CN111487398B - 血管瘤治疗的生物标记物 - Google Patents
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Abstract
本发明提供了血管瘤治疗的生物标记物，具体地，提供了使用生物因子的表达水平对伊曲康唑治疗婴幼儿血管瘤疗效进行有效评估和预测的方法和试剂盒，所述生物因子为VEGF‑A、PDGF‑A、PDGF‑D、HES、HEY1、HIF3A、IL‑8、TNF‑A或FGF中的一种或几种。对婴幼儿血管瘤疗效判定、预后评估和用药疗程指导有重要意义。
Description
技术领域
本发明涉及血管瘤治疗的生物标记物，具体地，涉及使用血液中生物因子作为标记物对伊曲康唑治疗血管瘤疗效进行有效评估和预测的方法和试剂盒，属于生物医药领域。
背景技术
血管瘤是婴幼儿最常见的皮肤良性肿瘤，其发生率为8％-10％。好发于头面部和四肢，可累及身体任何部位，在1岁以内快速增殖，随后逐渐进入自行消退期，持续时间长达5-9年。尽管多数血管瘤有自行消退的病理生理过程，但常遗留色素沉着、毛细血管扩张、纤维和脂肪组织沉积。10％-20％患儿瘤体随年龄增长而增大，甚至出现如溃疡、出血、感染等严重并发症，导致容貌损毁、功能丧失等后果：面部中央、气道、皮肤皱褶、***和肛周等容易摩擦或受汗液、尿液、粪便浸渍区域一旦溃疡形成后很难自愈；位于眼睑、结膜的血管瘤可影响视力导致视弱、散光及致盲；位于呼吸道则会导致呼吸障碍，影响心肺功能；部分血管瘤可导致Kasabach-Merritt综合征(卡萨巴赫-梅里特综合征)、充血性心力衰竭等而危及生命；皮肤血管瘤严重影响形象，导致患儿出现自卑、内向、自闭、极端性格等一系列心理障碍。
治疗血管瘤的有创治疗技术涉及到包括手术切除、染料脉冲激光、介入、冷冻、电灼等，因对手术医师临床技能、手术条件、仪器设备、患儿及家属的依从性、费用和方便程度要求不一，且容易继发感染、遗留瘢痕，具有一定副作用或局限性。无创治疗包括口服和外用药物，口服药物主要有糖皮质激素、α干扰素、长春新碱、***、伊曲康唑等，外用药物主要有噻吗洛尔、卡替洛尔等β受体阻滞剂。无创治疗优点在于减少了继发感染、遗留瘢痕的几率，操作简单，价格相对便宜，患儿及家属依从性高。但存在治疗时间长，药物本身副作用等等问题。申请人在临床接诊过程中发现，多数患儿家属更愿意从一开始先选择口服或者外用药物这些无创方法治疗血管瘤。一些特殊部位例如眼睑、鼻腔内、外阴、肛周部位的血管瘤，手术或外用药物均操作困难，只能选择口服药物。
长期使用口服药物所产生的副作用，例如对肝肾功能、心功能的影响是家属主要担心的问题。若顾及药物副作用，过早停药，可能导致皮损复发和反弹。因此，找到一些与血管瘤消退相关的生物标记因子，根据生物标记来评估治疗有效或无效，何时停药，是医生、家属都迫切关注的问题，目前尚无此类生物标记报道。
发明内容
本发明提供了一种评估、预测或优化伊曲康唑对血管瘤治疗有效性的方法，包括步骤：
a)获取来自血管瘤病人的测试样品，检测所述测试样品中生物因子的表达水平；
b)所述血管瘤病人接受有效剂量的伊曲康唑的治疗；
c)比较治疗前后，所述生物因子的表达水平的变化，当所述生物因子的表达水平上调或者下调时，达到治疗效果。
所述测试样品为正常组织、肿瘤组织、细胞系、血浆、血清、全血、脑脊髓液、淋巴液、循环肿瘤细胞、细胞裂解物、组织裂解物、尿和/或抽吸物，优选地，为血清或肿瘤组织；所述生物因子为VEGF-A、PDGF-A、PDGF-D、HES、HEY1、HIF3A、IL-8、TNF-A或FGF中的一种或几种。
在一些实施例中，所述生物因子的表达水平为相应生物因子在所述测试样品中的mRNA表达水平。
在一些优选例中，当治疗后PDGF-A、VEGF-A、HES、HEY1、IL-8或TNF-A中的一种或多种的mRNA表达水平较治疗前上调差异倍数大于或等于2、3、5、8、10或20时，更优地，上调差异倍数大于或等于10或20时，达到治疗效果；在另一些优选例中，当治疗后PDGF-D、HIF3A或FGF中的一种或多种的mRNA表达水平较治疗前下调差异倍数大于或等于2、3、5、8、10或20时，较优地，下调差异倍数大于或等于10或20时，达到治疗效果；较优地，所述生物因子为PDGF-A、PDGF-D或FGF中的一种或几种；更优地，所述生物因子为PDGF-D。
在另外一些实施例中，所述生物因子的表达水平为相应生物因子在测试样品中的蛋白水平，优选地，当治疗前后相应生物因子的蛋白水平下降比例大于或等于10％、30％、50％或70％时，较优地，下降比例大于或等于50％或70％时，达到治疗效果。更优地，所述生物因子为PDGF-D或PDGF-A。
在一些实施例中，所述血管瘤病人为婴幼儿。
在一些实施例中，所述血管瘤为增殖期血管瘤。
本发明还提供了一种检测试剂盒，包含用于测量受试者测试样品中生物因子的mRNA表达水平或蛋白水平的试剂，其特征在于，所述测试样品为正常组织、肿瘤组织、细胞系、血浆、血清、全血、脑脊髓液、淋巴液、循环肿瘤细胞、细胞裂解物、组织裂解物、尿和/或抽吸物，优选地，为血清或肿瘤组织，所述生物因子为VEGF-A、PDGF-A、PDGF-D、HES、HEY1、HIF3A、IL-8、TNF-A或FGF中的一种或几种。
在一些实施例中，所述检测试剂盒还包含用于选自下述的目的的说明书：通过所述生物因子表达水平或其变化测定血管瘤病人是否将响应伊曲康唑治疗，通过所述生物因子表达水平或其变化监控伊曲康唑治疗的血管瘤病人的疾病进展，通过所述生物因子表达水平或其变化证实在施用伊曲康唑的病人中伊曲康唑的生物活性，或其组合。
本发明还提供了所述检测试剂盒用于评估、预测或优化伊曲康唑对血管瘤病人治疗有效性的用途。
在一些实施例中，所述生物因子的表达水平为相应生物因子的mRNA表达水平；优选地，当治疗后PDGF-A、VEGF-A、HES、HEY1、IL-8或TNF-A中的一种或多种的mRNA表达水平较治疗前上调差异倍数大于或等于2、3、5、8、10或20时，更优地，上调差异倍数大于或等于10或20时，达到治疗效果；优选地，当治疗后PDGF-D、HIF3A或FGF中的一种或多种的mRNA表达水平较治疗前下调差异倍数大于或等于2、3、5、8、10或20时，更优地，下调差异倍数大于或等于10或20时，达到治疗效果；更优地，所述生物因子为PDGF-D、HIF3A或FGF中的一种或几种；更优地，所述生物因子为PDGF-D。
在另一些实施例中，所述生物因子的表达水平为相应生物因子的蛋白水平；优选地，当治疗后相应生物因子的蛋白水平较治疗前下降比例大于或等于10％、30％、50％或70％时，达到治疗效果；更优地，所述生物因子为PDGF-A或PDGF-D。
在一些实施例中，所述血管瘤为增殖期血管瘤。
在一些优选实施例中，所述血管瘤病人为婴幼儿。
本发明的有益效果是：目前尚无任何公开报道使用生物因子对婴幼儿血管瘤治疗疗效进行有效评估和预测的生物标记。而本发明所述的生物标记对婴幼儿血管瘤疗效判定、预后评估和用药疗程指导有重要意义。
附图说明：
图1治疗前后婴幼儿血清中VEGF与PDGF-AA水平变化趋势。
图2用伊曲康唑处理的婴儿血管瘤的mRNA表达谱及显著变化的前20个相关生物过程、细胞组分、分子功能及途径。(a)用伊曲康唑(10μM)和DMSO对照处理的来自婴儿血管瘤内皮细胞(HemEC)之间的mRNA表达水平的散点图。红点表示上调的mRNA，绿点表示下调的mRNA(倍数变化>2.0)。(b)分层聚类显示伊曲康唑和对照之间可区分的mRNA表达模式。在HemEC中，在伊曲康唑处理后，总共172个mRNA被上调(倍数变化>2.0)，819个mRNA被下调(倍数变化>2.0)。与伊曲康唑治疗的HemEC中差异表达显著的mRNA对应的，前20个生物过程的基因本体论术语(c)，前20个细胞组分的基因本体论术语(d)，前20个分子功能的基因本体论术语(e)，和前20个途径术语(f)。
图3婴儿血管瘤中的PI3K/Akt/mTOR信号传导在体外被伊曲康唑抑制。(a)通过逆转录PCR检测，用10μM伊曲康唑处理48小时的HemEC中PDGF-D mRNA的表达。(b)通过免疫印迹检测得到的，用10μM伊曲康唑处理指定时间的HemEC中PDGF-D蛋白的表达。(c)在用10μM伊曲康唑分别处理2小时，24小时和48小时的HemEC中检查p-Akt，p-p70S6K，p-4E-BP1和T-Akt的磷酸化水平。(d)用0.3μM或1μM伊曲康唑处理2小时和24小时的小鼠血管瘤内皮细胞(EOMA)中的p-Akt，p-p70S6K，p-4E-BP1和β-肌动蛋白水平。
图4伊曲康唑和PDGFR-β抑制剂CP-673451对婴儿血管瘤HemECs的影响的比较。(a)HemECs用指定浓度的伊曲康唑或CP-673451处理72小时，然后通过MTS测定法测定细胞活力。(b)用3μM伊曲康唑和5μM CP-673451处理48小时后，对HemEC进行Annexin V/PI染色。(c)通过管形式测定，10μM伊曲康唑和10μM CP-673451对HemEC的作用。比例尺＝0.1mm。(d)通过蛋白质印迹检测得到的，用10μM伊曲康唑或10μM CP-673451处理48小时的HemEC中p-PDGFR-β，p-Akt，p-p70S6K，p-4E-BP1，PDGFR-β和T-Akt的磷酸化水平。
具体实施方式：
本发明的方法与技术通常依据本领域已知的传统方法进行，除非另有说明。与本文中描述的生物学、药理学、及医学与医药化学相关的命名法，及实验方法与技术是本领域已知且常用的。化学合成法、化学分析法、医药制法、调配法与传送法，及检测或测试法均采用标准技术。
除非另有说明，否则本文中所使用的科学与技术术语应具有那些本领域普通技术人员通常理解的含义。但下列术语具有如下定义：
术语“有效剂量”，通常也称为“治疗有效剂量”，是指药物的任何如下所述的量，当单独使用或与另一种治疗剂组合使用该量的药物时，可促进疾病消退，疾病消退表现为疾病症状的严重度降低、无疾病症状期的频率和持续时间增加、或者防止由患病导致的障碍或失能。本发明药物的“有效剂量”也包括“预防有效剂量”，“预防有效剂量”是药物的任何如下所述的量，当将该量的药物单独施用或者与另一种治疗剂组合施用于具有发生疾病的风险或者遭受疾病复发的病人时，可抑制疾病的发生或复发。
如对本领域所属技术人员显而易见地，有效的体内给药剂量及具体的给药方式会根据所治疗的哺乳动物种类、体重和年龄，所使用的具体化合物及使用这些化合物的具体目的而变化。本领域所属技术人员根据常规的药理学方法可确定有效剂量水平(即达到所需效果所必需的剂量水平)。通常，产物的人体临床应用从较低的剂量水平开始，随后不断提高剂量水平直到达到所需的效果。可选择地，可通过现有的药理学方法采用可接受的体外研究来建立本方法鉴定的组合物的有用剂量和给药途径。
所述“药物”，是本领域认可的，作为生物、生理或药理活性物质的任何化学物质。药物也被称为“治疗剂”，其实例在已知参考文献中有描述(如Merck Index、PhysiciansDesk Reference和The Pharmacological Basis of therapeutics)，并且其包括(但不限于)药物、维生素、矿物质补充剂、用于治疗、预防、诊断、治愈或缓解疾病或病痛的物质、影响身体结构或功能的物质或前药，当将其置于生理环境后具有生物活性或活性更强。可使用各种形式的治疗药物，其中当给予受试者之后，组合物就能够从受试者中释放至邻近组织或流体中。本发明中的药物指伊曲康唑，包括以伊曲康唑为有效成分的各种药物制剂或药物组合物。
术语“血管瘤(hemangioma)”是指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，瘤内血管自成***，不与周围血管相连。血管瘤可发生于全身各处，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。血管瘤的血管内皮细胞具有增殖特性，自然病程可分为增生期、稳定期和消退期。根据组织学结构与临床表现，血管瘤又可分为毛细血管瘤、海绵状血管瘤、混合型血管瘤和蔓状血管瘤。
本发明所述生物因子的代码及其中英文名称对照见表1。
表1生物因子代码及其中英文名称对照表
术语“PDGF”，即血小板衍生生长因子，是一种生长因子，可以调控细胞的生长和分化，且在血管新生上扮演重要角色，未控制的血管新生常常导致癌症。存在由四个基因编码的四个已知的PDGF蛋白，PDGF-A，PDGF-B，PDGF-C和PDGF-D。PDGF由离散的细胞群体产生，并以二硫键连接的同二聚体或异二聚体形式分泌，包括PDGF-AA，PDGF-BB，PDGF-CC，PDGF-DD和PDGF-AB。PDGF二聚体通过结合PDGF受体主要以旁分泌的方式起作用。有两种已知的PDGF受体，PDGFR-α和PDGFR-β，它们可以同时形成异二聚体和同二聚体。配体结合促进受体二聚化，自磷酸化和激活下游的多个细胞内信号级联反应，从而刺激肌动蛋白丝重排，间隙连接通讯的破坏，基因转录的起始和细胞存活。
所述“测试样品”可以是从病人或受试者身体分离的任何生物材料，例如正常组织、肿瘤组织、细胞系、血浆、血清、全血、脑脊髓液、淋巴液、循环肿瘤细胞、细胞裂解物、组织裂解物、尿和抽吸物；优选地，测试样品源自正常组织、肿瘤组织、细胞系、循环肿瘤细胞、血清、血浆或全血；更优选地，测试样品源自血清、肿瘤组织。用于取出样品的方法是本领域众所周知的，并且它可以例如通过活组织检查从受试者中取出，例如通过穿刺活检、空芯针穿刺活检或细针抽吸活检、内窥镜活检或表面活检；或者通过静脉穿刺，收集全血、血浆或血清样品，并且根据标准技术进一步加工。从病人或受试者中获得测试样品后，对样品实施涵盖测量生物因子表达水平的方法步骤。在本发明中，生物因子表达水平主要指mRNA表达水平和蛋白水平。
实施例1伊曲康唑治疗婴幼儿血管瘤临床疗效观察及伊曲康唑对血管瘤患儿血清中VEGF及PDGF-AA水平的影响
门诊收集婴幼儿血管瘤患者17例，由患儿父母或法定监护人签署知情同意书后予以口服伊曲康唑5mg/kg/d，疗程2周至12周。从治疗起每4周记录其皮损变化情况(瘤体面积、颜色变平、变淡等)，从临床角度判断和评价治疗效果。采集血管瘤患儿服药前、服药4周、服药12周的血液样本17例，通过ELISA法检测血清中VEGF、PDGF-AA水平的变化，并进行定量分析。用SPSS软件对统计数据分析。研究结果参见图1。
本研究共纳入17名患儿，男6例，女11例，年龄1-12月(平均5.4月)，体重5-10.5kg，治疗前收集样本17个，治疗后样本17个，治疗前后间隔时间为天13-56天(平均31天)，内服伊曲康唑总剂量390-2240mg(平均1076mg)。总共随访时间为0.5-6月不等。这17例中，有12例为增殖期婴幼儿血管瘤，其中7例增殖期血管瘤在随访期间呈消退趋势，消退率为58.3％(7/12)。
(1)治疗前后血清VEGF(血管内皮细胞生长因子)检测(图1A)。
治疗前患儿血清VEGF浓度为214.82±135.58pg/mL，治疗后为390.81±649.18pg/mL，治疗后比治疗前上升175.98±551.17pg/mL(其中8名患者治疗后VEGF升高，9名患者下降)，经T检验分析，二组之间的差异不具有统计学意义(p>0.05)。治疗有效的7例增殖期血管瘤患儿中，治疗后VEGF水平上升有4人，下降有3人。
(2)治疗前后血清PDGF-AA(血小板衍生生长因子)检测(图1B)。
治疗前患儿血清PDGF-AA浓度为52879.92±31435.65pg/mL，治疗后为26289.34±26095.77pg/mL，治疗后比治疗前下降26590.57±35627.71pg/mL(其中2名患者上升，15名下降)，经T检验分析，二组之间的差异具有统计学意义(p＜0.05)。治疗有效的7例增殖期血管瘤患儿中，治疗后PDGF-AA水平上升有1人(14.3％)，下降有6人(85.7％)。
因此，以上17例婴幼儿血管瘤患儿在伊曲康唑治疗前后，血清中VEGF水平变化差异无统计学意义，而血清中PDGF-AA水平呈下降趋势，差异具有统计学意义。而在治疗有效的增殖期婴幼儿血管瘤中，PDGF-AA水平下降与血管瘤消退有密切相关性。由于PDGF-AA是PDGF-A的二聚体，PDGF-AA的变化反应PDGF-A蛋白水平的变化，因而可知PDGF-A蛋白水平的下降与血管瘤消退有密切相关性。
实施例2伊曲康唑治疗血管瘤的分子机制
(1)材料与方法
试剂和细胞培养
伊曲康唑，酮康唑和***购自Sigma-Aldrich。CP-67345从MedChem Express购买。EOMA获自哈佛医学院的儿童医院外科系(美国波士顿)的Catherine Butterfield博士的血管生物学计划。在含有10％胎牛血清和青霉素/链霉素/谷氨酰胺的RPMI 1640(GIBCO)中培养。从父母那里获得使用婴儿血管瘤标本的书面知情同意书后，我们收集了婴儿血管瘤的增生组织。他们接受过血管瘤外科手术治疗。用增生的IH组织的组织阻断方法成功培养了体外婴儿血管瘤内皮细胞(hemangioma endothel ial cel l，HemEC)。然后在含有10％胎牛血清，10ng/ml碱性成纤维细胞生长因子和青霉素/链霉素/谷氨酰胺的RPMI1640(GIBCO)中进行培养。将所有细胞在37℃含5％CO2孵箱中培养。
细胞活力测定
为了进行细胞活力分析，将细胞接种在96孔板中，并于第二天伊曲康唑或CP-673451开始处理。孵育72小时后，根据制造商的说明进行MTS分析(Promega，Madison，WD)，并使用GraphPad Prism软件(版本6)拟合归一化的细胞活力曲线。
凋亡分析
用伊曲康唑或CP-673451处理细胞48小时。使用Canto流式细胞仪(BectonDickinson，Franklin Lakes，NJ)，根据制造商的说明，通过膜联蛋白V/PI染色(Annexin VFITC和PI试剂盒，Thermo Scientific)分析凋亡细胞。此外用浓度为5μg/ml的Hoechst33258在37℃下将EOMA和HemEC的核染色5分钟。洗涤细胞以除去未结合的染料，随后通过荧光显微镜(Olympus)观察细胞活力。
血管生成测定
将HemECs以1.5×10细胞/孔的密度接种在Matrigel表面上的96孔板中。用0.1％DMSO、伊曲康唑、丙醇或CP-673451处理细胞。处理3小时后，拍摄了6个随机选择的显微镜视野(放大200倍)。总管长度和毛细管网络的密度通过Axiovision软件(马萨诸塞州内蒂克市The Math Works，Inc.)进行评估。
HemECs mRNA阵列和数据分析
用10μM伊曲康唑或0.1％DMSO处理2例婴儿的HemEC 48小时。根据制造商的说明，提取总mRNA进行安捷伦人类(V2)基因表达微阵列(8×60K芯片)的mRNA阵列分析。
逆转录PCR
从伊曲康唑处理的HemEC中提取总RNA。按照制造商的说明，使用Trizol试剂(Invitrogen，15596-026)用伊曲康唑或0.1％DMSO作为对照处理。引物序列如下：PDGF-D，5'-CCCAGGAATTACTCGGTCAA-3'(F)(SEQ ID NO.:1)和5'-ACAGCCACAATTTCCTCCAC-3'(R)(SEQ ID NO.:2)；ACTB，5'-GGACTTCGAGCAAGAGATGG-3'(F)(SEQ ID NO.:3)和5'-AGCACTGTGTTGGCGTACAG-3'(R)(SEQ ID NO.:4)。
蛋白质印迹
参照参考文献(Zheng et al.,2009)使用以下抗体进行蛋白质印迹：p-p70S6K(产品编号9272)，pAKT-T308(9275)，T-AKT(4691)，p-4E-BP1-S65(9451)，PDGFR(3169)，p-PDGFR-B(3161)和B-肌动蛋白(4970)购自Cell Signaling Technologies；PDGF-D购买自R&D Systems(产品编号AF1159)。
统计分析
定量数据表示为n＝3±标准差(S.D.)的平均值。两组通过学生t检验进行比较，多组通过不匹配的单因素方差分析与Tukey校正进行比较，使用GraphPad Prism中每列的平均值与其他每列的平均值进行比较(版本6：La Jolla.CA)。
(2)伊曲康唑作用的潜在靶向基因及生物因子
申请人通过使用全基因组基因表达谱(GEO登录号：GSE123108)探索了伊曲康唑在体外婴儿HemEC中的潜在靶向基因。进行散点图和层次聚类分析以鉴定差异表达的mRNA(参见图2)。HemECs mRNA阵列和数据分析来自2名婴儿患者的HemEC用10μM伊曲康唑或0.1％DMSO处理48小时。根据制造商的说明，用Agilent人类(V2)基因表达微阵列(8×60K芯片)提取总mRNA用于mRNA阵列分析。
根据MAS生物功能注释***手册，分别确定了前20个显著增强的生物过程基因本体论术语(GO terms)(图2c)，前20个显著增强的细胞成分基因本体论术语(图2d)，前20个显著增强的分子功能基因本体论术语(图2e)和前20个显著增强的途径术语(图2f)。
同时发现PDGF-A、VEGF-A、HES、HEY1、IL-8或TNF-A这些生物因子对应的基因在差异表达的mRNA中上调显著，差异倍数大于2；PDGF-D、HIF3A或FGF这些生物因子对应的基因在差异表达的mRNA中下调显著，差异倍数大于2，具体差异情况见表2：
表2表达差异显著的基因及其对应生物因子名称
注：以上差异倍数中，正数表示上调，负数表示下调，差异倍数以表中数值的绝对值表示。
(3)伊曲康唑显著降低了PDGF-D水平，从而抑制了PDGFR-β的活化并抑制了其下游效应物。
体外婴儿血管瘤内皮细胞(HemEC)在伊曲康唑处理后PDGF-D蛋白质水平被显著抑制(图3a、b)。进一步使用蛋白质印迹分析了PI3K/Akt/mTOR途径的活性，该途径是PDGF/PDGF受体-β(PDGFR-β)信号传导的重要下游和涉及婴儿血管瘤的关键信号传导途径。如图3c所示，发现伊曲康唑处理诱导Akt(p-Akt)磷酸化水平降低，并且Akt/mTOR，p70S6激酶(p-p70S6K)和4E结合蛋白1的两个下游效应物(p-4E-BP1)，在HemEC(图3c)和EOMA(图3d)细胞中。
(4)伊曲康唑对HemEC的活性与CP-673451(PDGFR-β抑制剂)类似
为了证实伊曲康唑是否可以通过抑制PDGF-D/PDGFR-β信号传导来抑制婴儿血管瘤，比较了HemEC中伊曲康唑与PDGFR-β抑制剂CP-673451的活性,结果表明，伊曲康唑和CP-673451均抑制细胞增殖(图4a)，促进细胞凋亡(图4b)，减少血管生成(图4c)，抑制磷酸化PDGFR-β及其下游效应物，包括p-Akt，p-p70S6K和HemEC中的p-4E-BP1(图4d)。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
参考文献：
Zheng B,Jeong JH,Asara JM,Yuan Y-Y,Granter SR,Chin L,et al.OncogenicB-RAF negatively regulates the tumor suppressor LKB1 to promote melanoma cellproliferation.Mol Cell 2009；33:237-47.
序列表
<110> 四川大学华西医院
<120> 血管瘤治疗的生物标记物
<130> P2020-0091
<150> CN201910074711.6
<151> 2019-01-25
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<170> PatentIn version 3.5
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Claims (3)
1. PDGF-A蛋白的检测试剂用于制备评估、预测或优化伊曲康唑对血管瘤病人治疗有效性的试剂盒的用途，所述血管瘤病人为婴幼儿。
2.如权利要求1所述的用途，其特征在于，当治疗后PDGF-A的蛋白水平较治疗前下降比例大于或等于10%时，达到治疗效果。
3.如权利要求1所述的用途，其特征在于，所述血管瘤为增殖期血管瘤。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1325407A (zh) * | 1998-11-10 | 2001-12-05 | 路德维格癌症研究所 | 血小板衍生生长因子d、其编码dna及其应用 |
| TW200803835A (en) * | 2006-01-11 | 2008-01-16 | Cleveland Clinic Foundation | Therapeutic compositions and methods useful in modulating protein tyrosine phosphatases |
| CN102159257A (zh) * | 2008-07-17 | 2011-08-17 | 米歇尔技术公司 | 药物递送医疗设备 |
| CN107109459A (zh) * | 2014-11-05 | 2017-08-29 | 加利福尼亚大学董事会 | 用于分层对阻断pd1/pdl1轴的疗法的无响应者的方法 |
| WO2018160889A1 (en) * | 2017-03-01 | 2018-09-07 | Achillion Pharmaceuticals, Inc. | Aryl, heteroary, and heterocyclic pharmaceutical compounds for treatment of medical disorders |
Family Cites Families (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7550143B2 (en) * | 2005-04-06 | 2009-06-23 | Ibc Pharmaceuticals, Inc. | Methods for generating stably linked complexes composed of homodimers, homotetramers or dimers of dimers and uses |
| MXPA01012385A (es) * | 1999-06-03 | 2002-09-02 | Human Genome Sciences Inc | Proteinas angiogenicas y usos de las mismas. |
| AU2002250032B2 (en) * | 2001-02-09 | 2008-06-05 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor (CCR5) HDGNR10 |
| CU23178A1 (es) * | 2002-04-15 | 2006-09-22 | Ct Ingenieria Genetica Biotech | INMUNOTERAPIA ACTIVA ANTIANGIOGéNICA |
| AU2003291631A1 (en) * | 2002-10-03 | 2004-04-23 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Method for regulating expression genes |
| US8906390B2 (en) * | 2006-03-24 | 2014-12-09 | Gencia Corporation | Synthetic lipid rafts and methods of use |
| US20110262517A1 (en) * | 2007-04-05 | 2011-10-27 | The Johns Hopkins University | Chirally pure isomers of itraconazole and inhibitors of lanosterol 14a-demethylase for use as angiogenesis inhibitors |
| US20080299077A1 (en) * | 2007-06-01 | 2008-12-04 | Nevada Cancer Institute | Isolation and growth of stem cells from hemangiomas |
| WO2010005046A1 (ja) * | 2008-07-09 | 2010-01-14 | 国立大学法人 北海道大学 | 血管新生抑制剤 |
| US20100029491A1 (en) * | 2008-07-11 | 2010-02-04 | Maike Schmidt | Methods and compositions for diagnostic use for tumor treatment |
| CN114262690A (zh) * | 2011-04-15 | 2022-04-01 | 吉恩勒克斯公司 | 减毒的痘苗病毒的克隆毒株及其使用方法 |
| DE112014005975T5 (de) * | 2013-12-17 | 2016-09-15 | Kymab Limited | Menschliche Ziele |
| EP2954934A1 (en) * | 2014-06-11 | 2015-12-16 | 3B Pharmaceuticals GmbH | Conjugate comprising a neurotensin receptor ligand and use thereof |
| CN105640957B (zh) * | 2014-11-28 | 2020-04-07 | 四川大学华西医院 | 伊曲康唑的新用途 |
| CN104651305A (zh) * | 2015-02-13 | 2015-05-27 | 沈阳澔源生物科技有限公司 | 一种利用脐带间充质干细胞获取生物活性蛋白的方法 |
| CN107561280B (zh) * | 2017-09-30 | 2019-07-26 | 四川大学华西医院 | 一种预测乳腺癌复发的试剂盒 |
-
2020
- 2020-01-20 WO PCT/CN2020/073250 patent/WO2020151666A1/zh active Application Filing
- 2020-01-20 CN CN202010067261.0A patent/CN111487398B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1325407A (zh) * | 1998-11-10 | 2001-12-05 | 路德维格癌症研究所 | 血小板衍生生长因子d、其编码dna及其应用 |
| TW200803835A (en) * | 2006-01-11 | 2008-01-16 | Cleveland Clinic Foundation | Therapeutic compositions and methods useful in modulating protein tyrosine phosphatases |
| CN102159257A (zh) * | 2008-07-17 | 2011-08-17 | 米歇尔技术公司 | 药物递送医疗设备 |
| CN107109459A (zh) * | 2014-11-05 | 2017-08-29 | 加利福尼亚大学董事会 | 用于分层对阻断pd1/pdl1轴的疗法的无响应者的方法 |
| WO2018160889A1 (en) * | 2017-03-01 | 2018-09-07 | Achillion Pharmaceuticals, Inc. | Aryl, heteroary, and heterocyclic pharmaceutical compounds for treatment of medical disorders |
Non-Patent Citations (5)
| Title |
|---|
| Contribution of vascular endothelial growth factor receptor-2 sialylation to the process of angiogenesis;P Chiodelli 等;《Oncogene》;20170807;第36卷;第6531-6541页 * |
| Itraconazole inhibits the PDGF-D/PI3K/Akt/mTOR pathway in vitro and down-regulates serum PDGF-A of infantile hemangioma via oral administration;Y Ran 等;《Journal of Investigative Dermatology》;20190419;第139卷(第5期);第S74页 * |
| Successful treatment of oral itraconazole for infantile hemangiomas: A case series;Ran, YP 等;《JOURNAL OF DERMATOLOGY》;20150304;第42卷(第2期);第202-206页 * |
| 伊曲康唑口服液治愈婴儿血管瘤1例:皮肤镜监测血管瘤消退情况;冉玉平 等;《中国皮肤性病学杂志》;20161216;第31卷(第01期);第45-47、49页 * |
| 血管内皮生长因子用于治疗性血管生成的问题及对策;黄新苗 等;《心血管病学进展》;20050825;第26卷(第4期);第413-417页 * |
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