1.1-fold(P＞0.05). Immunoreactivity for the A2aR protein was clearly observed in the cell membrane.
2.TSHR mRNA expression in FRTL-5
Results of the RT-PCR and real-time PCR showed that TSHR mRNA expression was decreased by 8.4%(P＞0.05) at 1.0 mU/ml and by 30.5%(P＜0.05) at 10 mU/ml TSH,while increased by 1.19-fold(P＞0.05) at 0.1μM and by 1.35-fold(P＜0.05) at 1.0μM CGS21680 compared with those of control cells.
3.VEGF gene transcription in FRTL-5
We successfully detected four isoforms in non-stimulated FRTL-5 cells:VEGF_(188), VEGF_(164),VEGF_(144) and VEGF_(120),while the VEGF_(204) isoform was not detected.Of them,the productions of both VEGF_(120) and VEGF_(164) isoforms appeared to be predominant.
①A2aR agonist CGS21680(1μM and 10μM) exposure predominantly enhanced the levels of VEGF_(188) and VEGF_(164) isoforms by approximate 2-fold(both P＜0.05,respectively),in contrast to the corresponding control.
②When cells were treated with TSH(10 mU/ml),VEGF_(164) and VEGF_(188) were decreased by 32.6%(P＜0.05) and 35.2%(P＜0.05) compared to the control.
③Isoforms of VEGF_(164) and VEGF_(120) were increased by 1.8-fold and 2.1-fold(P＜0.05,respectively) by 50ng/ml IGF-1.
4.VEGF protein production in FRTL-5
①Correlation analysis of VEGF protein contents intracellular and extracellular:The extracellular VEGF content in each group was approximately 22.3-fold higher than the intracellular VEGF,and the Pearson correlation analyses between them showed that the "R" value was 0.978(P＜0.05).
②Effect of A2aR on VEGF protein expression in FRTL-5:In comparison with the control,the media VEGF contents were increased by 23.3%(P＜0.05) at 1.0μM CGS21680 stimulation,while strongly inhibited by 31.6%(P＜0.05) with 10μM antagonist ZM241385 alone.In addition,in the presence of 1.0 or 10μM A2aR inhibitor ZM241385,1μM agonist CGS21680 induced effects could be reduced: the extracellular decreased by 16.2%(P=0.065)～24.2%(P=0.007), respectively,compared to 1μM agonist-treated FRTL-5 cells.
③Effect of TSH and IGF-1 on VEGF release in FRTL-5:After TSH incubation, there was 16.9%(P＜0.05) at 10 mU/ml decrease in the media VEGF content in contrast to non-stimulated cells.While there was 39.0%(P＜0.05) increase of VEGF content in media by 50ng/ml IGF-1 compared with the control.
5.IGF-1 expression in FRTL-5 cells
RT-PCR and real-time PCR showed that 10 mU/ml TSH(P＜0.05) diminished,while 1.0μM CGS21680(P＜0.05) pretreatment increased IGF-1 mRNA level.IP-Western blot results showed that agonist CGS21680(1.0μM) increased IGF-1 protein level by 1.6-fold(P＜0.05),but antagonist ZM241385(1.0μM) reduced it by 17.4%(P＜0.05).
Conclusions:
1.The mRNA and protein of adenosine A2aR is expressed in thyroid FRTL-5 cell line, especially this receptor protein is expressed in the cytomembrane.
2.Transcription of A2aR,TSHR and VEGF isoforms(especially VEGF_(164)) was enhanced by CGS21680 and diminished by TSH simultaneously,which can be inferred that there maybe a receptor cross-talk between the two G protein-coupled receptors,such as norientation associativity.
3.A2aR regulates mRNA and protein expression of VEGF and IGF-1.IGF-1 increases VEGF mRNA and protein expression in FRTL-5 cells,especially VEGF_(164), the most common form of VEGF in process of angiogenesis.Consequently,we can infer that A2aR induced upregulation of VEGF in the thyroid is tightly associated with IGF-1.A2aR might be involved in thyroid angoigenic goiter visa augmentation of VEGF and IGF-1.
引文
[1] Yang J, Wong RK, Park M, et al. Leucine regulation of glucokinase and ATP synthase sensitizes glucose-induced insulin secretion in pancreatic beta-cells. Diabetes. 2006 Jan;55(l):193-201.
[2] Yang J, Wong RK, Wang X, et al. Leucine culture reveals that ATP synthase functions as a fuel sensor in pancreatic beta-cells. J Biol Chem. 2004 Dec 24;279(52):53915-23.
[3] Anello M, Ucciardello V, Piro S, et al. Chronic exposure to high leucine impairs glucose-induced insulin release by lowering the ATP-to-ADP ratio. Am J Physiol Endocrinol Metab. 2001 Nov;281(5):E1082-7.
[4] Ashizawa S, Brunicardi FC, Wang XP. PDX-1 and the pancreas [J]. Pancreas, 2004, 28(2): 109-120.
[5] Jonsson J, Carlsson L, Edlund T, Edlund H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature. 1994 Oct 13;371(6498):606-9.
[6] Offield MF, Jetton TL, Labosky PA, et al. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development. 1996 Mar;122(3):983-95.
[7] Kaneto H, Miyatsuka T, Shiraiwa T, et al. Crucial role of PDX-1 in pancreas development, beta-cell differentiation, and induction of surrogate beta-cells. Curr Med Chem. 2007;14(16):1745-52.
[8] Leahy JL, Cooper HE, Deal DA, Weir GC. Chronic hyperglycemia is associated with impaired glucose influence on insulin secretion. A study in normal rats using chronic in vivo glucose infusions. J Clin Invest. 1986 Mar;77(3):908-15.
[9] Xiao CQ, Deng HM, Huang Y. Effects of supraphysiologic concentration glucose on pancreatic duodenal homeobox-1 expression and insulin secretion in rats. Chin Med J (Engl). 2007 Jun 5; 120(11): 1020-3.
[10] Sun Y, Zhang L, Gu HF, et al. Peroxisome proliferator-activated receptor-alpha regulates the expression of pancreatic/duodenal homeobox-1 in rat insulinoma (INS-1) cells and ameliorates glucose-induced insulin secretion impaired by palmitate. Endocrinology. 2008 Feb;149(2):662-71.
[11] Gremlich S, Bonny C, Waeber G, Thorens B. Fatty acids decrease IDX-1 expression in rat pancreatic islets and reduce GLUT2, glucokinase, insulin, and somatostatin levels. J Biol Chem. 1997 Nov 28;272(48):30261-9.
[12] Iype T, Francis J, Garmey JC, et al. Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes. J Biol Chem. 2005 Apr 29;280(17):16798-807.
[13] Marshak S, Totary H, Cerasi E, Melloul D. Purification of the beta-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26): 15057-62.
[14] Waeber G, Thompson N, Nicod P, Bonny C. Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1 /IDX-1 homeobox factor. Mol Endocrinol. 1996 Nov;10(11):1327-34.
[15] Melloul D, Marshak S, Cerasi E. Regulation of insulin gene transcription. Diabetologia. 2002 Mar;45(3):309-26.
[16] Garcia-Herrero CM, Galan M, Vincent O, et al. Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity. Diabetologia. 2007 Feb;50(2):325-33.
[17] Roche E, Assimacopoulos-Jeannet F, Witters LA, et al. Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1). J Biol Chem. 1997 Jan 31;272(5):3091-8.
[18] Kim WH, Lee JW, Suh YH, et al. Exposure to chronic high glucose induces beta-cell apoptosis through decreased interaction of glucokinase with mitochondria: downregulation of glucokinase in pancreatic beta-cells. Diabetes. 2005 Sep;54(9):2602-11.
[19] Guillam MT, Dupraz P, Thorens B. Glucose uptake, utilization, and signaling in GLUT2-null islets. Diabetes. 2000 Sep;49(9): 1485-91.
[20] Thorens B, Guillam MT, Beermann F, Burcelin R, Jaquet M. Transgenic reexpression of GLUT1 or GLUT2 in pancreatic beta cells rescues GLUT2-null mice from early death and restores normal glucose-stimulated insulin secretion. J Biol Chem. 2000 Aug 4;275(31):23751-8.
[21] Tiedge M, Lenzen S. Regulation of glucokinase and GLUT-2 glucose-transporter gene expression in pancreatic B-cells. Biochem J. 1991 Nov 1;279 ( Pt 3):899-901.
[22] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-8,2001.
[23] Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 285:194-204, 2000.
[24] Asfari M, Janjic D, Meda P, et al. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology. 1992 Jan; 130(l):167-178.
[25] Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology. 1992 Jan; 130(1): 167-78.
[26] Xiao J, Gregersen S, Pedersen SB, Hermansen K. Differential impact of acute and chronic lipotoxicity on gene expression in INS-1 cells. Metabolism. 2002 Feb;51(2): 155-62.
[27] Alstrup KK, Brock B, Hermansen K. Long-Term exposure of INS-1 cells to cis and trans fatty acids influences insulin release and fatty acid oxidation differentially. Metabolism. 2004 Sep;53(9):1158-65.
[28] Wang H, Maechler P, Ritz-Laser B et al (2001) Pdx1 level defines pancreatic gene expression pattern and cell lineage differentiation. J Biol Chem 276:25279-25286.
[29] Wang H, Iezzi M, Theander S, Antinozzi PA, Gauthier BR, Halban PA & Wollheim CB, Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells.Diabetologia,2005.48(4):p.720-31.
[30]Wang H,Maechler P,Ritz-Laser B,Hagenfeldt KA,Ishihara H,Philippe J,Wollheim CB.Pdx1 level defines pancreatic gene expression pattern and cell lineage differentiation.J Biol Chem.2001;276:25279-86.
[31]Wang H,Iezzi M,Theander S,Antinozzi PA,Gauthier BR,Halban PA,Wollheim CB:Suppression of Pdx-1 perturbs proinsulin processing,insulin secretion and GLP-1 signalling in INS-1 cells.Diabetologia 48:720-31,2005.
[32]徐彩棉,赵瑞景,朱铁年。PDX-1及其对胰岛β细胞的调控作用。国际病理科学与临床杂志。2006:26:55-58.
[33]贾延勃,周燕,董为伟,杨于嘉。高血糖对体外培养的胎鼠胰岛Pdx—1基因的影响。重庆医科大学学报,2003:28:559-561.
[34]Melloul D,Marshak S,Cerasi E.Regu lation of insulin gene transcription [J].Diabetologia,2002;45:309-326.
[35]Seijffers R,Ben-david O,Cohen Y,et al.Increase in Pdx-1 Levels suppresses insulin gene expression in RIN 1046-38 cells[J].Endocrinology,1999;140:3311-3317.
[36]Harmon JS,Gleason CE,Tanaka Y,et al.In vivo preven-Lion of hyperglycemia also prevents glucotoxic effects on Pdx-1 and insulin gene expression[J].Diabetes,1999;48:1995-1999.
[37]Macfadane WM,McKinnon CM,Felton-Edkins ZA,et al.Glucose stimulates translocation of the homeodomain transcription factor PDX1 from the cytoplasm to the nucleus in pancreatic beta-cells[J].JBiol Chem,1999,274(2):1011-1016.
[38]Melloul D.Transcription factors in islet development and physiology:role of PDX-1 in beta-cell function.Ann N Y Acad Sci.2004 Apr;1014:28-37.
[39]Leibowitz G,Melloul D,Yuli M,et al.Defective glucose-regulated insulin gene expression associated with PDX-1 deficiency in the Psammomys obesus model of type 2 diabetes.Diabetes.2001 Feb;50 Suppl 1:S138-139.
[40]Ahlgren,U.,Jonsson,J.,Jonsson,L.,et al.Beta- cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes Dev. 1998, 12,1763-1768.
[41] Ferber, S., Halkin, A., Cohen, H., et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nature Med., 2000, 6, 568-572.
[42] Horb, ME, Shen, CN, Tosh, D. et al. Experimental conversion of liver to pancreas. Curr. Biol., 2003, 13,105-115.
[43] Zalzman, M., Gupta, S., Giri, R.K., et al. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc. Natl Acad. Sci. USA, 2003, 100, 7253-7258.
[44] Peshavaria M, Cissell MA, Henderson E, et al. The PDX-1 activation domain provides specific functions necessary for transcriptional stimulation in pancreatic beta-cells. Mol Endocrinol. 2000 Dec; 14(12):1907-1917.
[45] McKinnon CM. and Docherty K. (2001) Pancreatic duodenal homeobox-1, PDX-1, a major regulator of beta cell identity and function. Diabetologia, 44, 1203-1214.
[46] Karlsson O, Edlund T, Moss JB, et al. A mutational analysis of the insulin gene transcription control region: expression in beta cells is dependent on two related sequences within the enhancer[J]. Proc Natl Acad Sci USA, 1987, 84(24): 8819-8823.
[47] Naya FJ, Stellrecht CM, Tsai MJ. Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor [J]. Genes Dev, 1995, 9(8): 1009-1019.
[48] Olbrot M, Rud J, Moss LG, et al. Identification of beta-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA [J]. Proc Natl Acad Sci USA. 2002; 99(10): 6737-6742.
[49] Samaras SE, Zhao L, Means A, et al. The islet beta cell-enriched RIPE3b1 / Maf transcription factor regulates pdx-1 expression[J]. J Biol Chem, 2003, 278, 12263-12270.
[50] Kawamori D. Kajimoto Y, Kaneto H, et al. Oxidative stress induces nucleo-cytoplasmic translocation of pancreatic transcription factor PDX-1 through activation of c-Jun NH(2)-terminal kinase[J]. Diabetes. 2003, 52(12): 2896-2904.
[51] Harmon JS, Stein R. Robertson RP. Oxidative stress-mediated, post-translational loss of MafA protein as a contributing mechanism to loss of insulin gene expression in glucotoxic beta cells[J]. J Biol Chem, 2005, 280(12): 11107-11113.
[52] Zhao L, Guo M, Matsuoka TA, et al. The islet beta cell-enriched MafA activator is a key regulator of insulin gene transcription[J]. J Biol Chem, 2005, 280(12): 11887-11894.
[53] Brissova M, Shiota M, Nicholson WE. et al. Reduction in Transcription factor pdx—1 impairs normal glucose sensing and insulin secretion by pancreatic islets[J]. J Bid Chem. 2002,277: 11225-11232.
[54] Gauthier BR, Brun T, Sarret EJ, et al. Oligonucleotide Microarray Analysis Reveals PDX1 as an Essential Regulator of Mitochondrial Metabolism in Rat Islets[J]. JBiolChem, 2004, 279: 31121-31130.
[55] Kulkarni RN, Jhala US, Winnay JN, et al. PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance[J]. J Clin invest, 2004, 114(6): 828-836.
[56] Thorens B, Gerard N and Deriaz N. Surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A. The Journal of Cell Biology, Vol 123, 1687-1694.
[57] Komatsu M, Sato Y, Yamada S, et al. Triggering of insulin release by a combination of cAMP signal and nutrients: an ATP-sensitive K+ channel-independent phenomenon. Diabetes. 2002 Feb; 51 Suppl l:S29-32.
[58] Guillam MT, Dupraz P, Thorens B: Glucose uptake, utilization, and signaling in GLUT2-null islets. Diabetes 49:1485-1491,2000.
[59] Iype T, Francis J, Garmey JC, et al. Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes. J Biol Chem. 2005 Apr 29;280(17): 16798-807.
[60] Marshak S, Totary H, Cerasi E, Melloul D. Purification of the beta-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26): 15057-62.
[61]Waeber G, Thompson N, Nicod P, Bonny C. Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol Endocrinol. 1996 Nov;10(11):1327-34.
[62] Melloul D, Marshak S, Cerasi E. Regulation of insulin gene transcription. Diabetologia. 2002 Mar;45(3):309-26.
[63] Wang H, Iezzi M, Theander S, et al. Suppression of Pdx-1 perturbs proinsulin processing, insulin secretion and GLP-1 signalling in INS-1 cells. Diabetologia. 2005 Apr;48(4):720-31.
[64] Dahlquist G, Bjork E, Eizirik D, Hagglof B, Kockum I, Persson LA. [Causes of diabetes in children and adolescents. A combination of heredity and environment]. Lakartidningen. 1996 Jul 24;93(30-31):2673.
[65] Ronningen KS, Stene LC, Rasmussen T, Wetlesen T, Magnus P. [Environmental causes of type 1 diabetes]. Tidsskr Nor Laegeforen. 2007 Sep 20;127(18):2405-8.
[1]熊正平,杨树仁.血管生成的调控[J].国外医学临床放射学分 2004;27(1):61-64.
[2]J D Ramsden.Angiogenesis in the thyroid gland[J].J Endocrinol,2000,166(3):475-480.
[3]Kucharz EJ,Kotulska A,Kopec M,et al.Serum level of the circulating angiogenesis inhibitor endostatin in patients with hyperthyroidism or hypothyroidism[J].Wien Klin Wochenschr,2003,115(5-6):179-181.
[4]Leung,D.W.,et al.,Vascular endothelial growth factor is a secreted angiogenic mitogen.Science,1989.246(4935):p.1306-9.
[5]Ferrara,N.and T.Davis-Smyth,The biology of vascular endothelial growth factor.Endocr Rev,1997.18(1):p.4-25.
[6]Kofidis,T.,et al.,Insulin-like growth factor promotes engraftment,differentiation,and functional improvement after transfer of embryonic stem cells for myocardial restoration.Stem Cells,2004.22(7):p.1239-45.
[7]Radcliff,K.,et al.,Insulin-like growth factor-Ⅰ regulates proliferation and osteoblastic differentiation of calcifying vascular cells via extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase pathways.Circ Res,2005.96(4):p.398-400.
[8]Poulaki,V.,et al.,Regulation of vascular endothelial growth factor expression by insulin-like growth factor Ⅰ in thyroid carcinomas.J Clin Endocrinol Metab,2003.88(11):p.5392-8.
[9]Ongini,E.and B.B.Fredholm,Pharmacology of adenosine A2A receptors.Trends Pharmacol Sci,1996.17(10):p.364-72.
[10]Hove-Madsen,L.,et al.,Adenosine A2A receptors are expressed in human atrial myocytes and modulate spontaneous sarcoplasmic reticulum calcium release.Cardiovasc Res,2006.72(2):p.292-302.
[11]Ryzhov,S.,et al.,Role of adenosine receptors in the regulation of angiogenic factors and neovascularization in hypoxia.J Pharmacol Exp Ther,2007.320(2): p. 565-72.
[12] Desai, A., et al., Adenosine A2A receptor stimulation increases angiogenesis by down-regulating production of the antiangiogenic matrix protein thrombospondin 1. Mol Pharmacol, 2005. 67(5): p. 1406-13.
[13] Leibovich, S.J., et al., Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin. Am J Pathol, 2002. 160(6): p. 2231-44.
[14] Ramanathan, M., et al., Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter. Mol Biol Cell, 2007. 18(1): p. 14-23.
[15] Olah, M.E. and F.L. Roudabush, Down-regulation of vascular endothelial growth factor expression after A(2A) adenosine receptor activation in PC12 pheochromocytoma cells. J Pharmacol Exp Ther, 2000. 293(3): p. 779-87.
[16] Ledent, C, et al., Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism. EMBO J, 1992. 11(2): p. 537-42.
[17] Ledent, C, et al., Transgenic models for proliferative and hyperfunctional thyroid diseases. Exp Clin Endocrinol Diabetes, 1996. 104 Suppl 3: p. 43-6.
[18] Ledent, C, [Transgenic models of human thyroid pathologies: hyperfunctional adenoma, anaplastic cancer, differentiated cancer, hypothyroidism]. Bull Mem Acad R Med Belg, 1996. 151(2): p. 195-201.
[19] Gerard, A.C., et al., Relationships between cell division, expression of growth factors and microcirculation in the thyroids of Tg-A2aR transgenic mice and patients with Graves' disease. J Endocrinol, 2003. 177(2): p. 269-77.
[20] Goffard, J.C., et al., Gene expression profile in thyroid of transgenic mice overexpressing the adenosine receptor 2a. Mol Endocrinol, 2004. 18(1): p. 194-213.
[21]Pons S, Torres-Aleman I 2000 Insulin-like growth factor-I stimulates dephosphorylation of ikappa B through the serine phosphatase calcineurin (protein phosphatase 2B).J Biol Chem 275:38620-38625
[22]Ambesi-Impiombato FS,Parks LA,Coon HG 1980 Culture of hormone-dependent functional epithelial cells from rat thyroids.Proc Natl Acad Sci U S A 77:3455-3459.
[23]Miyagi E,Braga-Basaria M,Hardy E,Vasko V,Burman KD,Jhiang S,Saji M,Ringel MD 2004 Chronic expression of RET/PTC 3 enhances basal and insulin-stimulated PI3 kinase/AKT signaling and increases IRS-2 expression in FRTL-5 thyroid cells.Mol Carcinog 41:98-107.
[24]Kim H,Lee TH,Park ES,Sub JM,Park S J,Chung HK,Kwon OY,Kim YK,Ro HK,Shong M 2000 Role of peroxiredoxins in regulating intracellular hydrogen peroxide and hydrogen peroxide-induced apoptosis in thyroid cells.J Biol Chem 275:18266-18270.
[25]Vainio M,Fredholm BB,Tomquist K 2000 Thyrotropin regulates adenosine A(1)receptor expression in rat thyroid FRTL-5 cells.Br J Pharmacol 130:471-477.
[26]黄亿华,翁星河,周曾铨.胞外ATP的作用、来源和命运[J].生理科学进展,1998,29(2):115-9.
[27]Huang Y H,Weng X H,Zhou Z Q.Extracellular ATP:Effects,Sources,Fate[J].Sheng Li Ke Xue Jin Zhan,1998,29(2):115-9.
[28]Stein,M.,P.Kimbel,and R.L.Johnson,Pulmonary Function In Hyperthyroidism.J Clin Invest,1961.40(2):p.348-63.
[29]Pino-Garcia,J.M.,et al.,Regulation of breathing in hyperthyroidism:relationship to hormonal and metabolic changes.Eur Respir J,1998.12(2):p.400-7.
[30]Fukui,H.,et al.,Enhanced activity of the purine nucleotide cycle of the exercising muscle in patients with hyperthyroidism.J Clin Endocrinol Metab,2001.86(5):p.2205-10.
[31]Muller,M.J.and H.J.Seitz,Thyroid hormone action on intermediary metabolism.Part Ⅰ:respiration,thermogenesis and carbohydrate metabolism.Klin Wochenschr,1984.62(1):p.11-8.
[32]Daval,J.-L.,A.Nehlig,and F.Nicolas,Life Sci,1991.49:p.1435-1453.
[33]Deleu,S.,et al.,IGF-1 or insulin,and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis. Mol Cell Endocrinol, 1999. 149(1-2): p. 41-51.
[34] Burikhanov, R., et al., Thyrotropin via cyclic AMP induces insulin receptor expression and insulin Co-stimulation of growth and amplifies insulin and insulin-like growth factor signaling pathways in dog thyroid epithelial cells. J Biol Chem, 1996.271(46): p. 29400-6.
[35]Ongini E et al. 腺苷A2a受体的药理学. 国外医学药学分册 , 1997, 24(5), 297-231.
[36] Fredholm BB, Chen JF, Cunha RA, et al. Adenosine and brain function. Int Rev Neurobiol, 2005, 63:191-270.
[37] Phillis, J.W., The effects of selective A1 and A2a adenosine receptor antagonists on cerebral ischemic injury in the gerbil. Brain Res, 1995. 705(1-2): p. 79-84.
[38] Jones, P.A., R.A. Smith, and T.W. Stone, Protection against kainate-induced excitotoxicity by adenosine A2A receptor agonists and antagonists. Neuroscience, 1998. 85(1): p. 229-37.
[39] Michael, Y., et al., Characterisation of the aggregation behaviour in a salmeterol and fluticasone propionate inhalation aerosol system. Int J Pharm, 2001. 221(1-2): p. 165-74.
[40] Ohta A, Sitkovsky M. Role of G protein-coupled adenosine receptors in down-regulation of inflammation and protection from tissue damage. Nature, 2001, 414 : 916-920.
[41] Maddock, H.L., et al., Role of endothelium in ischaemia-induced myocardial dysfunction of isolated working hearts: cardioprotection by activation of adenosine A(2A) receptors. J Auton Pharmacol, 2001. 21(5-6): p. 263-71.
[42] Varani, K., et al., Changes of peripheral A2A adenosine receptors in chronic heart failure and cardiac transplantation. FASEB J, 2003. 17(2): p. 280-2.
[43] Day, Y.J., et al., Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow-derived cells. J Clin Invest, 2003. 112(6): p. 883-91.
[44] Montesinos, M.C., et al., Adenosine promotes wound healing and mediates angiogenesis in response to tissue injury via occupancy of A(2A) receptors.Am J Pathol,2002.160(6):p.2009-18.
[45]Tebano MT,Pintor A,Frank C,et al.Adenosine A2A receptor blockade differentially influences excitotoxic mechanisms at pre-and postsynaptic sites in the rat striatum.J Neurosci Res.2004,77:100-107.
[46]Yu L,Huang Z,Mariani J,et al.Selective inactivation or reconstitution of adenosine A2a receptors in bone marrow cells reveals their significant contribution to the development ofischemic brain injury.Nat Med,2004,10:1081-1087.
[47]Neary J T,Rathbone M P,Cattabeni F,et al.Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells[J].Trend Neurosci,1996,19(1):13-8.
[48]Vainio,M.,B.B.Fredholm,and K.Tornquist,Thyrotropin regulates adenosine A(1) receptor expression in rat thyroid FRTL-5 cells.Br J Pharmacol,2000.130(2):p.471-7.
[49]仲霞,赵家军,戴晓华,高聆,粱军,王汝霞。VEGF、IGF-Ⅰ与Graves病患者甲状腺内血管形成的关系。中华内分泌代谢杂志,2006,22(2),119-120.
[50]Cameliet P,Collen D,Molecular basis of angiogenesis.Role of vegf and ve-cadherin[J].Ann N Y Acad Sci,2000,902:249-264.
[51]Veikkola T,Karkkainen M,Claesson-Welsh L,et al.regulation of angiogenesis via vascular endothelial growth factor receptor[J].Cancer Res,2000,60:202-212.
[52]Giatromanolaki A,Sivridis E,Athannasson N et al.The angiogenic pathway "vascular endothelial growth factor/flk-1(KDR) receptor" in rheumatoid arthritis and osteoarthritis.[J].J Pathnol,2001,194(1):101-108.
[53]Shibuya M,.Structure and function of VEGF/VEGF-receptor system involved in angiogenesis[J]Cell Struct Funct,2001,26(1):25-35.
[54]Josko J,Gwozdz B,Jedrzejow ska-Szypulka H,et al.Vascular endothelial growth factor(VEGF) and its effect on angiogenesis[J].Med Sci Monit,2000,6(5):1047-1052.
[55] Miyagi, E., et al., Thyroid stimulating hormone downregulates vascular endothelial growth factor expression in FRTL-5 cells. Thyroid, 2001. 11(6): p. 539-43.
[56] Hoffmann, S., et al., Thyrotropin (TSH)-induced production of vascular endothelial growth factor in thyroid cancer cells in vitro: evaluation of TSH signal transduction and of angiogenesis-stimulating growth factors. J Clin Endocrinol Metab, 2004. 89(12): p. 6139-45.
[57] Van Keymeulen A, Dumont JE, Roger PP. TSH induces insulin recptors that mediate insulin costimulation of growth in normal human thyroid ceils. Biochem Biophys Res Commun. 2000. 279: 202-207.
[58] Johansson GS, Arnqvist HJ. Insulin and IGF-1 action on insulin receptors, IGF-1 receptors, and hybrid insulin / IGF-1 receptors in vascular smooth muscle ceils. Am J Physiol Endocrinol Metab. 2006. 291: E1124-E1130.
[59] Ren, M., et al., Phosphatidylinositol 3-kinase/nuclear factor-kappaB signaling pathway is involved in the regulation of IGF-I on Fas-associated death domain-like interleukin-1-converting enzyme-inhibitory protein expression in cultured FRTL thyroid cells. J Mol Endocrinol, 2007. 38(6): p. 619-25.
[60] Kimura T, Van Keymeulen A,Golstein J,et al. Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models[J]. Endocr Rev. 2001, 22(5): 631-56.
[61] T Mitsiades CS, Poulaki V, Mitsiades N.The role of apoptosis-inducing receptors of the tumor necrosis factor family in thyroid cancer[J]. J Endocrinol. 2003;178(2):205-16.
[62] Poulaki V, Mitsiades CS, McMullan C,et al.Regulation of vascular endothelial growth factor expression by insulin-like growth factor I in thyroid carcinomas[J].J Clin Endocrinol Metab. 2003,88(11):5392-8.
[63] Mincione G, Esposito DL, Di Marcantonio MC, et al. TGF-beta 1 modulation of IGF-I signaling pathway in rat thyroid epithelial cells[J].Exp Cell Res. 2003,287(2):411-23.
[64] Fan J, Wojnor MM,Theodorakis M, et al. Regulation of insulin-like growth factor (IGF-I) mRNA and peptide and IGF-binding protein by interleukin-1[J]. Am J Physiol, 1996, 270 (4): 621-629.
[65] Inukai T, Takanashi K, Takebayashi K, et al. Thyroid hormone modulates insulin-like growth factor-I(IGF-I) and IGF-binding protein-3, without mediation by growth hormone, in patients with autoimmune thyroid diseases[J]. Horm Metab Res. 1999,31(10):576-579.
[66] Nagura, S., et al., Expression of vascular endothelial growth factor (VEGF) and VEGF receptor-1 (Fit-1) in Graves disease possibly correlated with increased vascular density. Hum Pathol, 2001. 32(1): p. 10-7.
[67] Deleu, S., et al., IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis. Mol Cell Endocrinol, 1999. 149(1-2): p. 41-51.
[68] Ferrara, N., Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev, 2004. 25(4): p. 581-611.
[69] Houck, K.A., et al., Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem, 1992. 267(36): p. 26031-7.
[70] Hofmann, A., et al., Vascular endothelial growth factor in thyroid cyst fluids. Wien Klin Wochenschr, 2007. 119(7-8): p. 248-53.
[71] Tanaka, K., et al., Expression of vascular endothelial growth factor family messenger RNA in diseased thyroid tissues. Surg Today, 2002. 32(9): p. 761-8.
[72] Salabe, GB., Pathogenesis of thyroid nodules: histological classification? Biomed Pharmacother, 2001. 55(1): p. 39-53.