CN114984219A - Pd1抑制剂在制备心脏成纤维细胞转分化抑制剂中的用途 - Google Patents
Pd1抑制剂在制备心脏成纤维细胞转分化抑制剂中的用途 Download PDFInfo
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Abstract
本发明涉及生物医药领域，本发明公开了一种PD1抑制剂在制备心脏成纤维细胞转分化抑制剂中的用途，本发明通过细胞试验和动物试验发现PD1抑制剂可降低心肌梗死后成心脏纤维细胞的转分化水平。不仅为心肌梗死提供了一种治疗药物或者治疗策略，同时也为心脏成纤维细胞的转分化研究提供了一种工具药。
Description
技术领域
本发明涉及生物医药领域，尤其涉及PD1抑制剂在制备心脏成纤维细胞转分化抑制剂中的用途。
背景技术
心肌梗死是一种严重威胁人类健康的常见的心血管疾病，尽管近年来随着医疗技术水平的提高许多急性心肌梗死患者得以生存，但心肌梗死后心肌重构可以导致心力衰竭，严重心力衰竭是患者死亡最主要原因。心肌梗死后各个时相都有着其特征性病理生理改变，其梗死过程由多种细胞相互作用并共同参与，包括早期心肌细胞大量死亡，免疫细胞浸润，同时伴有血管新生，成纤维细胞激活，心肌代偿肥厚以及慢性病理性心肌重构等一系列病理生理过程。近年来一系列研究表明：心肌梗死导致心肌慢性重构过程中，免疫细胞参与了整个过程，利用免疫细胞调控血管新生以及成纤维细胞活化是改善心肌梗死预后的重要靶点。
PD1/PD-L1作为T细胞抗原TCR以外的第二刺激信号通路，阻断PD1信号活性具有重新激活被耗竭的T细胞的生物活性并发挥其免疫活性的功能，这也是当今肿瘤治疗的研究热点。然而PD1/PD-L1信号通路在非肿瘤病变中对免疫细胞功能的调控机制却并不明了。
此外，在不同种类的疾病以及不同组织器官中PD1抑制剂的生物效应有明显的差异。例如，在神经系统中，PD1抑制剂可削弱吗啡的镇痛效果；而在运用于治疗肿瘤的过程中，PD1抑制剂可引起神系统的副作用，主要是神经肌肉病变；在阿尔茨海默病方面，有研究表明，PD1信号通路阻滞可改善该症状；这说明了在不同疾病、不同器官中，PD1抑制剂最终发挥的作用是具有显著差异的，并没有明显的相通性。在现有的报道中，尚鲜有关于将PD1抑制剂应用于心脏类疾病中的报道。在心肌梗死中，心脏成纤维细胞过度纤维化(过度转分化)可恶化心功能。因此对于与心脏成纤维细胞过度纤维化相关的疾病而言，寻求可抑制心脏成纤维细胞转分化的药物是一种潜在有效的治疗途径。
发明内容
为了解决上述技术问题，本发明提供了一种PD1抑制剂在制备心脏成纤维细胞转分化抑制剂中的用途。本发明通过细胞试验和动物试验发现PD1抑制剂可降低心肌梗死后成心脏纤维细胞的转分化水平。不仅为心肌梗死提供了一种治疗药物或者治疗策略，同时也为心脏成纤维细胞的转分化研究提供了一种工具药。
本发明的具体技术方案为：
本发明提供了一种PD1抑制剂在制备心脏成纤维细胞转分化抑制剂中的用途。
作为优选，所述心脏成纤维细胞转分化抑制剂为用于科学研究的工具药，或者是用于治疗心肌梗死的药物。
作为优选，PD1抑制剂选自纳武单抗、帕博利珠单抗和T药（Tecentrip）。
进一步优选，所述PD1抑制剂为纳武单抗。
作为优选，所述工具药或药物包括所述PD1抑制剂，以及药学上可接受的载体和/或赋形剂。
作为优选，所所述工具药或药物为注射制剂。
进一步优选，所所述注射制剂为静脉注射制剂。
与现有技术相比，本发明具有以下技术效果：本发明通过建立小鼠心肌梗死后心力衰竭动物模型，进行了PD1抑制剂干预，利用心脏超声以及天狼星红评估心功能，发现PD1抑制剂可降低心肌梗死后成心脏纤维细胞的转分化水平。不仅为心肌梗死提供了一种治疗药物或者治疗策略，同时也为心脏成纤维细胞的转分化研究提供了一种工具药。
附图说明
图1为本发明实施例中PD1+T细胞与成纤维细胞共培养对成纤维细胞转分化水平的影响图。
图2为本发明实施例中PD1+T细胞对心脏炎症反应和纤维化的影响图。
图3为本发明实施例中PD1抑制剂对心梗后心功能的影响图。
图4为本发明实施例中PD1抑制剂对心梗后纤维化的影响图。
图5为本发明实施例中PD1抑制剂通过抑制炎症因子分泌抑制纤维化的影响图。
图6为本发明实施例中PD1抑制剂对非人灵长类动物食蟹猴心梗后远端区域的纤维化水平的影响图。
具体实施方式
下面结合实施例对本发明作进一步的描述。
总实施例
本发明提供了一种PD1抑制剂在制备心脏成纤维细胞转分化抑制剂中的用途。具体可用于科学研究的工具药，或者是用于治疗心肌梗死的药物。
作为优选，PD1抑制剂选自纳武单抗、帕博利珠单抗和T药（Tecentrip）；进一步优选为纳武单抗。
作为优选，所述工具药或药物包括所述PD1抑制剂，以及药学上可接受的载体和/或赋形剂。进一步优选，所述工具药或药物为注射制剂，最优选为静脉注射制剂。
实施例1（体外细胞实验）
（1）分离乳鼠心脏成纤维细胞，将第1代乳鼠心脏成纤维细胞在37℃、CO2体积浓度5％、空气体积浓度95％的混合气体的条件下常规培养于含10％FBS(v/v的低糖DMEM培养基中，一旦汇集，采用0.25％胰酶(w/v)-0.02％乙二胺四乙酸(EDTA)(w/v)消化传代。
（2）待乳鼠心脏成纤维细胞生长至50％汇合度，去除常规的含10％FBS的细胞培养基，采用PBS洗涤3次以上，置于37℃，含5％CO2和饱和湿度的细胞培养箱(Forma 3111，美国Thermo Fisher 公司) 内培养1天，每日更换培养基。
（3）流式分选心肌梗死中PD1+ T 和PD1-T 细胞，收集心肌梗死后7天T细胞，红细胞裂解液（索莱宝）裂红10分钟，于室温1200rpm离心5分钟，弃去上清，并添加 PBS 缓冲液离心洗涤2次，最后重悬于含1%BSA的PBS缓冲液中，制备成5×105/100μl 细胞悬液，常温封闭20分钟，随后依次添加抗小鼠 CD45，CD3，CD279抗体及其同型对照抗体 (美国BIOLEGEND公司)，避光常温孵育20分钟，1500rpm离心5 分钟去除抗体，添加PBS缓冲液离心洗涤2 次，最后添加 500μl PBS缓冲液重悬后进行流式细胞仪(美国BD公司) 分选。
（4）分选后的T细胞分别与乳鼠心脏成纤维细胞共培养，将T细胞置于Tran'swell( 美国 Costar 公司，孔径为 0.4μm)的上室，浓度为2*105/ml，培养基为1640培养基。同时在tran’swell的上室上清中10ug/ml 的PD1抑制剂(BIoX cell)，处理48小时，乳鼠心脏成纤维细胞放于transwell板的下室。
（5）共培养48小时后利用收集下室的乳鼠心脏成纤维细胞生长，通过WesternBlot检测乳鼠心脏成纤维的转分化水平。
结果见图1所示：PD1+T细胞较PD1-T细胞能明显促进乳鼠心脏成纤维细胞转分化蛋白Collagen 3 、Periostin、Asma的高表达，PD1抑制剂能够抑制PD1+T引起的乳鼠心脏成纤维细胞转分化；因此PD1+T细胞较能够促进乳鼠心脏成纤维细胞转分化。
实施例2（小鼠体内实验）
（1）治疗小鼠心肌梗死后的模型用药包括：建立心肌梗死模型提前24小时给予腹腔注射400ug/20g的PD1抑制剂(BIoX cell)，并对小鼠前胸进行脱毛备皮。建立心肌梗死模型，将小鼠用4%戊巴比妥（10mg/0.8ml）麻醉，待其处于麻醉状态后固定于鼠板上，然后使用气管插管，并连接呼吸机。调整呼吸机频率为98次/分，潮气量为1.2-1.5ml。用剪刀剪开小鼠左侧胸廓表面皮肤，使用镊子钝性分离胸前肌肉，用剪刀沿左侧3-4肋剪开，然后使用小动物胸撑逐步撑开肋骨，充分暴露心脏。撕开心包，可见左冠状动脉前降支走行于左心耳与肺动脉圆锥交界处。使用7-0 prolene线在左心耳下2mm处结扎前降支。心肌梗死模型成功建立后，分别每间隔3天通过腹腔注射给与PD1抑制剂(BIoX cell)，总疗程时间28天。并且在心肌梗死术后3天，7天，14天，28天，通过心脏超声检测心功能。
（2）心梗术后28天后，将小鼠使用4%戊巴比妥（10mg/0.8ml）麻醉，固定在鼠板上。开腹，暴露下腔静脉，静脉注射5mg/ml 氯化钾100ul，使心脏停跳在舒张期。开胸，使用棉签轻轻剥离梗死处与肋骨粘合的心脏。用剪刀将心脏右心耳剪开。用1ml注射器将 PBS缓冲液从左心室心尖注入小鼠心脏进行灌流，直到小鼠肝脏和肺脏颜色变白为止。沿主动脉根部取出心脏。放入30%蔗糖包埋，做组织冰冻病理切片用。病理切片马松染色后用使用体式显微镜拍照来评估心脏梗死面积。使用Imagepro plus 软件画出蓝色和红色的内膜和外膜长度。心肌梗死面积为：（心梗内膜+外膜）/（总内膜+外膜）。每个心脏取4个section进行统计。
如图2所示，回输PD1+T能够明显增加心脏的梗死面积，并且使心功能恶化；图3显示：建立心肌梗死模型后，给予PD1抑制剂治疗后能够改善心脏的收缩功能；图4显示建立心肌梗死模型后，给予PD1抑制剂治疗后，减少心肌梗死的面积，同时能够减少心肌梗死后心脏远端区的纤维化，这说明PD1抑制剂能够抑制心肌梗死炎症反应和心肌梗死后心脏纤维化；机制探讨方面如图5所示；通过分选PD1+T和PD1-T细胞进行炎症因子测序，发现PD1+T细胞高表达促成纤维细胞转分化的分子MIG；进一步通过用MIG抑制CXCR3来阻滞MIG的功能，发现阻滞了PD1+T分泌的炎症因子能够明显改善成纤维细胞的转分化水平，这说明PD1抑制剂能够通过抑制炎症因子分泌进一步抑制心脏成纤维细胞转分化。
实施例3（食蟹猴体内试验）
（1）食蟹猴心肌梗死模型建立：食蟹猴备皮，诱导麻醉后将其仰卧位固定于操作台上，随后气管插管，连接呼吸机辅助通气。手术野皮肤以碘伏消毒后，逐层开胸，于第3肋间隙进胸去心包后暴露心脏，用6-0尼龙线永久结扎冠状动脉前降支，可见结扎线以下区域颜色立即变苍白，局部心肌活动度降低，此时可视为心梗模型建立成功。手术结束后，用4-0聚乙烯缝合线缝合心包及胸膜，用10-0丝线缝合胸骨，然后用1-0丝线缝合皮肤切口。在自主呼吸恢复后拔出猴子气管内插管。
（2）体内动物实验分组：a、MI（心肌梗塞）组：MI模型前静脉注射生理盐水；b、MI+(纳武单抗，10 mg/kg) ，给药时间（造模前24小时、造模后第14天，静脉注射）
（3）指标评价：
A. 心功能评价：分别于模型构建前 1 天，纳武单抗治疗后 3 天、28 天，采用心脏MRI及心脏超声检测各组猴子左室射血分数（EF）和左室短轴缩短率（FS）评价心功能情况，左室收缩末直径和舒张末直径评价心腔扩张情况，通过心脏MRI延迟后显像扫描评价心肌梗死面积情况；
B.术前/抑制剂注射后1天、3天、7天、14、28天抽取血液，检测血常规、心肌酶谱、肝肾功能、甲状腺功能、抑制剂血药浓度，进一步评估抑制剂治疗心梗的安全性；
C. 取28天的心脏组织，将心脏从心尖-至心底等距离分成5份，利用常光显微镜进行拍照，评估心脏梗死面积大小。随后将心脏每等分按照梗死区域、梗死周边区域、梗死远端区域进行分割，分别于液氮、福尔马林、30%蔗糖各保存一份。
利用Western blot（asma periostin fibronectin）、Masson、天狼猩红等方法比较上述各组的心脏组织纤维化的情况，评价抑制剂治疗心肌梗死过程中对心脏纤维化的影响。
图6信息提示在食蟹猴模型中，在建立心肌梗死模型同时给予PD1抑制剂治疗，发现PD1抑制剂能够明显改善食蟹猴心肌梗死的心功能，通过天狼星红评估心肌梗死远端区的纤维化，发现PD1抑制剂能够改善心肌梗死后远端区域的纤维化水平。通过Western blot检测到给予PD1抑制剂后心肌梗死远端区域的转分化表达水平（asma periostinfibronectin）明显下降，这说明PD1抑制剂同样也能够抑制食蟹猴中心脏重塑后的过度纤维化。
本发明中所用原料、设备，若无特别说明，均为本领域的常用原料、设备；本发明中所用方法，若无特别说明，均为本领域的常规方法。
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制，凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效变换，均仍属于本发明技术方案的保护范围。
Claims (7)
1.PD1抑制剂在制备心脏成纤维细胞转分化抑制剂中的用途。
2.如权利要求1所述的用途，其特征在于：所述心脏成纤维细胞转分化抑制剂为用于科学研究的工具药，或者是用于治疗心肌梗死的药物。
3.如权利要求1所述的用途，其特征在于：所述PD1抑制剂为纳武单抗、帕博利珠单抗或T药。
4.如权利要求1所述的用途，其特征在于：所述PD1抑制剂为纳武单抗。
5.如权利要求2-4之一所述的用途，其特征在于：所述工具药或药物包括所述PD1抑制剂，以及药学上可接受的载体和/或赋形剂。
6.如权利要求5所述的用途，其特征在于：所述工具药或药物为注射制剂。
7.如权利要求6所述的用途，其特征在于：所述注射制剂为静脉注射制剂。
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