CN105943526B - Pd0325901在维持动脉导管开放中的应用 - Google Patents

Pd0325901在维持动脉导管开放中的应用 Download PDF

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CN105943526B
CN105943526B CN201610330333.XA CN201610330333A CN105943526B CN 105943526 B CN105943526 B CN 105943526B CN 201610330333 A CN201610330333 A CN 201610330333A CN 105943526 B CN105943526 B CN 105943526B
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洪海筏
刘锦纷
叶霖财
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Shanghai Childrens Medical Center Affiliated to Shanghai Jiaotong University School of Medicine
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Abstract

本发明涉及PD0325901在维持动脉导管开放中的应用。在乳鼠出生前或出生后给予注射PD0325901，乳鼠动脉导管均能维持开放；实验结果显示PD0325901主要通过以下三个机制来维持DA开放：抑制DASMC增殖，减少迁移和抑制胞内钙浓度，说明PD0325901可用于制备维持动脉导管的药物或试剂。

Description

PD0325901在维持动脉导管开放中的应用
技术领域
本发明涉及医药技术领域，具体地说，是PD0325901在维持动脉导管开放中的应用。
背景技术
动脉导管开放(patent ductus arteriosus,PDA)是指胎儿时期连接肺动脉和主动脉之间的动脉导管在出生后未能正常关闭。正常新生儿生后10-15h可达到功能性关闭，3个月达解剖学上的关闭。如果DA关闭机制异常，即为动脉导管未闭。PDA是常见的先天性心脏病，发病率在先天性心脏畸形中占第2位。早产儿因发育不成熟，出生后动脉导管多不能及时关闭。胎龄越小，出生体重越低，PDA发生率越高，对早产儿危害也越大。
动脉导管未闭的临床表现取决于主动脉至肺动脉分流血量的多少以及是否产生继发肺动脉高压和其程度。轻者可无明显症状，重者可发生心力衰竭。常见的症状有劳累后心悸、气急、乏力，易患呼吸道感染和生长发育迟缓。晚期肺动脉高压严重，产生逆向分流时可出现下半身发绀。PDA是由多种因素参与的复杂的病理生理过程，目前仍缺少对PDA发病机制和病理变化足够认识。
PD0325901是一种可以口服的，细胞穿透力强的第二代非ATP竞争的MEK抑制剂，对于治疗晚期的恶性肿瘤细胞具有非常好的疗效，例如：黑色素瘤，乳腺癌，非小细胞肺癌(NSCLC)和结肠癌。而关于PD0325901在维持动脉导管开放中的应用，目前还未见报道。
发明内容
本发明的目的是针对现有技术中的不足，提供PD0325901的新用途。
为实现上述目的，本发明采取的技术方案是：
PD0325901在制备维持动脉导管开放的试剂中的应用。
PD0325901在制备维持动脉导管开放的药物中的应用。
所述抑制动脉导管平滑肌细胞增殖，减少细胞迁移和抑制胞内钙浓度。
本发明优点在于：
本发明提供了PD0325901的新用途，用于制备维持动脉导管开放的试剂/药物。在乳鼠出生前或出生后给予注射PD0325901，乳鼠动脉导管均能维持开放；实验结果显示PD0325901主要通过以下三个机制来维持DA开放：抑制DASMC增殖，减少迁移和抑制胞内钙浓度，说明PD0325901可用于制备维持动脉导管的药物或试剂。
附图说明
附图1为对照组及给药组大体标本，其中给药组动脉导管开放。
附图2为对照组及给药组HE染色，出生前给药或出生后给药，PD0325901均能维持DA开放。其中，DA：动脉导管；Ao：主动脉。
附图3为细胞迁移实验、细胞增殖实验结果。图3A：DASMC表达成纤维细胞的标志蛋白vimentin；图3B PD0325901减少透明质酸(HA)的分泌；图3C和D：PD0325901减少迁移的平滑肌细胞的数量；图3E和F和G：PD0325901抑制DASMC细胞的增殖。
附图4为细胞内钙离子检测结果。PD0325901减少DASMC细胞内的钙离子浓度。
具体实施方式
下面结合具体实施方式，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1 PD0325901具有维持动脉导管(DA)开放的作用
一、材料
实验动物：C57/BL6孕鼠E16.5天，刚出生乳鼠P1天。人开放或闭合动脉导管组织。MEK抑制剂PD0325901(Tocris Bioscience，CAS No：391210-10-9)。DMSO(Sigma，D2650)。胶原酶II型(sigma，C6885)。DMEM/F12(Thermo)。
二、方法
1.动物实验
出生前给药：母鼠怀孕第E16.5天，腹腔注射PD0325901 10mg/kg，连续注射3-5天，直至幼鼠出生。幼鼠出生12小时后，解剖取心脏(包括主动脉和动脉导管)和肺，剔除周围***后，拍照。将心脏和肺放入4％多聚甲醛，4度过夜。
出生后给药：正常未给药的幼鼠在生后2小时左右，腹腔注射PD0325901 10mg/kg，8小时后取心脏和肺，剔除周围***后，拍照。将心脏和肺放入4％多聚甲醛，4度过夜。
2.苏木精-伊红染色
4度4％多聚甲醛过夜的心脏或肺组织，OCT包埋，液氮速冻，冰冻切片机切片，厚度8um。蒸馏水洗涤2分钟，苏木***染色10分钟，过蒸馏水1秒，1％盐酸乙醇1秒，蒸馏水10分钟，伊红染色3分钟，过蒸馏水1秒，80％乙醇脱水1秒，95％乙醇脱水3秒，无水乙醇(I)脱水5分钟，无水乙醇(II)脱水5分钟，二甲苯(I)2分钟，二甲苯(II)2分钟，中性树脂封片，拍照。
3.人动脉导管平滑肌细胞(DASMC)的分离
DA组织在手术室切除后，用无菌DMEM/F12冲洗2遍，转移至超净台，用无菌棉签刮除内膜，小心撕去外膜。将血管剪碎，转移至新鲜配制的消化液(含胶原酶II型1.8mg/kg，弹性蛋白酶III型0.3mg/kg，胰蛋白酶抑制剂I型0.44mg/kg，2mg/ml牛血清白蛋白)，37度消化30分钟后，取上清液，10％胎牛血清中和；剩余组织添加新鲜消化液继续消化30分钟，取上清，10％胎牛血清中和。将两次消化收集的上清混合，1000rpm离心5分钟，将收获的细胞种植于10cm培养皿，20％胎牛血清培养扩增。
4.细胞迁移实验
将用或不用PD0325901处理24小时的人DASMC以1*105个/ml种植于TranswellChamber的上室，下室添加含有20％血清的培养液。细胞培养4小时后，小心吸取上室液体，在膜上层的细胞用棉签刮除，下层的细胞用4％多聚甲醛固定，考马斯蓝染色，计数。
5.细胞增殖实验
增殖实验采用CCK-8(Dojindo Laboratories,Kumamoto,Japan)和EdU试剂盒(广州锐博)。具体实验步骤依照厂家说明书进行。
6.细胞内钙离子的检测
细胞用PD0325901处理48小时后，用Fluo-3AM(上海碧云天)检测。具体实验步骤按照试剂盒说明书进行。
三、实验结果
图1：大体标本，给药组动脉导管开放。图2：HE染色，出生前给药或出生后给药，PD0325901均能维持DA开放。DA：动脉导管；Ao：主动脉。PD0325901主要通过以下三个机制来维持DA开放：抑制DASMC增殖，减少迁移和抑制胞内钙浓度。图3A：DASMC表达成纤维细胞的标志蛋白vimentin；图3B：PD0325901减少透明质酸(HA)的分泌；图3C和D：PD0325901减少迁移的平滑肌细胞的数量；图3E和F：PD0325901抑制DASMC细胞的增殖。图4：PD0325901减少DASMC细胞内的钙离子浓度。以上结果表明，出生前或出生后给予注射PD0325901的乳鼠动脉导管均能维持开放，PD0325901可用于制备维持动脉导管的药物或试剂。
实施例2
对10只C57/BL6孕鼠腹腔注射PD0325901 10mg/kg，连续注射3-5天；10只幼鼠在生后2小时左右腹腔注射PD0325901 10mg/kg。实验条件及检测同实施例1，结果表明给予注射PD0325901的乳鼠动脉导管均能维持开放，而对照乳鼠(未给药)动脉导管均闭合。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (2)

1.PD0325901在制备维持动脉导管开放的试剂中的应用，所述试剂抑制动脉导管平滑肌细胞增殖，减少细胞迁移和抑制胞内钙浓度。
2.PD0325901在制备维持动脉导管开放的药物中的应用，所述药物抑制动脉导管平滑肌细胞增殖，减少细胞迁移和抑制胞内钙浓度。
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CN102027105A (zh) * 2008-03-17 2011-04-20 斯克里普斯研究所 用于产生诱导的多能干细胞的组合化学遗传方法

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CN102027105A (zh) * 2008-03-17 2011-04-20 斯克里普斯研究所 用于产生诱导的多能干细胞的组合化学遗传方法

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