【作者】 卢忠燕；

【导师】 邹全明；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2009， 博士

【摘要】 2型糖尿病（T2DM）是一种严重危害人类健康的代谢性疾病,胰岛素抵抗和胰岛β细胞功能受损是T2DM发病的中心环节[1]。胰岛β细胞是机体胰岛素的唯一内分泌细胞,其数量、体积及功能状态决定了胰岛素的分泌水平,对维持血糖恒定起重要作用[1]。胰岛β细胞数量的维持主要取决于β细胞新生、增殖、肥大及凋亡之间的动态平衡。T2DM患者,β细胞数量显著低于非糖尿病者,最终导致胰岛素分泌量不足,其机体能量代谢障碍,但其发生机制尚未完全阐明[2-5]。FOXO1是调节细胞氧化应激反应及增殖、凋亡的重要转录因子,广泛表达于糖、脂代谢的重要器官,如肝脏、脂肪和骨骼肌等[6-7]。最近研究显示FOXO1亦表达在成人胰岛中,Guerra等[8]从13例人的组织标本中报道,2型糖尿病人的胰岛中FOXO1的mRNA表达量显著高于非糖尿病对照组,提示高表达FOXO1可能造成β细胞功能损伤。然而,对FOXO1在成熟β细胞中的作用尚不清楚。为此,本研究采用免疫组化方法结合激光共聚焦技术观察FOXO1在胰岛的表达及细胞定位;通过病毒介导的基因转移技术和siRNA干预技术,在培养的大鼠胰腺癌β细胞系（INS-1E）中特异高表达组成性活性的FOXO1（FOXO1-AAA）或抑制其表达水平,观察FOXO1表达水平的改变对β细胞增殖、凋亡的影响;进一步采用基因芯片技术分析了INS-1E中受FOXO1调控的下游靶基因,并对其功能进行了初步的分析预测。具体的研究工作如下:1.首先观察了FOXO1在成年SD大鼠胰腺中的表达及分布:4-6月成年SD大鼠胰腺进行冰冻切片免疫组化分析,结果显示FOXO1特异性表达在胰腺的胰岛组织中。为了进一步确定FOXO1在胰岛组织中的细胞定位,采用免疫荧光标记观察不同荧光标记的FOXO1（绿色）和胰岛素（红色）在胰岛中的分布,激光共聚焦观察发现绝大多数FOXO1蛋白与胰岛素的定位一致（橙色）,表明FOXO1在生理情况下主要高表达在分泌胰岛素的β细胞中。2.为了阐明FOXO1在成熟胰岛β细胞中的作用,我们首先构建了高表达FOXO1的腺病毒载体:利用基因同源重组原理,通过复制缺陷型pAdEasy腺病毒系统构建了磷酸化位点突变的具有组成活性的全长FOXO1（T24A\S256A\S319A）重组腺病毒,纯化后病毒滴度约为5×10¹²U/ml。病毒感染肝癌细胞系HepG2,通过观察GFP荧光、Western blot等方法证明Ad-FOXO1-AAA重组腺病毒能有效的介导FOXO1蛋白的表达,经灰度扫描及定量分析发现使FOXO1蛋白表达水平升高9.8倍。3.为了比较内源性FOXO1蛋白表达水平降低对成熟β细胞功能的影响,我们构建了针对FOXO1的siRNA腺病毒载体:利用www.ambion.com网站在线设计,并辅助设计软件,选择了三个针对FOXO1的siRNA位点,分别为si-FOXO1-1、si-FOXO1-2和si-FOXO1-3;一个针对FOXO1和FOXO3同源序列的siRNA位点,命名为si-FOXO-pan;以及一个不针对任何基因的随机序列作为阴性对照。人工合成si-FOXO1s的cDNA序列,退火形成的双链cDNA克隆到腺病毒表达载体pAdTrack-CMV上,经PmeI线性化后与pAdEasy-1细胞内同源重组,最后在HEK293细胞中包装为重组腺病毒,分别命名为Ad-si-FOXO1-1、Ad-si-FOXO1-2、Ad-si-FOXO1-3、Ad-si-FOXO-pan和Ad-NC。扩增后测定病毒滴度均约为5×10¹²U/ml。重组腺病毒感染肝癌细胞系HepG2,Western blot检测结果显示,重组腺病毒能有效的抑制FOXO1蛋白的表达,经灰度扫描及定量分析发现抑制效果达45-65%,其中si-FOXO1-3的抑制效果最显著,达到65%。4.为了确定FOXO1表达降低是否影响其转录功能,选取了FOXO1的靶基因磷酸烯醇式丙酮酸羧激酶（PEPCK）,检测si-FOXO1s对其启动子转录活性的影响:提取HepG2基因组DNA,PCR扩增得到PEPCK基因长约592bp的5′侧翼区启动子序列,插入pGL3-Basic荧光素报告质粒中。加入Ad-si-FOXO1s重组腺病毒感染HepG2细胞后,检测si-FOXO1s对PEPCK启动子转录活性的影响,结果显示,与阴性对照si-NC相比,si-FOXO1s显著降低PEPCK启动子的荧光素酶活性,其中Ad-si-FOXO1-1降低40%,Ad-si-FOXO1-2降低55%,Ad-si-FOXO1-3降低67%,Ad-si-FOXO-pan降低68%,表明构建的Ad-si-FOXO1s能显著抑制FOXO1靶基因的表达,具有功能活性。si-FOXO1-3抑制蛋白表达最低,抑制后FOXO1的功能活性也最低,所以后续实验均选择si-FOXO1-3。5.为了观察FOXO1表达水平的改变对胰岛β细胞功能的影响,首先检测si-FOXO1和FOXO1-AAA重组腺病毒能否有效的下调或上调胰腺癌β细胞系中FOXO1表达水平:将Ad-si-NC、Ad-si-FOXO1-3和Ad-FOXO1-AAA腺病毒感染胰腺癌β细胞系INS-1E,发现有明显绿色荧光表达,进一步采用Western Blot分析各组细胞中FOXO1蛋白质的表达水平,经扫描及定量分析发现Ad-si-FOXO1对FOXO1的表达抑制达74%,FOXO1-AAA导入细胞内使FOXO1表达升高8.7倍。6.采用3H-TdR掺入实验观察FOXO1表达水平的改变对胰岛β细胞增殖的影响,结果显示,降低FOXO1的表达显著促进了β细胞的增殖,而高表达FOXO1则显著抑制了β细胞的增殖;与之相应,MTT检测结果显示,降低FOXO1的表达对β细胞存活有显著促进作用,高表达FOXO1对β细胞存活有显著抑制作用;进一步采用流式细胞仪检测细胞凋亡,结果显示降低FOXO1的表达使β细胞凋亡率降低,反之高表达FOXO1使β细胞凋亡率增加。采用放射免疫法检测感染重组腺病毒后INS-1E细胞胰岛素的分泌情况,实验结果显示,FOXO1表达水平的改变对胰腺癌β细胞系INS-1E的胰岛素分泌没有显著影响。7.为了深入揭示FOXO1在胰岛β细胞中的作用机制,我们进一步采用基因芯片技术研究受FOXO1调节的下游靶基因表达变化情况:提取Ad-si-NC和Ad-FOXO1-AAA重组腺病毒感染的胰腺癌β细胞系INS-1E的总RNA,纯化后制成cDNA探针,与Agilent大鼠全基因组芯片杂交,经过信号检测、微阵列图像分析、计算机软件数据处理,将所得的数据进行比较,筛选出差异表达基因。结果显示,INS-1E细胞中高表达FOXO1与对照组有显著差异（基因表达水平差异>2为显著升高,<0.5为显著降低）的基因254条,其中表达序列标签（expressed sequence taq,EST）有107条,全长基因有147个。表达上调的基因有135个,表达下调的基因有12个。分析其功能后,从差异表达基因中初步筛选出参与调节细胞增殖、凋亡的基因16个。本研究显示在SD大鼠的胰岛中,FOXO1主要高表达在分泌胰岛素的胰岛β细胞中;在培养的胰腺癌β细胞中初步证实,FOXO1转录因子表达水平的变化对分化成熟的β细胞的增殖及凋亡具有调节作用,高表达FOXO1显著抑制了β细胞的增殖,同时使β细胞凋亡率增加,对胰岛β细胞分泌胰岛素的能力没有显著影响;通过基因芯片初步筛选出了受FOXO1调控的与细胞增殖和凋亡相关的基因。这些结果为深入揭示FOXO1调节β细胞的增殖及凋亡的分子机制奠定了基础,为揭示2型糖尿病发生发展中β细胞代偿性增生受损的发生机制提供了重要的线索。更多还原

【Abstract】 Type 2 diabetes is characterized by a progressive decline inβ-cell function and chronic insulin resistance. Pancreaticβ-cells produce insulin and secrete it in response to elevations in circulating blood glucose and to other signaling molecules, providing a key contribution to glucose homeostasis and to the coordination of metabolism within the body. Defects ofβ-cell function in diabetes are complex and include reduced insulin secretion and alterations ofβ-cell mass. Regulation of theβ-cell mass involves a balance ofβ-cell replication and apoptosis, as well as development of new islets from exocrine pancreatic ducts. Disruption of any of these pathways ofβ-cell formation or increased rates ofβ-cell death could cause a decrease inβ-cell mass. Previous studies showed a reduction of islet and/or insulin-containing cell mass or volume in type 2 diabetes, indicating the importance of apoptosis as the cause of pancreaticβcell loss in the development of insulin deficiency and the onset and/or progression of the disease. However, the mechanisms regulatingβcell mass and their adaptations to inRsulin resistance are not complelely defined.FOXO transcription factors control several fundamental cellular processes, including metabolism, cell differentiation, cell cycle arrest, DNA repair, and cellular stress. FOXO1, one of the O subfamily members of FOXO factors, was identified in liver, adipose tissue andβ-cells. Quantitative PCR experiments of FOXO1 mRNA level were significantly higher in islets isolated from type 2 diabetic compared to the matched nondiabetic cadaveric organ donors, indicating that increased FOXO1 was associated withβcell dysfunction. However, the exact funtion of FOXO1 inβcells remains to be further characterized. In this study, the expression and cellular localization of FOXO1 were detected by immunohistochemistry and laser confocal microscopy. Adenoviral vectors expressing a constitutively active form of FOXO1 (FOXO1-AAA) and its small interfering RNA (siRNA) were prepared. The effects of FOXO1 onβcell proliferation and apoptosis were investigated with rat insulinoma cell line (INS-1E) by introducing either active FOXO1 or its siRNA. Furthermore, DNA arrays were carried to reveal the possible mechanisms mediated by the downstream target genes under the control of FOXO1. The major results are summarized below:1. To detect the distribution and subcellular localization of FOXO1 protein in adult rat pancreatic islets, SD rats aged 4-6 weeks were fed for one mouth, then sacrificed and their pancreatic islets were subjected for cryostat section. Distributions of FOXO1 protein in rat pancreatic islets were observed by immunohistochemical staining using anti-FOXO1 antibody. Subcellular localization of FOXO1 was further analyzed by double immunofluorescent staining with anti-FOXO1 and anti-insulin antibody under a laser confocal microscope. Results showed that FOXO1 proteins expressed in pancreatic islet and co-localized with insulin inβcells.2. To evalue overexpression of FOXO1 onβcell function, a constitutively active mutant FOXO1 (FOXO1-AAA) with single-amino-acid-substitution on the three main phosphorylation sites, Thr24/Ala, Ser256/Ala, and Ser319/Ala corresponding to human FOXO1 was used. FOXO1-AAA was subcloned into the pAd-Track vector coexpressing GFP. Adenoviruses carrying the FOXO1-AAA constructs were produced using the pAdeasy adenovirus-packaging system. Expression of FOXO1 on the recombinatant adenovirus can thus be easily detected by fluorescent microscope. The titer of recombinant adenovirus was assayed (about 5×1012U/ml). To test the expression capabilities of recombinant adenoviruses, HepG2 cells were infected with adenovirus carring FOXO1 and the cell lysates were assayed by Western blot, results demonstrated that the FOXO1 remarkablely overexpressed to 9.8 times.3. To investigate down regulation of FOXO1 onβcell function, four siRNA sites specifically targeting FOXO1, named si-FOXO1-1, si-FOXO1-2, si-FOXO1-3 and si-FOXO-pan, were designed using an online software (www.ambion.com). The corresponding double strands of oligodeoxynucleotides sequences for si-FOXO1s and a negative control, a stretch of scrambled sequence not targeting any gene, were synthesized by automatic DNA synthesis machine. After annealing and disgesting with restriction enzymes, the four si-FOXO1s DNA sequences were inserted into the shuttle vector pAd-Track-CMV carrying green fluorescent protein (GFP). Recombinant adenovirus encoding si-FOXO1s were generated after linearisation of pAd-Track-CMV-si-FOXO1s and cotransformed with the adenoviral plasmid pAd-easy-1 in E. coli BJ5183. Kanamycin was used for selection. After linearisation by restriction enzyme PacI, the recombinant plasmids were transfected into HEK 293-cells and amplified to give large quantities of infectious recombinant adenovirus carrying the sequences of si-FOXO1s, designated Ad-si-FOXO1-1, Ad-si-FOXO1-2, Ad-si-FOXO1-3, Ad-si-FOXO-pan and Ad-NC, respectively. The titers as potency of infection were determined in HEK 293 cells (about 5×1012pfu/ml). The efficiency of si-FOXO1s to downregulate the endogenous FOXO1 gene expression were detected by Western blotting in HepG2 cells. Results demonstrated that endogenouse FOXO1 was efficiently suppressed by 45-65% with Ad-si-FOXO1s and 65% with Ad-si-FOXO1-3.4. To test downregulation of FOXO1 on its transcriptional activity, an established FOXO1 target gene, phosphoenolpyruvate carboxykinase (PEPCK), was tested. The 592bp 5′flanking region of PEPCK promoter was amplified with genome DNA of HepG2 by PCR and the segment was cloned into the eukaryotic expression vector pGL3-Basic. HepG2 cells were transfected with plasmid PGL3-PEPCK, followed by infection with Ad-si-NC and Ad-si-FOXO1s recombinant adenoviruses. Effects of si-FOXO1s on the transcriptional activity of PEPCK promoter were analyzed by measuring luciferase activity. The expression level of PEPCK was decreased to 40% by Ad-si-FOXO1-1, 55% by Ad-si-FOXO1-2, 67% by Ad-si-FOXO1-3 and 68% by Ad-si-FOXO-pan.5. To test the expression activities of Ad-si-FOXO1 and Ad-FOXO1-AAA inβcells function, INS-1E cells, a rat insulinoma cell line, were infected with Ad-si-NC, Ad-si-FOXO1-3 or Ad-FOXO1-AAA recombinant adenoviruses for 24h. Endogenous FOXO1 expression levels were determined with Western blotting. Results demonstrated that FOXO1 decreased by 74% with si-FOXO1-3, no statistical change with Ad-si-NC and increased about 8.7 times with Ad- FOXO1-AAA.6. To test effects of up- or down-regulation of FOXO1 onβcell proliferation or its apoptosis, INS-1E was assessed. By infection of the cells with Ad-FOXO1-AAA or Ad-si-FOXO1, 3H-TdR incorporation experiment was performed as a test for cell growth. Results showed that decreased expression of FOXO1 significantly promotes INS-1E growth, whereas increased expression of FOXO1 significantly inhibits cell growth. MTT experiment showed the same effects of FOXO1 expression levels on the survival of INS-1E cells. Furthermore, the effect of FOXO1 on INS-1E apoptosis was assessed by fluorescence activated cell sorter (FACS) and results showed an increased apoptosis with higher levels of FOXO1 expression and decreased apoptosis with lower levels of FOXO1. Insulin secretion of INS-1E cells by radioimmunoassay did not signaficantly change with infection of Ad-si-NC, Ad-si-FOXO1-3 or Ad-FOXO1-AAA.7. To further understand the molecular mechanisms mediating FOXO1 effects onβcell functions, gene array studies were carried out. INS-1E cells were infected with Ad-si-NC or Ad-FOXO1-AAA, and total RNAs were isolated. The cRNA probes prepared from total RNA were hybridized with Agilent rat gene array. After signal detection, image comparison and data analysis, the differentially expressed genes were screened. 254 significantly differentially expressed (with the fold differences of >2, or <0.5) genes were detected by genechips, 107 of them were ESTs (Expressed Sequenee Tag) and the others were genes with complete sequences. 135 showed higher expressions and 33 showed lower expressions. Of them, 16 target genes with a potential role in cell proliferation or apoptosis were discussed.These data demonstrates that FOXO1 proteins expressed in pancreatic islet and co-localized with insulin inβcells. Increased expression of FOXO1 significantly inhibits INS-1E cell growth and survival, but increases cell apoptosis. Sixteen target genes with a potential role in cell proliferation or apoptosis were detected by genechips. Taken together, these data demonstrates that FOXO1 involves in the regulation of survival, proliferation and apoptosis of pancreaticβcells. Dysregulation of FOXO1 expression may thus conttributes to pathogenesis of type 2 diabetes which is closely linked to decreasedβcell mass.更多还原