【作者】 梁光萍；

【导师】 罗向东；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 研究背景烧伤早期脏器损害是大面积烧伤患者严重的并发症,组织缺血缺氧是其关键始发因素。烧伤后由于血管通透性增加,导致有效血容量和外周组织血液供应急剧减少,局部氧分压下降引发组织细胞缺氧,造成细胞代谢障碍和结构损伤,最终导致全身各脏器功能受损。血管内皮细胞作为血管壁最内一层衬里,与血液直接接触,是最早感受氧浓度变化的细胞,在烧伤早期脏器损伤的发病中起核心作用。血管内皮细胞在受到缺氧刺激后被激活,通过基因表达谱的改变,对细胞多种功能产生调节。在缺氧初期,血管内皮细胞对缺氧的反应主要表现为适应性反应,但随着缺氧时间延长和缺氧程度的加深,这种反应将转变为使血管内皮细胞功能降低,进而造成细胞损伤。研究缺氧后血管内皮细胞产生的适应性或病理性反应,对阐明严重烧伤后血管内皮细胞缺氧性损害的分子机制具有重要意义。但截止当前,有关缺氧后血管内皮细胞适应性或病理性反应的研究多偏重于特定的基因,从整体上研究缺氧对血管内皮细胞基因网络的改变并不多见,缺氧引起血管内皮细胞反应机制尚不完全清楚。从基因表达谱水平研究缺氧后血管内皮细胞差异表达基因,对阐明血管内皮细胞缺氧反应机制,并从中寻找内源性保护因子有重要意义。在众多基因表达谱研究手段中,基因系列表达分析(SAGE)具有高重复性和高精确性,可定量检测差异基因表达丰度等诸多优点。Long SAGE在原有SAGE基础上经过改进,大大提高了其特异性。因此,在预实验检测缺氧后内皮细胞活化标志ICAM-1和缺氧反应基因HIF-1α表达变化的基础上,我们确定以缺氧3h作为时相点,以正常细胞为对照,构建了常氧和缺氧3 h组EA.hy926细胞Long SAGE文库,并从生物信息学分析、基因及基因功能研究入手,系统研究了脐静脉内皮细胞缺氧早期转录组变化,旨在发现血管内皮细胞缺氧早期基因表达模式和功能状态。一、方法1.制作脐静脉内皮细胞株(EA.hy926)缺氧模型,检测常氧和缺氧1 h、3 h、6 h、12 h各时相点ICAM-1、HIF-1α蛋白表达情况。2.将细胞分为常氧组和缺氧3 h组,分别提取总RNA,依照Invitrogen公司Long SAGE kit说明,构建常氧和缺氧3 h脐静脉内皮细胞Long SAGE文库,具体操作简述如下:制备mRNA,合成cDNA双链,锚定酶酶切后的cDNA片段等分两份,分别与A、B接头连接;然后以标签酶酶切,并连接形成双标签;PCR扩增,分离纯化双标签;双标签随机连接形成串联子并将其克隆至pZero载体中,测序。测序结果经软件分析,获得标签序列及丰度信息,通过与Genbank数据库比对确认基因。3.生物信息学分析:通过NCBI ftp://ftp1.nci.nih.gov/pub/ SAGE/HUMAN/Hs_long.best_gene.gz平台分析去除多重匹配标签中的冗余标签,统计差异表达P值<0.05的有意义标签,纳入后续分析(信号途径活化分析除外);根据单一匹配有意义标签在2个Long SAGE文库中差异表达,总结缺氧后表达明显上调和下调的基因;通过制作MA散点图了解缺氧和常氧两个SAGE文库中标签的分布情况;制作常氧和缺氧脐静脉内皮细胞基因表达图谱;采用在线数据库(http://www.discover.nci.nih.gov/ gominer/),基于GO (Gene Ontology,有译为“基因本体”或者“基因注释”)生物信息学系统,对SAGE文库中P值小于0.05的差异表达基因进行了基因功能注释和分类;基因富集分析采用PERL程序(geneid2path.pl),通过KEGG(Kyoto Encyclopedia of Genes and Genomes)平台预测分析缺氧早期血管内皮细胞信号通路与生化途径改变。4.常氧和缺氧脐静脉内皮细胞基因表达谱的验证:RNA水平的验证采用荧光定量PCR,对SAGE文库中随机挑选的5个已知基因进行验证,比较缺氧后其在RNA水平变化是否与SAGE文库中所得结果相符。采用结合标签的基因序列延伸(GLGI)技术对从文库中选取的3个无匹配标签进行扩增,确定其是否属于未知新基因。与常氧和缺氧基因表达谱相关蛋白质验证采用Western blot检测缺氧后VEGF、PCNA蛋白表达变化。与常氧和缺氧基因表达谱相关细胞功能验证采用细胞计数绘制缺氧前后细胞生长曲线、流式细胞仪分析缺氧后细胞周期改变、以及Annexin V方法检测短暂缺氧后细胞凋亡发生率,对缺氧早期促细胞增殖和凋亡效应等方法进行验证。5. Western blot检测缺氧后血管内皮细胞Integrinβ1蛋白表达变化;转染Integrinβ1全长启动子荧光素酶报告基因至细胞,转染48 h后行缺氧3 h处理,检测缺氧前和缺氧后不同时间Integrinβ1启动子活性变化;构建Integrinβ1荧光小RNA干扰载体,转染EA.hy926细胞,筛选稳定低表达细胞株,通过绘制常氧、缺氧状态下正常EA.hy926细胞和下调表达细胞株生长曲线,对Integrinβ1在缺氧早期促血管内皮细胞增殖效应中的作用进行了初步研究。二、结果1.通过Long SAGE技术构建了常氧和缺氧脐静脉内皮细胞Long SAGE文库,缺氧SAGE文库测得30,756个标签,其中识别出的基因(即unique tag)总数为13879,比例为45.13%;重复的双标签体总数为762,比例为2.40%。常氧SAGE文库测得标签数为31213,其中识别出的基因总数为13665,比例为43.78%;重复的双标签体总数为758,比例为2.40%。文库中共有112个基因表达量出现明显差异(P<0.05),涵盖编码细胞代谢酶类,核糖体蛋白,生长因子,转录和翻译调节因子等,其中缺氧后明显上调的基因为62个,包括信号转导和转录调节因子GPCR5A和C11ORF13,以及促血管生成因子ANGPLT4等;而显著下调基因为50个,包含参与离子结合和防御反应的基因如HMOX1、SOD1、FTH 1、CTGF、OTUB1等;这些基因被视为缺氧后有显著差异表达的基因,纳入后续生物信息学分析中。2.生物信息学分析:综合基因富集分析和GO分析结果,发现缺氧早期由于负调控增殖基因群的表达受到明显抑制(共有4个负调控基因出现显著差异表达,其中CAV1,GJA1,HMOX1表达下调,NME1表达上调),总趋势是促进细胞增殖;另外,缺氧早期PI3K通路被显著激活,而TGF-β受体和Notch信号通路被抑制。由于抗凋亡基因(HSPA1A, ANXA1,CFL1,HMOX1)表达明显下调,促使缺氧早期凋亡发生;另有71个参与细胞周期调节的基因表达发生改变,使细胞周期进程受到影响;由于血管生成素样蛋白4(ANGPTL4)表达显著上调,促进了血管内皮细胞的血管发生潜能;由于ITGB1,ITGA6,THBS1,LAMB3,CD47,COL5A1,FNDC4,ITGA2,ITGA10黏附分子表达上调,促进ECM受体相互作用;由于脂代谢相关基因ANGPTL4表达明显上调,脂代谢加强,但ATP合成和转运却减少;虽然细胞对温度反应能力增强,但总体而言细胞对外界刺激反应减弱,使脐静脉内皮细胞对外界防御能力降低。3.荧光定量PCR结果表明EA.hy926细胞缺氧处理3 h后,ANGPTL4、HNRPDL mRNA表达上调;FTH1,HMOX1,CTGF mRNA表达下调,其变化趋势和SAGE文库中丰度变化情况一致。4.从SAGE文库中挑选的3个无匹配标签通过GLGI均得到不同程度延伸,经BLAST后与已知基因匹配,分别是微管蛋白β2C、核糖体蛋白SA、核糖体蛋白L41。5.据生物信息学分析对缺氧早期细胞增殖和凋亡改变的功能验证,证实缺氧早期此两项细胞功能的改变与文库生物信息学分析结果吻合。缺氧早期血管内皮细胞生长曲线左移,增殖指数于缺氧1 h即明显升高,持续到缺氧6 h,缺氧12 h较正常对照下降; VEGF和PCNA表达均于缺氧早期升高,提示缺氧早期促进细胞增殖。缺氧后3 h,12 h细胞凋亡发生率明显增高,提示缺氧早期促凋亡发生。6.缺氧后Integrinβ1启动子活性明显增强,其蛋白表达明显增加;Integrinβ1 RNA干扰可以明显下调Integrinβ1蛋白的表达,抑制EA.hy926细胞的增殖;缺氧处理后,,Integrinβ1下调细胞株和正常EA.hy926细胞增殖曲线均发生明显左移,但正常EA.hy926细胞生长曲线左移程度较Integrinβ1下调细胞株更大,两者差别具有统计学意义,提示缺氧早期通过上调Integrinβ1表达,促进EA.hy926细胞的增殖。三、结论1.采用Long SAGE技术成功构建了人脐静脉内皮细胞常氧和缺氧3 h Long SAGE文库,获得涵盖上万个转录本信息的表达谱数据,鉴定出112个差异表达基因(P<0.05)。2.缺氧早期以多种基因的激活或抑制方式,调节多种信号分子的表达,促进细胞增殖、脂代谢、血管生成,有利于启动血管内皮细胞自身保护性机制;同时细胞凋亡增加,细胞周期进程受影响,细胞防御能力降低;文库所筛选出的这些差异表达基因,为日后研究缺氧细胞保护性机制提供了思路和依据。3.对Long SAGE文库中5个差异表达基因,以及增殖和凋亡功能的验证结果与文库结果相符,说明文库标签丰度定量及后续生物信息学分析具有良好的置信度。3个无匹配标签3’EST延伸片段经BLAST后证实均为已知基因。4.缺氧早期通过激活Integrinβ1启动子促使其蛋白表达量增加,参与缺氧早期促脐静脉内皮细胞增殖效应。更多还原

【Abstract】 Background:The organ failure at early postburn stage is one of the most common and severe complications of burn patients, in which the hypoxia plays a key role in initiation of this pathological process. Due to the increase of vascular permeability immediately after burns, and the sharp decrease of blood volume and supply, the cells will subjected to hypoxia. This will cause the injury of cell structure and dysfunction of cell metabolism, and finally promote the development of systemic organ dysfunction.As a intima lining of the blood vessels, the vascular endothelial cells is one of the most oxygen sensitive cells and regulate a variety of functions, which has been identified to be pivotal in the onset of early organ dysfunction after burns. In common occasions, the vascular endothelial cells maintain a quiescent condition. But when subjected to extracellular stimulation, it is activated and then participated in various biological and pathological process. The endothelial activation may exhibit protective effects on the body through initiation of a series of cell defensive and adaptive mechanisms early after hypoxia, but it may turn to be distructive to cell function as time prolongs and the degree of hypoxia aggravates. So the research on the adaptive or pathological response of vascular endothelial cells and its mechanism after hypoxia is vital for the management of hypoxic injury after burns. As to now, most of reserches in this field focued on the study of the changes in certain special genes, but not on the regulation of the whole genomics, yet the mechanism of endothelial response is not fully understood.Because the cell function is determined by the changes in the gene expression, the study of genomic changes in vascular endothelial cells after hypoxia is an important and effective way for the research of mechanism of endothelial response after hypoxia, and the hooking of significant endogenous protective factors. Among so many genormic research methods, serial analysis of gene expression (SAGE) has many advantages, including high accuracy and reproducibility, ability to quantitate the differentially expressed genes, and so on. Long SAGE is more powerful for its improvement in the identification of gene specificity. So in this study, human cord vein endothelial cell model of hypoxia (3 h) was established based on the preliminary experiment, and normal EA.hy926 cells were enrolled as controls. Then we used Long SAGE technology to construct normoxic and hypoxic endothelial SAGE libraries, and make wide research on the changes and mechanism of endothelial response to short-term hypoxia through bioinformatic and special gene functional studies.Methods:1. To establish hypoxia model of EA.hy926 cells, and detect the changes in ICAM-1 and HIF-1αprotein expression before hypoxia and at 1,3,6,12 post-hypoxia hours(PHH).2. The cells were divided into normoxia and short-hypoxia(with hypoxic treatment for 3 hrs)groups. Total RNA from the 2 groups were extracted for the construction of Long SAGE libaries. The process is briefly described as follows: poly(A)-RNA was isolated using oligo(dT) magnetic beads and converted to cDNA. The resulting cDNA library was digested with NlaIII (anchoring enzyme), and divided into two populations following extensive washing. Each population was ligated with one of the two annealed linker pairs. The tags besides the NlaIII restriction site (CATG) of each transcript were released from the magnetic beads by cleavage with MmeI (tagging enzyme). The two tag populations were then blunted and ligated to produce ditags. The Ditags were amplified and isolated with PCR, and then self-ligated to produce long concatemers. After the resulting concatemers were ligated into the pZErO-1, the ligated products were transformed into One Shot- TOP10 electrocompetent Escherichia coli, seeded onto a low salt LB plate containing 50ug/ml zeocin, and cultured at 37℃overnight. White colonies were screened by PCR to select long inserts (>600 bp) for automated sequencing. SAGE tags of 17 bp were extracted, filtered, and tabulated using SAGE2000 software.3. Bioinformatics was used to analyze the Long SAGE libraries, including gross analysis of tags in SAGE libraries; Draw the gene expression maps according to the gene data of the 2 libraries; Application of Gominer platform to make classification of gene function categories, as well as gene enrichment analysis for the study of signaling pathway changes after hypoxia. 4. Realtime PCR was used to determine the changes in the expression of 5 selected differentially expressed genes , so as to check the reliability of SAGE libraries; Generation of longer cDNA fragments from SAGE tags for gene identification(GLGI) was used to prolong 3’EST fragment of 3 no-matched tags in SAGE libraries; At the same time, the protein expression of VEGF and PCNA by western blot, growth curve by cell counting, cell cycle by flowcytometry, as well as apoptosis by Annexin V methods were also determined to verify the endothelial functional changes in proliferation and apoptosis compared with the result in SAGE libaries.5. According to the bioinformatic analysis, we examined the changes in protein expression of Integrinβ1 after hypoxia with western blot. Then we determined the changes in the promoter activity of Integrinβ1 under hypoxic conditions 48 hrs after transfection of luciferase reporter plasmid containing full-length of Integrinβ1 promoter. Meanwhile, we constructed fluorescent Integrinβ1 siRNA plasmid, transfected it into EA.hy926 cells, and got stably expressed clones after G418 selection. The growth curves of the normal cells without hypoxia, normal cells with 3 hrs of hypoxia, si- Integrinβ1 cells without hypoxia, and si- Integrinβ1 cells with 3 hrs of hypoxia were drawn by cell counting for the study of the influence of Integrinβ1 on the proliferation of EA.hy926 cells under hypoxic conditions.Results:1. Totally 31213 tags, and 13665 unique tags were got from library of normoxia EA.hy926 cell line, while 30756 tags and 13879 unitags were got from hypoxia (3 h) library. The repeated Ditags in the 2 libraries were 762 and 758, respectively. There were 112 genes with obvious differential expression(P<0.05), among which 62 were evidently up-regulated, and other 50 were markedly down-regulated. In order to avoid the possibility of sequencing errors and to perform a valuable comparison between two libraries, the unique tags with a combination of 1:0, 0:1, or1:1 tags were removed. The remaining tags were designated as meaningful unique tags. These genes were used in our bioinformatic analysis.2. Bioinformatic analysis revealed that short-term hypoxia could promote ECM receptor interaction and lipid metabolism; It could also promote cell proliferation by down regulation of the expression of genes (CAV1,GJA1,HMOX1) which exhibit negative effect in regulation of proliferation. In addition, we found that PI3K-Akt,TGF-β, and Notch signaling pathway participated in the positive regulation of proliferation after hypoxia. We also found that short-term hypoxia promote apoptosis and angiogenesis, affect the cell cycle progress, and decrease the defence ability. Furthermore, we observed that HIF-1 tags was not in our SAGE library, indicating endothelial gene expression after short-term hypoxia was mainly modulated by HIF-independent way.3. Realtime PCR results showed that after 3 hrs of hypoxia, the mRNA expression of ANGPTL4, HNRPDL were up-regulated, while that of HMOX1, FTH1 and CTGF were down-regulated. The results were in concord with that of SAGE libraries.4. GLGI results showed that each selected tags was prolonged to some extent. BLAST showed that all of them were known genes.5. In the study of influence of hypoxia on the proliferation and apoptosis of endothelial cells, left move of the growth curve occurred after hypoxia. The proliferation index obviously increased at 1 PHH, maintained to 6 PHH, and decreased at 12 PHH. The expression of VEGF and PCNA increased at 1 PHH and 3 PHH, respectively. The VEGF expression decreased at 12 PHH, while PCNA still maintained at high level at this time-point. All these results indicated that short-term hypoxia could promote endothelial proliferation. Additionally, the occurance of apoptosis was significantly increased at 3 and 12 PHH, indicating pro-apoptotic effect of short-term hypoxia on endothelium.6. Hyoxia could markedly upregulate the protein expression of Integrinβ1, promote the luciferase reporter gene activity. After hypoxia, the growth curve of both cell lines turned to left, though the proliferation of siIntegrinβ1 cells was enhanced compared with that of normal cells, it was obviously decreased when compared with that of hypoxic Eahy926 cells, indicating that the increase of Integrinβ1 expression is benefitial for the EA.hy926 cell proliferation early after hypoxiaConclusion:1. We successfully constructed two Long SAGE libraries: One is about normoxia EA.hy926 cells, the other is about short-term hypoxic(3 hrs) EA.hy926 cells. Each library contains more than 10,000 transcripts, among which 112 were obvious differentially expressed genes(P<0.05).2. Short-term hypoxia promotes cell proliferation, lipid metabolism and angiogenesis, etc through regulation of various signaling pathways and genes. These effects are benefitial for the initiation of self-adaptive system. Meanwhile, short-term hypoxia also promotes apoptosis, affect cell cycle process and decreases the defence ability, which represents harmful effects to endothelial cells. The endothelial gene expression after hypoxia may be regulated through HIF-1 independent way. The differentially expressed genes aquired from our libraries provide direction and basis for the deep study in the future.3. The verification of SAGE libraries from single gene expression and special gene function show the good quality of our SAGE library and good reliability of our bioinformatic analysis.4. Integrinβ1 protein expression is up-regulated early after hypoxia through activation of its promoter, and which make it partly participate in the pro-proliferative effect early after hypoxia.更多还原