CN111067895A - Ite在制备预防和/或治疗心肺循环系统疾病的药物中的应用 - Google Patents
Ite在制备预防和/或治疗心肺循环系统疾病的药物中的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及ITE在制备预防和/或治疗心肺循环系统疾病的药物中的应用,属于医药技术领域。
背景技术
肺血管重构(Pulmonary artery remodeling,PAR)是肺动脉在受到低氧或各种损伤刺激之后,内皮细胞损伤、增殖以及平滑肌细胞增殖导致血管中膜增厚、小血管管腔闭塞等病理改变,是低氧性肺动脉高压(Hypoxic pulmonary hypertension,HPH)持续且难以逆转的主要原因,其中正常无肌型小肺动脉肌化是低氧引起的PAR的重要特征之一。目前,肌化过程中平滑肌样细胞的来源和机制尚未阐明。过去认为α-SMA阳性细胞来源于常驻血管中膜SMC的迁移、增殖和去分化以及循环祖细胞的募集,并在此基础上进行增殖与扩张。随着病理生理学研究的不断深入,越来越多的研究表明内皮细胞在受到低氧等刺激下,通过转分化失去了内皮细胞原有结构特征和表型并进行细胞骨架重排,向平滑肌样细胞表型转化并参与肌化的形成,是肺血管重构的重要原因。
小分子化合物ITE,其结构式如式(Ⅰ)所示,ITE是人体必需氨基酸——色氨酸代谢产物之一,作为芳香烃受体(AhR)内源性天然配体参与细胞功能的调节。近年来的研究发现ITE在肿瘤、免疫领域发挥重要的调控作用,作为人体内源性小分子化合物并且无明显的毒理作用,ITE受到越来越多的重视与研究。然而,ITE在肺动脉高压疾病的发生、发展中的作用尚不明确。
中国专利CN106588908A公开了用于介入治疗和根除癌症的ITE及其结构类似物,通过将ITE的对于芳基烃(Ah)受体(AhR)的内源性配体或其类似物之一(活性成分)至患有癌症的受试者,用于介入治疗或根除癌症;专利CN102573470A公开了通过给药有效量的命名为ITE的对于芳基烃(Ah)受体(AhR)的内源性配体或其类似物之一(活性成分)至患有癌症的受试者,介入治疗或根除癌症的方法,在所述受试者摆脱癌症后,提供维持给药以确保根除癌症,患有前列腺癌、肝癌、肺癌、卵巢癌和乳癌的受试者优选地接受治疗。专利CN106588908A和专利CN102573470A公开的均为ITE在肿瘤防治领域的应用,均未涉及ITE在心肺循环系统疾病的新应用。
发明内容
本发明的目的在于克服现有技术的不足,提供ITE在制备预防和/或治疗心肺循环系统疾病的药物中的应用。
为实现上述目的,本发明采取的技术方案为:ITE在制备预防和/或治疗心肺循环系统疾病的药物中的应用,所述ITE的结构式如式(Ⅰ)所示:
作为本发明应用的优选实施方式,在低氧条件下,所述ITE能促使人肺动脉内皮细胞失去内皮特有的结构,能诱导人肺动脉内皮细胞(HPAECs)转分化为平滑肌样细胞。
在低氧条件下,本发明所述ITE能显著促进HPAECs转分化为平滑肌样细胞,并且可能通过上调TGF-β1参与HPAECs转分化,表明ITE在HPAECs转分化为平滑肌样细胞的过程中发挥重要的调控作用,由此说明ITE可作为预防和/或治疗心肺循环系统疾病的药物靶点,具有重要的应用价值。
本发明所述ITE能显著抑制HPAECs转分化所致的肺血管肌化、抑制肺动脉血管重构、抑制肺动脉管壁增厚、抑制肺动脉管腔狭窄、抑制肺动脉压力增高、降低右心室肥厚和改善肺动脉高压所致的靶器官损伤;或者通过制备HPAECs转分化的动物模型、制备HPAECs转分化相关疾病的动物模型,本发明所述ITE能抑制HPAECs转分化所致的肺血管肌化、抑制肺动脉血管重构、抑制肺动脉管壁增厚、抑制肺动脉管腔狭窄、抑制肺动脉压力增高、降低右心室肥厚和改善肺动脉高压所致的靶器官损伤。
作为本发明应用的优选实施方式,在低氧条件下,所述ITE能促进所述平滑肌样细胞的增值。
所述ITE能促进HPAECs转分化为平滑肌样细胞的增殖,证实了HPAECs是肺动脉血管平滑肌细胞过度增殖和中膜异常增厚中的关键因素,表明ITE在肺动脉高压血管重构过程发挥重要作用,可作为抑制和防控肺动脉高压发生发展的有效靶点。
作为本发明应用的优选实施方式,在低氧条件下,所述ITE能抑制人肺动脉内皮细胞标记蛋白VE-cad和CD31的表达,所述ITE能促进平滑肌样细胞标记蛋白OPN和α-SMA的表达。
作为本发明应用的优选实施方式,在低氧条件下,所述ITE能抑制人肺动脉内皮细胞的成管能力。
作为本发明应用的优选实施方式,在低氧条件下,所述人肺动脉内皮细胞的成管能力与时间呈正相关。
所述ITE处理HPAECs后,HPAECs的内皮功能和在肺动脉高压保护中的作用显著下降,说明ITE在HPAECs内皮功能形成过程发挥重要作用,可作为抑制和防控肺动脉血管重构的有效靶点。
作为本发明应用的优选实施方式,以体积浓度计,所述低氧条件为含有氧气的体积浓度低于21%的气体环境。
作为本发明应用的优选实施方式,所述心肺循环系统疾病包括人肺动脉内皮细胞转分化相关的疾病。
作为本发明应用的优选实施方式,所述人肺动脉内皮细胞转分化相关的疾病包括肺动脉高压、右心衰竭、心脑血管疾病、慢性阻塞性肺疾病、先天性心血管畸形、肺心病以及肺动脉高压所致的心衰并发症
所述肺动脉高压疾病包括:①动脉性肺动脉高压;②左心疾病所致肺动脉高压;③缺氧和/或肺部疾病引起的肺动脉高压;④慢性血栓栓塞性肺动脉高压;⑤多种机制和/或不明机制引起的肺动脉高压。
另一方面,本发明还提供一种用于预防和/或治疗心肺循环系统疾病的药物组合物,所述药物组合物的有效成分包括本发明所述的ITE,所述药物组合物还包括药学上可接受的载体。
与现有技术相比,本发明的有益效果为:本发明提供ITE在制备预防和/或治疗心肺循环系统疾病的药物中的应用,在低氧(氧浓度低于21%)条件下,ITE能显著促进HPAECs转分化为平滑肌样细胞,能显著促进平滑肌样细胞的增值,显著抑制HPAECs的成管能力,说明了ITE可作为预防和/或治疗肺动脉血管重构的有效靶点,ITE能有效治疗心肺循环系统疾病,为心肺循环系统疾病的预防和/或治疗提供了新的药物选择途径。
附图说明
图1为ITE对低氧诱导HPAECs转分化细胞形态学特征的作用图;
图2为ITE对低氧诱导HPAECs转分化相关指标的作用图;
图3为ITE对低氧诱导HPAECs转分化中VE-cad、CD31、OPN、α-SMA蛋白的表达图;
图4为ITE对低氧诱导HPAECs转分化细胞成管能力的影响结果图;
图5为ITE对低氧诱导HPAECs转分化细胞增殖的影响结果图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如《分子克隆》(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
实施例1
本实施例为ITE在肺动脉内皮细胞转分化细胞表型变化中的作用。
一、实验材料
1.实验细胞:人肺动脉内皮细胞(HPAECs)购自ScienCell公司。
2.主要试剂:ITE购买于Tocris Bioscience(San Diego,CA),内皮培养基(ECM,Cat.#1001)购自ScienCell公司,胎牛血清(fetal bovine serum,FBS)、胰酶购于美国Gibco公司产品,总蛋白提取液、蛋白酶抑制剂、BCA蛋白浓度测定试剂盒、SDS-PAGE蛋白上样缓冲液(5x)、ECL化学发光超敏显色试剂盒均购于碧云天(上海)生物公司,Alexa488标记抗小鼠荧光二抗、Alexa555标记抗兔荧光二抗购自(Cell signaling公司),兔抗α-tublin抗体、小鼠抗CD31抗体、兔抗VE-cad抗体购自,兔抗α-SMA抗体、小鼠抗OPN抗体、兔抗TGF-β1抗体购自Abcam公司。
3.主要实验仪器:酶标仪为Thermo公司产品,激光共聚焦显微镜(日本OLYPUS公司),流式细胞仪(BD公司)。
二、实验方法
1.HPAECs培养及实验分组
将HPAECs接种于25cm2的培养皿中,加入采用含10%FBS的ECM培养基培养HPAECs,每两天给细胞换液,待细胞传至第3代供实验使用,将HPAECs分为4组:常氧组:置于含21%O2,5%CO2的培养箱中培养;常氧+ITE(5μmol/L)组;低氧组:置于含1%O2,5%CO2,94%N2的培养箱中培养;低氧+ITE(5μmol/L)组。以上各组分别在常氧以及低氧条件下培养1、4、7d,每组重复3次实验。
2.免疫荧光双标记法鉴定HPAECs转分化细胞表型变化
取对数生长期的第3代HPAECs,培养于激光共聚焦专用培养皿中,将上述各组处理结束后弃培养基,用PBS冲洗3次,加入免疫染色固定液固定细胞30min,0.5%TritonX-100溶液通透30min,免疫染色封闭液封闭1h,加羊抗鼠CD31(1:200稀释)、羊抗兔α-SMA(1:100稀释);羊抗兔VE-cadhein(1:200稀释)、羊抗鼠OPN(1:100稀释),孵育4℃过夜,次日加入对应的两种二抗(羊抗兔Alexa、羊抗鼠Alexa),37℃避光孵育1h;DAPI染核,防淬灭剂封片,采用激光共聚焦显微镜(奥林巴斯FV3000)观察荧光镜下图片获取。
3.组织病理学检查
在上述3个时间点,每亚组随机取3只小鼠断颈处死,完整切取心肺和主动脉弓,主动脉弓存放于-80℃冰箱保证组织新鲜。
剖开腹腔,胸腔,清除胃部,肠道等组织,小心分离心脏,尽量保留冠状动脉完整性,分离主动脉,清除主动脉周围结缔组织,保留髂总动脉以及分支,将主动脉放入液氮保存。将主动脉分离取下,用镊子尽可能地去除血管周围的脂肪组织,固定液固定24h后用解剖剪沿血管壁小心地将血管纵向剖开。油红染液中37°染色60min,蒸馏水洗2次后拍照,结果判读:脂滴呈橘红色或鲜红色,其它部位近无色。
4.蛋白质印迹法(Western blotting)检测α-SMA、OPN、CD31、VE-cadherin和TGF-β1蛋白在ITE对低氧诱导HPAECs转分化表达变化
将HPAECs置于六孔板中培养,按照上述实验方法分组,提取分别提取不同时间段细胞总蛋白,用含有蛋白酶抑制剂(PMSF)细胞裂解液,收集细胞于冰上裂解30min,12000r/min离心15min,取上清,根据BCA法测定蛋白浓度。加入上样缓冲液,加热变性后按照Western-blot操作步骤及要求进行操作,制备10%分离胶、5%浓缩胶,经SDS-PAGE恒压电泳、转膜,并用脱脂牛奶封闭孵育一抗,4℃孵育过夜。一抗使用碧云天生物公司生产的一抗稀释液稀释,比例为CD31、VE-cad、OPN、α-tublin 1:1000;α-SMA 1:2000,TBST漂洗后,相应二抗按1:2000比例稀释,室温孵育1h后,放入Tanon 5200全自动化学发光图像分析系统光仪中曝光显影,用Image J软件对目的条带的灰度值进行半定量分析,相对灰度值=(目的蛋白灰度值-背景灰度)/(内参蛋白灰度值-背景灰度)。
三、实验结果
1.ITE对低氧诱导HPAECs转分化细胞形态学特征
由图1可知,倒置相差显微镜下观察HPAECs约24h后形成细胞集落,呈小簇状分布,细胞扩增培养,呈典型的铺路石样形态,低氧7d后,铺路石样内皮细胞向多角形细胞改变,低氧下给予5μmol/L ITE处理后,细胞失去铺路石样,ITE联合低氧处理7d后细胞形态明显呈梭形或多角形。
2.免疫荧光双标记检测ITE对低氧诱导HPAECs转分化相关指标
由图2可知,免疫荧光双染结果显示与常氧组相比,低氧组和低氧+ITE组随培养时间延长,内皮细胞标记物(CD31、VE-cad)表达下调而平滑肌样细胞标记物(α-SMA、OPN)表达上调,以低氧7d最显著,与低氧组相比,ITE联合低氧共同培养HPAECs 7d后,α-SMA和OPN表达明显上调而CD31和VE-cad表达明显下调,其中常氧和低氧1d组均未见α-SMA、OPN的表达,表明低氧能够促进低HPAECs转分为平滑肌样细胞并且ITE能够明显促进这一过程。
3.ITE对低氧诱导HPAECs转分化中VE-cad、CD31、OPN、α-SMA蛋白的表达
由图3可知,Western blot结果显示,上述四组(常氧组、常氧+ITE(5μmol/L)组、低氧组、低氧+ITE(5μmol/L)组)在常氧组和低氧组培养1d的人肺动脉内皮细胞中VE-cad、CD31、OPN、α-SMA蛋白表达无显著差别。
VE-cad、CD31蛋白表达:VE-cad、CD31蛋白在低氧组和低氧+ITE组培养4d、7d的人肺动脉内皮细胞中的表达,均分别低于在常氧组培养4d、7d的人肺动脉内皮细胞的表达。
α-SMA、OPN蛋白表达:α-SMA、OPN蛋白在低氧组和低氧+ITE组培养4d、7d的平滑肌样细胞中的表达,均分别高于在常氧组培养4d、7d的平滑肌样细胞的中的表达,而常氧+ITE组培养4d、7d的平滑肌样细胞与常氧组培养4d、7d的平滑肌样细胞中的α-SMA、OPN蛋白表达无明显差异。
VE-cad、CD31蛋白表达:VE-cad、CD31蛋白在低氧组和低氧+ITE组培养7d的人肺动脉内皮细胞中的表达,低于在低氧组培养7d的人肺动脉内皮细胞中的表达。
α-SMA、OPN蛋白表达:α-SMA、OPN蛋白在低氧组和低氧+ITE组培养7d的平滑肌样细胞中的表达,高于在低氧组培养7d的平滑肌样细胞中的表达;α-SMA、OPN蛋白在低氧组培养7d的平滑肌样细胞中的表达,高于在低氧组培养4d的平滑肌样细胞中的表达;α-SMA、OPN蛋白在低氧+ITE组的培养7d的平滑肌样细胞中的表达,高于在低氧+ITE组培养7d的平滑肌样细胞中的表达。
由以上说明了,低氧能够促进HPAECs转分为平滑肌样细胞并且ITE能够明显促进这一过程。
四、结论:
ITE处理促使HPAECs失去内皮特有结构,促进低氧诱导HPAEC转分化为平滑肌样细胞,表明ITE在HPAECs转分化和肺无肌细动脉肌化过程发挥重要作用,可作为抑制和防控肺动脉血管重构的有效靶点。
实施例2:
本实施例为ITE对低氧诱导HPAECs转分化细胞成管能力的作用
一、实验材料
1.实验细胞:人肺动脉内皮细胞(HPAECs)购自ScienCell公司。
2.主要试剂:ITE购买于Tocris Bioscience(San Diego,CA),内皮培养基(ECM,Cat.#1001)购自ScienCell公司,胎牛血清(fetal bovine serum,FBS)、胰酶购于美国Gibco公司产品,BrdU抗体购于Sigma公司。
3.主要实验仪器:酶标仪为Thermo公司产品,激光共聚焦显微镜(日本OLYPUS公司),流式细胞仪(BD公司)。
二、实验方法
1.基质胶体外成管实验检测ITE对低氧诱导HPAECs转分化细胞成管能力
在4℃条件下融化Matrigel胶过夜,预冷24孔板和枪头。取24孔板在冰上操作,每孔缓慢加入250μL液态Matrigel胶。37℃孵育30~60min,使Matrigel胶凝固;将按上述方法处理的各组HPAECs釆用胰酶消化并用ECM培养基重悬细胞后计数,将HPAECs细胞稀释为2×105个/mL细胞悬液,每组加入250μL细胞悬液,孵育12h后在显微镜下观察。
三、实验结果
1.ITE对低氧诱导HPAECs转分化细胞成管能力的影响
由图4可知,基质胶体外成管实验结果显示,与常氧组相比,HPAECs随着低氧及低氧+ITE处理时间延长在基质胶上形成的管腔数显著减少;与低氧组相比,ITE+低氧共同处理HPAECs后,管腔数量明显减少,其中以低氧+ITE 7d组HPAECs管腔数抑制最显著,表明ITE对HPAECs的成管抑制效果与时间呈正相关。
四、实验结论
ITE处理抑制HPAECs的成管能力,表明其内皮功能和在肺动脉高压保护中的作用显著下降,说明ITE在HPAECs内皮功能形成过程发挥重要作用,可作为抑制和防控肺动脉血管重构的有效靶点。
实施例3:
本实施例为ITE对低氧诱导HPAECs转分化细胞增殖的作用
一、实验材料
1.实验细胞:人肺动脉内皮细胞(HPAECs)购自ScienCell公司。
2.主要试剂:ITE购买于Tocris Bioscience(San Diego,CA),内皮培养基(ECM,Cat.#1001)购自ScienCell公司,胎牛血清(fetal bovine serum,FBS)、胰酶购于美国Gibco公司产品,BrdU抗体购于Sigma公司。
3.主要实验仪器:酶标仪为Thermo公司产品,激光共聚焦显微镜(日本OLYPUS公司),流式细胞仪(BD公司)。
二、实验方法
1.BrdU掺入率检测ITE对低氧诱导HPAECs转分化细胞增殖的影响
分别将上述各组细胞收集,接种于激光共聚焦培养皿中,细胞数为每孔1×104,培养24h后,在培养基中添加BrdU,浓度为10μmol/L,继续培养24h后,进行免疫荧光染色检测。弃掉培养液,用PBS洗涤3次后,4%多聚甲醛固定30min,2M HCl 37℃孵育30min,0.1mol/L硼酸溶液洗3次,0.3%Triton-100透膜20min,5%BSA37℃封闭30min,再加入小鼠抗人BrdU抗体(1︰100),4℃孵育过夜。加入荧光二抗37℃孵育1h,再用DAPI染核5min,荧光显微镜下计数,计数10个视野。BrdU掺入率=BrdU阳性细胞数/DAPI阳性细胞数。
三、实验结果
1.ITE对低氧诱导HPAECs转分化细胞增殖的影响
由图5可知,对各组中BrdU阳性的细胞和DAPI阳性的总细胞计数,与常氧组比较,低氧组及低氧+ITE组培养7d的人肺动脉内皮细胞(HPAECs)中,BrdU阳性的数目比例增加;与低氧组相比,ITE+低氧组共同处理HPAECs 7d后,BrdU阳性的数目比例显著增加,表明ITE能够显著促进低氧诱导HPAECs转分化为平滑肌样细胞的增殖。
四、实验结论
ITE处理促进HPAECs转分化为平滑肌样细胞的增殖,说明其是肺动脉血管平滑肌细胞过度增殖和中膜异常增厚中的关键因素,表明ITE在肺动脉高压血管重构过程发挥重要作用,可作为抑制和防控肺动脉高压发生发展的有效靶点。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
2.如权利要求1所述的应用,其特征在于,在低氧条件下,所述ITE能诱导人肺动脉内皮细胞转分化为平滑肌样细胞。
3.如权利要求2所述的应用,其特征在于,在低氧条件下,所述ITE能促进所述平滑肌样细胞的增值。
4.如权利要求1所述的应用,其特征在于,在低氧条件下,所述ITE能抑制人肺动脉内皮细胞标记蛋白VE-cad和CD31的表达,所述ITE能促进平滑肌样细胞标记蛋白OPN和α-SMA的表达。
5.如权利要求1所述的应用,其特征在于,在低氧条件下,所述ITE能抑制人肺动脉内皮细胞的成管能力。
6.如权利要求5所述的应用,其特征在于,在低氧条件下,所述人肺动脉内皮细胞的成管能力与时间呈正相关。
7.如权利要求2-6任一所述的应用,其特征在于,以体积浓度计,所述低氧条件为含有氧气的体积浓度低于21%的气体环境。
8.如权利要求1所述的应用,其特征在于,所述心肺循环系统疾病包括人肺动脉内皮细胞转分化相关的疾病。
9.如权利要求8所述的应用,其特征在于,所述人肺动脉内皮细胞转分化相关的疾病包括肺动脉高压、右心衰竭、心脑血管疾病、慢性阻塞性肺疾病、先天性心血管畸形、肺心病以及肺动脉高压所致的心衰并发症。
10.一种用于预防和/或治疗心肺循环系统疾病的药物组合物,其特征在于,所述药物组合物的有效成分包括如权利要求1所述的ITE,所述药物组合物还包括药学上可接受的载体。
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CN116148471A (zh) * | 2022-11-01 | 2023-05-23 | 中南大学 | 肺动脉高压的生物标志物及其应用 |
CN116148471B (zh) * | 2022-11-01 | 2024-05-07 | 中南大学 | 肺动脉高压的生物标志物及其应用 |
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