CN111643515A - 靶向hmga2基因的药物及应用 - Google Patents
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Abstract
本发明提供一种靶向HMGA2基因的药物，其特征在于，包括：沉默HMGA2基因的siRNA。本发明的靶向HMGA2基因的药物及应用，靶向HMGA2基因，有能够有效抑制血管新生。
Description
技术领域
本发明涉及一种靶向HMGA2基因的药物及应用，属于生物医学领域。
背景技术
血管在整个生命过程中都发挥着重要的作用，与胚胎发育、生殖、肿瘤的发生发展等多种生理病理过程都密切相关。血管形成主要包括两个阶段：血管发生和血管新生。血管发生是指在胚胎发育的早期，从中胚层分化出的血管内皮前体细胞形成新血管的过程；它是在胚胎发育过程中主要血管的形成方式。血管新生则是指在已经形成的血管基础上通过内皮细胞活化、增殖、迁移和延伸以“出芽”的方式形成新血管的过程。血管新生受到多种调节因子的共同调控，取决于调节内皮细胞增殖和迁移的促血管生成和抗血管生成分子共同维持血管稳态，包括VEGF、 FGF和DLL4等，但具体的调控机制并不清楚。血管新生与疾病之间有着重要的关系，血管生长异常会对人体健康造成影响并参与多种疾病发生过程，包括一些先天性疾病和遗传疾病都与血管异常有关。最为熟悉的诸如：癌症、动脉粥样硬化性疾病、血管瘤等。血管瘤(hemangioma) 是较常见的良性血管性肿瘤，是内皮细胞的真性肿瘤，其特征是血管内皮细胞异常增殖。目前常见将血管瘤分为良性、恶性及侵袭性血管瘤；其具体的发病机制至今尚未阐明，但其与血管生成因子及血管抑制因子间的调控失衡有关。
高迁移族蛋白A家族(High-mobility group A，HMGA)是一组染色质相关蛋白，包含4种蛋白：HMGA1a、HMGA1b、HMGA1c和 HMGA2，由109个氨基酸组成，在已发现的所有哺乳动物中具有高度同源性，其中包括一个相同的特征：拥有多个高度保守的AT-hooks(蛋白N-末端包含3个可以与DNA结合的基本短重复序列)，上面具有 2个磷酸化位点，此位点上的丝氨酸可被磷酸化从而影响其与DNA的亲和力，导致DNA弯曲、拉伸、成环或者解链，调节相关基因的表达，因此被称为“构架转录因子”。本课题组前期在完全性肺静脉异位引流 (TAPVC)患者外周血DNA的全外显子测序中的研究结果表明， HMGA2可能作为TAPVC致病基因。此外，qRT-PCR结果显示在 TAPVC患者肺静脉中其表达较对照组病人有显著的升高，在小鼠肺静脉和HUVECs中表达较高，提示其可能参与血管形成。HMGA2基因全长200kb，在人类基因位于12号染色体(12q14)，而在斑马鱼则位于4号染色体。HMGA2广泛表达于胚胎发育的过程中，而在成熟组织中不表达或者低表达。动物实验表明敲除HMGA2基因的小鼠生长发育迟缓，身材矮小，出现矮小症。HMGA2基因的表达在胚胎发育15 d后即被关闭，除肺和肾脏外，成年组织中几乎检测不到其表达。然而，在肿瘤组织或转化细胞中HMGA2的表达出现异常激活。HMGA2在恶性肿瘤中高表达，HMGA2经常在各种癌症中被重新激活，被认为是重要的致癌基因，参与肠癌、非小细胞肺癌等恶性肿瘤的发生。并且HMGA2高水平与癌症组织中转移和预后不良有关。基于HMGA2在胚胎期的高表达，有人研究了其与干细胞的关系，发现在胚胎期干细胞中HMGA2高表达，而随着年龄的增长，其表达逐渐下降。最近有研究发现，HMGA2可参与上皮-间质转化(Epithelial-mesenchymal transition,EMT)过程，与胚胎发育、伤口愈合及细胞迁移、转移有关。此外，有文献支持HMGA2参与正常心脏的发育，在心脏形成中发挥重要的作用。并且HMGA2作为Micro RNA 10A下游，具有调控内皮祖细胞的血管功能。但是，HMGA2是否可影响斑马鱼的血管发育及其调节血管新生的具体下游靶基因等均有待进一步深入研究。
血管瘤(hemangioma)是由胚胎期间成血管细胞增生而形成的常见于皮肤和软组织内的先天性良性肿瘤或血管畸形，多见于婴儿出生时或出生后不久。残余的胚胎成血管细胞，活跃的内皮样胚芽向邻近组织侵入，形成内皮样条索，经管化后与遗留下的血管相连而形成血管瘤。血管瘤可发生于全身各处，发生于口腔颌面部的血管瘤占全身血管瘤的60％，其次是躯干(25％)和四肢(15％)。其中大多数发生于颜面皮肤、皮下组织及口腔黏膜、如舌、唇、口底等组织，少数发生于颌骨内或深部组织。女性多见，男女比例约为1：3～1∶4。目前对于血管瘤的发生机制研究还未透彻，在治疗方面可供选择的治疗药物种类也非常有限。本发明旨在通过研究HMGA2在血管生成方面的作用，确证其是否参与血管瘤的发病，从而为血管瘤早期诊断及治疗提供理论指导。本发明结果表明HMGA2可以作为药物作用的靶基因在血管瘤或血管生成异常类的疾病中应用。
发明内容
本发明的目的在于提供靶向HMGA2基因的药物及应用，为治疗血管瘤提供新的选择。
本发明采用了如下技术方案：
本发明提供一种靶向HMGA2基因的药物，其特征在于，包括：沉默HMGA2基因的siRNA。
进一步，本发明的靶向HMGA2基因的药物，其特征在于：siRNA 通过慢病毒进行转染。
本发明还提供siHMGA2在制备调控IGFBP2的试剂中的应用。
本发明还提供siHMGA2在制备治疗血管瘤的药物中的应用。
本发明还提供siHMGA2在制备抑制肿瘤细胞血管形成的药物中的应用。
上述应用中，还包括在外周血淋巴细胞中检测HMGA2 mRNA表达水平的步骤。
发明的有益效果：本发明的靶向HMGA2基因的药物及应用，靶向 HMGA2基因，能够有效抑制血管新生。本发明对于针对HMGA2基因所作的药物开发工作也具有重要的指导意义。
附图说明
图1HMGA2在斑马鱼原位杂交图。
图1分别为WT型斑马鱼在24hpf、48hpf、72hpf原位杂交。A-E为侧面观，F、G为头部的俯视图，H为不同时间的mRNA相对表达量。
图2为HMGA2敲降影响斑马鱼头部中央动脉的血管生成结果。
图2A为正常对照组斑马鱼在明场下，白色方框表示右边放大的部位。B、E、H为对照组斑马鱼脑部血管；图2C、图2D、图2F、图2G、图2I、图2J为HMGA2-MO组，*表示P<0.05,**表示P<0.01，均为与对照组比较的结果，图2K为出现异常表型胚胎比例；图2L为每组中央动脉的条数统计，图2M为用蛋白印迹法检测HMGA2-Morpholinos的敲降效率的结果，图2N为实时荧光定量PCR(N)检测HMGA2-Morpholinos 的敲降效率。
图3为RNA-sequence测序分析结果。
图3A显示差异基因(P<0.05)；图3B显示GO富集分析显示与血管发育管径、心脏发育等相关的基因改变较显著(P<0.05)。图3C是实时荧光定量PCR验证结果，显示IGFBP2a,LMLN,TINAGL1,和ANO6在 HMGA2-Morpholinos组出现显著下调，*P<0.05。
图4为IGFBP2a回补HMGA2敲降导致的斑马鱼中央动脉异常的表型。
图4A-图4C为IGFBP2a原位杂交表达谱。对照组为图4D，图4E、图4F为两种Morpholinos显微注射组，图4G、图4H为Morpholinos和 IGFBP2a mRNA混合显微注射组，图4I为对有缺陷表型的胚胎进行定量统计的结果。**表示P<0.01，与对照组比较的结果；#表示P<0.05，分别与MO组相比较。
图5为HMGA2敲降抑制人脐静脉内皮细胞(HUVECs)迁移、成管，而IGFBP2可以回补这些表型。
图5A为HUVECs划痕后20小时的结果；图5B为将划痕后愈合面积进行定量统计的结果。图5C为基质胶上模拟小管生成实验。图5D为对管腔的长度的统计结果、图5E为对分支进行定量统计的结果。图中 **表示P<0.01，是与对照组比较；#表示P<0.05，是与siHMGA2组相比较。
图6为球体出芽实验和小鼠皮下基质胶栓塞实验结果。
图6A、B、C显示24h时三组细胞球体在胶原上出现的分支，图6J 为对分支的长度进行定量统计的结果。图6J中**表示P<0.01，与对照组比较的结果；#表示P<0.05，与siHMGA2组相比。将基质胶与内皮细胞混悬液用注射器打在小鼠皮下，12天后取出胶栓。图6D-F为对照组细胞，图6G-I为HMGA2过表达组细胞。图6D、G为体式显微镜下大体图；图6E、H为胶栓的HE切片染色；图6F、I为抗CD31的免疫组化。图6K为对单位视野出现血管的个数进行定量统计的结果。**表示P<0.01，与对照组比较的结果。
图7为HMGA2调控IGFBP2的表达。
图7A显示HMGA2在蛋白水平调控IGFBP2；图7B为IGFBP2启动子区保守序列示意图；图7C为mRNA水平调控IGFBP2；图7D荧光素酶报告基因实验显示HMGA2可以激活IGFBP2的转录；图7E为ChIP 实验显示HMGA2直接结合在IGFBP2启动子区，调控IGFBP2；图7F、图7G为HMGA2和IGFBP2分别在血管瘤病人外周血mRNA表达水平 (n＝18)；图7H为HMGA2通过调控IGFBP2参与血管新生过程。*表示 P<0.05，**表示P<0.01，与对照组比较的结果。
具体实施方式
以下通过具体实施来进一步说明本发明的技术方案。
以下实施方式中未具体描述的方法，均按照《生物化学实验技术》和《分子生物学实验技术》所记载的方法或者在商用试剂供应商所建议的实验操作下进行。
实验材料与方法
1.动物
实验动物使用TU系斑马鱼和Tg(fli1a:EGFP)转基因斑马鱼，喂养及杂交方式严格按照Westerfield编著的《实验室斑马鱼实验指南》进行，饲养于长海医院斑马鱼饲育室。所有的小鼠都是5-7周的C57BL/6 野生型，饲养于上海交通大学医学院附属新华医院动物房。
2.斑马鱼胚胎获取
繁育收胚胎前一晚将斑马鱼成鱼按雌雄比为2:1放入杂交缸，次日早晨亮灯后拔去挡板，交配后收集胚胎，于28.5℃系统水中进行培养。分别在24hpf、36hpf和48hpf收集胚胎，然后在4℃条件下，4％多聚甲醛固定过夜，PBST洗脱后甲醇梯度脱水，-20℃存储备用。
3.原位杂交(详细步骤)
斑马鱼胚胎整体原位杂交实验步骤如下：
第一天：
复水：从-20℃中拿出保存的胚胎，直接用PBST润洗3次，每次5min。
蛋白酶K消化：根据胚胎发育时期的不同，蛋白酶K消化的时间也有区别：24hpf胚胎，用10μg/ml蛋白酶K消化1min；48hpf胚胎，用10μg/ml 蛋白酶K消化2min，依此类推。再固定：蛋白酶K消化的胚胎用4％多聚甲醛室温下再固定1h,PBST室温洗3次，每次5min。
预杂交：去掉上述的PBST，300μl杂交缓冲液预润胚胎后，去除上述杂交缓冲液，加入300μl新杂交缓冲液，68℃预杂交2-4小时。
杂交：移去预杂交液，换成300μl杂交液，68℃过夜。
预杂交液mix配制方法(HM):(保存在-20℃)
第二天：
回收探针后，按以下步骤操作：
1)100％HMS at 68℃,快速洗涤；
2)66％HMS/33％2 x SSC at 68℃,20min；
3)33HMS/66％2 x SSC at 68℃,20min；
4)2x SSC at 68℃,20min；
5)0.1x SSC at 68℃,40min；
6)0.05x SSC at 68℃,40min。弃液，冷却至室温；
7)66％0.05x SSC/33％PBS磷酸缓冲盐溶液室温洗涤,10min；
8)33％0.05x SSC/66％PBS磷酸缓冲盐溶液室温洗涤,10min；
9)PBS磷酸缓冲盐溶液室温洗涤,10min；
10)封闭：加入适量的封闭液，室温孵育2h；
11)抗体孵育：弃去封闭液，加入用封闭液以1：4000稀释的碱性磷酸酶标记的抗地高辛抗体(Anti-Digoxigenin Fab-alkaline phosphatase)，4℃孵育过夜。
第三天：
1)漂洗：用PBST洗涤胚胎6次，每次20min；
2)平衡：用原位杂交显色液洗涤胚胎3次，每次5min；
3)显色:原位杂交显色液中加入NBT/BCIP；
4)配制成染色液，在避光情况下进行显色反应。每30min在体视显微镜下观察。观察发现染色完成后，弃染色液，PBST洗涤数次后用 4％多聚甲醛再固定，拍照。
4.斑马鱼显微注射Morpholinos
(1)反义寡核苷酸的设计：根据NCBI报道的基因序列，由 GeneTools公司设计并合成了HMGA2基因的两种MO，分别抑制转录和剪接。从GeneTools公司购买的standard MO作为对照。一般将购买来的MO粉末用无菌水溶解为2mM的储存液，-20℃保存备用，使用时稀释至适当浓度既可。
(2)显微注射：用2％的琼脂糖制成用于固定斑马鱼胚胎的槽型结构，将准备好的注射液，用移液器吸至注射针的顶部，注射针自带有毛细管，注射液会自动下下流到底部。调整显微注射设备，在解剖镜下将封闭的毛细管从头部适当的位置用镊子掐断，调整好注射压和平衡压，试打几下，到针头处出现合适大小液滴为止。收集胚胎，解剖镜下观察发育时期，在1-2细胞期均可进行注射。把胚胎排列于注射槽中。注射时，保证每枚胚胎得到的溶液量在1nl左右。注射完成的胚胎28.5℃培养。实时观察胚胎的表型变化并拍照。
5.RNA-Sequence
提取样品总RNA进行转录组测序，并使用DNase消化DNA后，用带有Oligo(dT)的磁珠富集真核生物mRNA；加入打断试剂将mRNA 打断成短片段，以打断后的mRNA为模板,用六碱基随机引物合成一链 cDNA，然后配制二链成反应体系合成二链cDNA，并使用试剂盒纯化双链cDNA；纯化的双链cDNA再进行末端修复、加A尾并连接测序接头，然后进行片段大小选择，最后进行PCR扩增；构建好的文库用Agilent 2100 Bioanalyzer质检合格后，使用Illumina HiSeqTM 2500或Illumina HiSeq X Ten等测序仪进行测序，产生125bp或150bp的的双端数据。质检合格后，使用Illumina测序仪进行测序。
6.IGFBP2 mRNA的合成和显微注射
选择在pCS2+载体上正向插入的克隆，提取质粒后用合适的内切酶线性化，线性化质粒纯化后直接作为体外合成mRNA的模板。mRNA 的合成用的是mMessagemMachine kit(Ambion)试剂盒，反应体系完全按照说明书推荐的添加，如下所示：10×Buffer 2ul,ATP、GTP、CTP、UTP 各1ul,sp6酶2ul，DNA模板1ug,depc水补足至20ul，37℃培养2小时。然后加入Add 1μL TURBO DNase 1充分混匀，37℃消化剩余模板15分钟。用醋酸铵纯化试剂盒纯化。电泳，观察条带。将IGFBP2 mRNA与 HMGA2-Morpholinos混合一起在斑马鱼1-2个胚胎阶段行显微注射。
7.内皮细胞球体出芽实验步骤:
参考文章(Ying Wang.et al，Journal of Visualized Experiments，2018， 12；(134))。将细胞接种在U型底的96孔板上培养3天，待聚集成细胞球体，将球体接种在铺有鼠尾胶原培养基的普通96孔板上，添加生长因子，37℃，5％CO2培养箱，观察12-24h细胞球体出芽的个数和长度，倒置显微镜下拍照计数。
8.HMGA2对人脐静脉内皮细胞(HUVECs)迁移及成管的影响
(1)慢病毒转染HUVECs：用Scramble、sh-HMGA2病毒液分别转染内皮细胞，48h后观察荧光强度，用嘌呤霉素筛选，构建敲降HMGA2 稳转株。
(2)细胞迁移实验：铺板之前先用marker笔在6孔板背后，用直尺比着，均匀得划横线，大约每隔0.5～1cm一道，横穿过孔。每孔至少穿过5条线。每孔铺约5×105个细胞，掌握过夜能铺满。第二天用枪头比着直尺，尽量垂至于背后的横线划痕，枪头要垂直，不能倾斜。用PBS 洗细胞3次，去处划下的细胞，加入无血清培养基。放入37度5％CO2培养箱，培养。按12，24小时取样，拍照。
(3)体外血管生成实验：提前将Matrigel(BD)置于冰盒中，使胶能过夜缓慢融化。取出预冷的ibidi血管生成载玻片，每孔中加入10μl Matrigel，放入4℃冰箱平衡10min。将培养皿放入培养箱中，静置30 分钟左右，等待胶凝结。等待同时准备细胞悬液，充分混匀。取出已凝固的Matrigel，每孔加入50μl的细胞悬液，盖上盖，静置放回培养箱，观察4h、8h血管生成的情况。
9.小鼠皮下成管试验体内验证HMGA2对血管生成的影响
将基质胶与生长因子(VEGF)、肝素及各种稳转株细胞混匀后注射植入小鼠皮下，7d后取出基质胶栓，显微镜下计算微血管密度(MVD)。随后进HE染色和免疫组织化学染色，观察血管生成的效果，Tukey检验法进行定量分析，比较其MVD的差异。具体方法参考Shen等(Shen et al.,Circulation Research,2016,118(8):1194-1207)。
10.HMGA2在婴幼儿血管瘤外周血中表达量
收集18例确诊为婴幼儿血管瘤患者外周血，同时收集18例同等年龄段的非婴幼儿血管瘤患者外周血作为正常对照。分离外周血淋巴细胞培养，提取RNA，用qPCR检测HMGA2表达含量。
11.数据分析
结果用平均值±标准误差(SEM)表示。采用16.0版本SPSS软件对数据进行统计学分析,用t检验或方差分析。分析中小于0.05的P值认为有统计学意义。
结果
1.HMGA2在斑马鱼胚胎发育过程中血管部位表达。
图1分别为WT型斑马鱼在24hpf、48hpf、72hpf原位杂交。A、C 为侧面观，B、D为头部的俯视图。在24hpf(A)，HMGA2主要表达在头部，及躯干的背动脉，后主静脉部位。在48，72hpf时，在中央动脉(CtA)、基底动脉(BA)有较明显的分布。(H)为各个时间点HMGA2 表达量的定量统计。箭头指示中央动脉有明显的信号。实时荧光定量PCR 结果显示HMGA2在不同时间节点的mRNA定量。说明HMGA2可能参与斑马鱼血管的发育。
2.HMGA2敲降影响斑马鱼头部中央动脉的血管生成。
用HMGA2-Morpholinos，敲降Tg(fli1a:EGFP)品系斑马鱼，观察在胚胎发育过程中是否出现异常的表型。如图2，HMGA2-MO组较对照组相比，大体表型正常；图2A为正常对照组斑马鱼在明场下，白色方框表示右边放大的部位。图2B、E、H为对照组斑马鱼脑部血管；HMGA2-MO组(图2C、D、F、G、I、J)较对照组相比，在48hpf时，斑马鱼头部中央动脉出现明显的出芽减少及中央动脉尚未完全和基底动脉相连。红色线条指示中央动脉部位，白色箭头指示中央动脉的对数。 K为出现异常表型胚胎比例；L为每组中央动脉的条数统计。用蛋白印迹法(M)和实时荧光定量PCR(N)检测HMGA2-Morpholinos的敲降效率。*表示P<0.05,**表示P<0.01，均为与对照组比较的结果。每个试验都进行三次生物重复试验。
3.RNA-sequence测序分析发现IGFBP2作为HMGA2的潜在调控靶点。
如图3，收集48hpf HMGA2-Morpholinos组VS对照组斑马鱼的胚胎做RNA-seq，GO富集分析显示与血管发育管径、心脏发育等相关的基因改变较显著。收集48hpf HMGA2-Morpholinos组和对照组斑马鱼的胚胎做RNA-seq，图3A显示差异基因(P<0.05)；图3B，GO富集分析显示与血管发育管径、心脏发育等相关的基因改变较显著(P<0.05)。实时荧光定量PCR验证，图3C结果显示，IGFBP2a,LMLN,TINAGL1,和 ANO6在HMGA2-Morpholinos组出现显著下调，每个试验都进行三次生物重复试验。*P<0.05。我们挑选了一些与血管发育相关的基因，并且用实时荧光定量PCR验证，结果显示，IGFBP2a,LMLN,TINAGL1,和ANO6 在HMGA2-Morpholinos组出现显著下调。
4.IGFBP2a回补HMGA2敲降对斑马鱼中央动脉表型的影响。
如图4，IGFBP2a在斑马鱼原位杂交结果显示，其在48hpf时在脑部有表达。合成的IGFBP2a的mRNA可以部分回补HMGA2敲降引起的表型。图4A-图4C为IGFBP2a原位杂交表达谱。对照组为图4D，图 4E、图4F为两种Morpholinos显微注射组，图4G、图4H为Morpholinos和IGFBP2a mRNA混合显微注射组，图4I为对有缺陷表型的胚胎进行定量统计的结果。**表示P<0.01，与对照组比较的结果；#表示P<0.05，分别与MO组相,。每个试验都进行三次生物重复试验。
5.HMGA2敲降抑制人脐静脉内皮细胞(HUVECs)迁移、成管，而 IGFBP2可以回补这些表型。
如图5，通过HMGA2敲降的慢病毒(siHMGA2)转染HUVECs，构建si HMGA2稳转株。图5A为HUVECs划痕后20小时的结果；图 5B为将划痕后愈合面积进行定量统计的结果。图5C为基质胶上模拟小管生成实验。图5D为对管腔的长度的统计结果、图5E为对分支进行定量统计的结果。图中**表示P<0.01，是与对照组比较；#表示P<0.05，是与si HMGA2组相比较。每个试验都进行三次生物重复试验。划痕实验显示si HMGA2抑制HUVECs的迁移，这种迁移能力可以被外源性人重组蛋白IGFBP2(rh IGFBP2)回补；基质胶上模拟血管生成实验表明，si HMGA2抑制小管形成，并且也可以被外源性人重组蛋白rh IGFBP2 回补。
6.球体出芽实验和小鼠皮下基质胶栓塞实验。
si HMGA2抑制内皮细胞球的出芽，这种出芽的能力可以被外源性人重组蛋白IGFBP2(rh IGFBP2)回补。在构建过表达HMGA2的 HUVECs，将细胞与基质胶混悬液注射小鼠皮下形成栓子，2周后栓子取出后发现HMGA2过表达组栓子里长入较多的毛细血管。如图6，图6A、B、C显示24h时三组细胞球体在胶原上出现的分支，图6J为对分支的长度进行定量统计的结果。**表示P<0.01，与对照组比较的结果； #表示p<0.05，与si HMGA2组相比。将基质胶与内皮细胞混悬液用注射器打在小鼠皮下，12天后取出胶栓。图6D-F为对照组细胞，图6G-I为 HMGA2过表达组细胞。图6D、G为体式显微镜下大体图；图6E、H 为胶栓的HE切片染色；图6F、I为抗CD31的免疫组化。图6K为对单位视野出现血管的个数进行定量统计的结果。**表示P<0.01，与对照组比较的结果。每个试验都进行三次生物重复试验。
因而，我们得出结论，HMGA2可以促进血管的新生，因此可以作为制备抑制血管异常生成的药物靶点。
7.HMGA2调控IGFBP2的表达。
通过Western blotting检测HMGA2过表达时分别在mRNA和蛋白水平提高IGFBP2表达。如图7，其中，图7A显示HMGA2在蛋白水平调控IGFBP2；图7B为IGFBP2启动子区保守序列示意图；图7C为mRNA 水平调控IGFBP2；图7D荧光素酶报告基因实验显示HMGA2可以激活IGFBP2的转录；图7E为ChIP实验显示HMGA2直接结合在IGFBP2 启动子区，调控IGFBP2；图7F、图7G为HMGA2和IGFBP2分别在血管瘤病人外周血mRNA表达水平(n＝18)；图7H为HMGA2通过调控 IGFBP2参与血管新生过程。*表示P<0.05，**表示P<0.01，与对照组比较的结果。每个试验都进行三次生物重复试验。荧光素酶报告基因测定， HMGA2表达增加而导致IGFBP2启动子活性改变。使用ChIP实验结果显示HMGA2可以直接结合IGFBP2启动子。在血管瘤患者外周血淋巴细胞中检测HMGA2，IGFBP2的mRNA表达水平，较对照病人相比，均显著上调。
因而，HMGA2可能参与血管瘤的发病。本发明从斑马鱼、细胞及人群样本全面研究HMGA2影响血管生成的具体过程及其分子机制，初步阐明其在血管瘤中的作用，为血管瘤早期诊断及治疗提供理论指导。
序列表
<110> 上海交通大学医学院附属新华医院
<120> 靶向HMGA2基因的药物及应用
<130> JSP12000131
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial
<220>
Claims (6)
1.一种靶向HMGA2基因的药物，其特征在于，包括：
沉默HMGA2基因的siRNA，序列为：agGAGGAAACTGAAGAGACAT。
2.如权利要求1所述的靶向HMGA2基因的药物，其特征在于：siRNA通过慢病毒进行转染。
3.siHMGA2在制备调控IGFBP2的试剂中的应用。
4.siHMGA2在制备治疗血管瘤的药物中的应用。
5.siHMGA2在制备抑制肿瘤细胞血管形成的药物中的应用。
6.如权利要求3-6中所述的应用，其特征在于，还包括：
在外周血淋巴细胞中检测HMGA2 mRNA表达水平的步骤。
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| CN102174514A (zh) * | 2010-11-24 | 2011-09-07 | 华侨大学 | 一种抑制Hmga2基因表达的双链siRNA及其应用 |
| US20190093106A1 (en) * | 2015-10-06 | 2019-03-28 | University Of Virginia Patent Foundation | Compositions and methods for treating diabetic retinopathy |
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| CN102174514A (zh) * | 2010-11-24 | 2011-09-07 | 华侨大学 | 一种抑制Hmga2基因表达的双链siRNA及其应用 |
| US20190093106A1 (en) * | 2015-10-06 | 2019-03-28 | University Of Virginia Patent Foundation | Compositions and methods for treating diabetic retinopathy |
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