CN114832091A - Neogenin在制备预防、缓解和/或治疗心肌梗死及其相关疾病药物中的应用 - Google Patents
Neogenin在制备预防、缓解和/或治疗心肌梗死及其相关疾病药物中的应用 Download PDFInfo
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Abstract
本发明公开了Neogenin在制备预防、缓解和/或治疗心肌梗死及其相关疾病药物中的应用,属于基因的功能与应用领域。本发明通过研究发现Neogenin(Neo1)中和抗体或过表达Neo1能加重或减轻心肌梗死后心肌重构、恶化或改善心脏功能,表明Neo1在心肌梗死中表现为保护作用,以Neo1为靶点或Neo1可用于制备预防、缓解和/或治疗心肌梗死及其相关疾病的药物,其中心肌梗死及其相关疾病包括急性ST段抬高型心肌梗死、急性非ST段抬高型心肌梗死、缺血性心肌病、心力衰竭等;制药应用包括Neo1、Neo1激活剂或促进Neo1表达的物质作为药物。本发明为预防、缓解和/或治疗心肌梗死提供了新药物。
Description
技术领域
本发明属于基因的功能与应用领域,涉及一种免疫球蛋白超家族的多功能跨膜受体 neogenin(Neo1),具体来说,是以Neo1为干预靶点在制备预防、缓解和/或治疗心肌梗死及其相关疾病药物中的应用。
背景技术
心肌梗死(简写MI)是指因冠脉血流急剧减少或中断引起心肌细胞持久缺血缺氧,最终导致心肌细胞不可逆坏死。目前,我国急诊PCI治疗率的明显增加并没有明显遏制死亡率的上升,心肌梗死后患者的生存质量仍然不容乐观[1]。心肌梗死后病理性心肌重构可导致恶性心律失常、心力衰竭及猝死等不良事件的发生,是影响心肌梗死患者预后的重要因素[2,3]。因此,干预心肌梗死后病理性心肌重构在改善心肌梗死患者预后方面具有重要作用。
Neogenin(Neo1)属于免疫球蛋白(Ig)超家族的多功能跨膜受体,由4个免疫球蛋白样区域和6个纤维蛋白FNIII功能区组成的胞外域、1个跨膜区和1个胞内域,是神经导向蛋白家族中神经导向因子Netrins和排斥性导向分子RGMs的受体[4]。Neo1与Netrin1结合可诱导趋化性轴突导向反应,而与RGMa相互作用则导致排斥性轴突导向反应[5]。研究表明,Neo1在多种免疫细胞中也有表达[6,7]。然而,Neo1在心肌梗死中的作用尚不明确。
参考文献:
1.中国心血管健康与疾病报告编写组.中国心血管健康与疾病报告2019概要.中国循环杂志.2020.35:833-854.
2.Anderson,J.L.and D.A.Morrow.Acute Myocardial Infarction.N Engl JMed.2017.376: 2053-2064.
3.Thygesen K,Alpert JS,Jaffe AS,Chaitman BR,Bax JJ,Morrow DA,WhiteHD,and Executive Group on behalf of the Joint European Society of Cardiology/American College of Cardiology /American Heart Association/World HeartFederation Task Force for the Universal Definition of Myocardial.FourthUniversal Definition of Myocardial Infarction(2018).J Am Coll Cardiol.2018.72:2231-2264.
4.Yamagishi S,Bando Y and Sato K.Involvement of Netrins and TheirReceptors in Neuronal Migration in the Cerebral Cortex.Front Cell DevBiol.2020.8:590009.
5.De Vries M,Cooper HM.Emerging roles for neogenin and its ligands inCNS development.J Neurochem.2008.106:1483-92.
6.Muramatsu R,Kubo T,Mori M,Nakamura Y,Fujita Y,Akutsu T,Okuno T,Taniguchi J, Kumanogoh A,Yoshida M,Mochizuki H,Kuwabara S and YamashitaT.RGMa modulates T cell responses and is involved in autoimmuneencephalomyelitis.Nat Med.2011.17:488-94.
发明内容
目前临床上治疗心肌梗死仍存在不足,仍需要新颖的治疗手段改善心肌梗死患者预后。本发明阐述并明确了Neo1中和抗体可加重心肌梗死后病理性心室重构,恶化心脏功能;过表达Neo1可减轻心肌梗死后病理性心室重构,改善心脏功能,因此Neo1在心肌梗死中起保护作用。提供了一种以Neo1为靶点在制备预防、缓解和/或治疗心肌梗死及其相关疾病药物中的新应用。
本发明的目的通过下述技术方案实现:
本发明以C57BL/6小鼠为实验对象,通过永久性结扎左前降支动脉构建心肌梗死模型,通过术前尾静脉注射给予Neo1中和抗体(Anti-Neo1),以完成Neo1在体功能抑制,心肌梗死3天后评估心脏功能和心肌重构。本发明还构建了Neo1过表达腺病毒(Ad-Neo1),以C57BL/6小鼠为实验对象,通过永久性结扎左前降支动脉构建急性心肌梗死模型,通过心肌注射腺病毒,实现心脏Neo1过表达,心肌梗死2周后评估心脏功能和心肌重构。结果表明,Neo1中和抗体加重心肌梗死后心肌重构,恶化心脏功能;Neo1过表达减轻心肌梗死后心肌重构,改善心脏功能,因此Neo1在心肌梗死中表现为保护作用。本发明提供了一种以Neo1为靶点在制备预防、缓解和/或治疗心肌梗死及其相关疾病药物中的新应用。本发明还提供Neo1在制备预防、缓解和/或治疗心肌梗死及其相关疾病药物中的新应用。上述制药应用包括Neo1作为药物、Neo1激活剂作为药物、促进Neo1表达的物质作为药物等。
一种预防、缓解和/或治疗心肌梗死及其相关疾病的药物,包含Neo1、Neo1激活剂或促进Neo1表达的物质等,以及药学上可接受的载体或赋形剂。
所述心肌梗死及其相关疾病包括但不限于:急性ST段抬高型心肌梗死,急性非ST段抬高型心肌梗死,缺血性心肌病,心力衰竭等。
本发明所提供的Neo1为靶点的预防、缓解和/或治疗心肌梗死及其相关疾病药物具有如下优点:
(1)中和抗体敲低Neo1可加重心肌梗死后心肌重构和心脏功能,Neo1过表达减轻心肌梗死后心肌重构、改善心脏功能,Neo1在心肌梗死中表现为显著的保护作用。
(2)以Neo1为靶点,开发特异性激活剂或过表达Neo1有望为心肌梗死的预防、缓解和/或治疗提供新的药物。
附图说明
图1是Neo1中和抗体加重心肌梗死后心脏功能的结果图。A:心脏超声代表图像;B:左心室舒张末径(LVEDD);C:左室收缩末径(LVESD);D:左室射血分数(LVEF);E:左室射短分数(LVFS)。
图2是Neo1中和抗体增加梗死面积、加重梗死后心肌重构的结果图。A:心重(HW) /体重(BW);B:心重(HW)/胫骨长度(TL);C:TTC染色梗死面积效果图;D:梗死面积柱状图;E-F:ANP、BNP的mRNA水平;G-H:胶原Ⅰ、胶原Ⅲ的mRNA水平;I-L:胶原Ⅰ、胶原Ⅲ、α-SMA的Western blot条带和统计结果。
图3是Neo1中和抗体加重心肌梗死后心肌细胞凋亡的结果图。A-D:凋亡相关蛋白Bax、Bcl-2代表性WB条带以及定量结果柱状图;E:c-caspase3荧光图;F:TUNEL+凋亡细胞荧光图。
图4是Neo1过表达改善心肌梗死后心脏功能的结果图。A:左心室舒张末径(LVEDD); B:左室收缩末径(LVESD);C:左室射血分数(LVEF);D:左室射短分数(LVFS)。
图5是Neo1过表达减轻梗死后心肌重构的结果图。A:HE染色显示心肌梗死面积(左心室乳头肌水平);B:心肌梗死面积统计结果;C:PSR染色显示梗死后心肌纤维化面积; D:梗死后心肌纤维化面积统计结果。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实验动物及饲养:
C57BL/6小鼠,雄性,6周龄,购自江苏集萃药康。饲养在武汉大学人民医院标准化实验动物中心(SPF级),饲养条件:温度在22-24℃之间,湿度在40-70%之间,明暗交替照明时间为12h,自由饮水摄食。
实验例1 Neo1中和抗体加重心肌梗死后心脏功能
1、注射Neo1中和抗体
Neo1中和抗体(货号AF1079)购买自美国RD公司,能够有效结合受体Neo1,阻断Neo1与其相关配体的相互作用,完成在体Neo1功能抑制。心肌梗死术前2小时,固定小鼠,通过尾静脉注射Neo1中和抗体(溶剂为生理盐水),剂量2μg/小鼠,对照组给予安慰剂(vehicle,生理盐水)。实验遵循随机、盲法原则分为4组:未手术(sham)+vehicle、 sham+Anti-Neo1、心肌梗死组(MI)+vehicle、MI+Anti-Neo1。
2、心肌梗死模型获得
(1)3%戊巴比妥钠90mg/kg腹腔注射麻醉,用小鼠剃毛器剃除小鼠胸部及腋下毛发(充分暴露手术区),用碘酒和75%乙醇手术区消毒。
(2)气管插管:麻醉后,夹趾检测无反应即可进行MI手术。打开外置光源、显微镜开关,打开呼吸机,设置好各参数(呼吸频率110bpm),将气管插管沿声门插入气管,取下小鼠接上呼吸机,观察小鼠呼吸状况,胸廓起伏与呼吸机频率一致表示插管成功,即可进行 MI手术。
(3)小鼠采用右侧卧位,用眼科剪在左前肢腋下,用显微剪于三、四肋间打开胸腔充分暴露心脏,显微直镊轻轻夹起少量心包并于左心耳下撕开少许心包,充分暴露左冠状动脉前降支(LAD)或所在区域。
(4)结扎冠状动脉:于显微镜下找到LAD走向或可能所在位置,持针器持取7-0带针缝合线,于左心耳下缘2mm处进针,缝线穿过LAD,以完全阻断LAD血流。
(5)关胸:结扎完成后,6-0缝线完全缝合胸腔开口(保证无缝隙、无错位)关闭胸腔,由内向外逐层缝合各层肌肉和皮肤。
(6)术后管理:术后密切关注小鼠状态,有无呼吸异常等。待小鼠自然苏醒后将小鼠从呼吸机上取下并取下气管插管,正常饲养。于MI术后3天取材。
3、心脏功能评估
(1)心肌梗死术后3天,小鼠处死前,用剃毛刀剔除胸前鼠毛。
(2)1.5%异氟烷麻醉,使用超声仪(VINNO 6,飞依诺科技有限公司,苏州,中国)行小鼠超声心脏检查;短轴测心率(HR)、左室舒张末径(LVEDD)、左室收缩末径(LVESD)、左室射血分数(LVEF)左室射短分数(LVFS)。
心脏超声结果如图1所示。Neo1中和抗体加重心肌梗死后心脏功能(图1A-E)。
实验例2 Neo1中和抗体增加梗死面积,加重心肌梗死后心肌重构
1、2,3,5-三苯基氯化四氮唑(2,3,5-Triphenyltetrazolium chloricej,TTC)染色
(1)心肌梗死术后3天处死小鼠后,快速取出心脏组织,置于10%KCl溶液。
(2)清洗心脏后,置于-20℃冰箱30分钟。
(3)取出心脏组织,由心尖至心底部沿心脏长轴均匀切成切片,将切片立即置于10mL 2%TTC溶液中,37℃恒温孵育20min。正常心脏组织染色后呈鲜红色,而梗死区呈苍白色。
(4)用10%中性福尔马林溶液固定脑组织切片,大体拍照,计算梗死面积。
2、心肌梗死后心肌重构评估
2.1 qRT-PCR检测心肌重构
2.1.1 RNA提取
(1)准备物品:将提取RNA所需剪刀、镊子、研磨EP管、EP管、纱布、钢珠、枪头等物品提前高压蒸汽灭菌,然后放置在55℃烘箱烘干,最后保存在无菌除酶柜里。
(2)提前准备所需数量的研磨管和钢珠,每个研磨管中放入4颗钢珠,放置在冰上预冷。同时提前预冷研磨适配器。将心脏组织从-80℃冰箱取出放在干冰上,借助剪刀和镊子,称取30mg组织放置在研磨管中,并标记编号。每管加入1mL TRIzol,并混合均匀。
(3)研磨:使用研磨仪研磨组织,频率30Hz/s,时间180s。同时提前预冷高速离心机。
(4)研磨结束后转移研磨液,然后冰上静置10min。
(5)抽提:三氯甲烷200μL/EP管。充分混匀,室温静置5min。之后,12000g、4℃离心15min。缓慢吸取上层清液(上层为RNA)转移至新的1.2mL EP管中,记录体积,注意枪头不能碰到中间层的白色蛋白质,如果碰到重新离心。
(6)RNA沉淀:加入异丙醇(等体积),混匀静置5min,再次离心10min,参数同上。同时,配制75%乙醇备用。
(7)漂洗RNA沉淀:离心后可见白色RNA沉淀。弃去上清液,加入75%乙醇加入,每管500μL或1mL。再次离心5分钟,条件同上。重复此步骤2-3次。
(8)干燥RNA沉淀:离心后尽量吸净上清液(推荐依次使用1mL移液器、100μL移液器和10μL移液器)。为避免RNA降解,PE手套盖住管口,室温干燥10min。干燥同时打开水浴锅,温度设置为55℃。
(9)溶解RNA沉淀:12-15μL DEPC水/EP管,55℃水浴10min。
(10)检测RNA的浓度和纯度:使用NanoDrop2000分光光度计和软件。使用DEPC 水清洗点样孔并校正仪器,直到DEPC水检测浓度处于±1ng/μL,曲线基本处于底部水平位置。每管1μL样品,检测RNA浓度,A260/A280比值和A260/A230比值。
2.1.2逆转录RNA合成cDNA
(1)提前解冻Roche公司的RNA逆转录试剂盒。
(2)第一步反应体系:共13μL,依次加入RNA模板xμL(2000ng/RNA浓度),PCR 水(10-x)μL,⑤dT primer 1μL和⑥Random primer 2μL。
(3)涡旋混匀器充分混匀,离心,PCR仪中行第一步PCR反应,程序为70℃,10min。注意:反应结束后应立即取出置于冰上,10min。
(4)第二步反应体系(7μL):冷却同时,配制第二步反应体系:②RT-bμffer 4μL,④dNTP 2μL,③inhibitor 0.5μL,①RTase 0.5μL。配制混合液,然后将7μL混合液加入每个PCR 管中。行第二步逆转录反应,Roche推荐程序为:25℃10min→50℃60min→90℃5min→4℃ 1min。反应结束后,每管加入60μL PCR水(也可以根据情况不加PCR水,先行保存备用) -80℃冰箱保存。
2.1.3 qRT-PCR
(1)设计引物,引物序列见表1:
表1
基因 | Forward premier(5’-3’) | Reverse premier(5’-3’) |
ANP | ACCTGCTAGACCACCTGGAG | CCTTGGCTGTTATCTTCGGTACCGG |
BNP | GAGGTCACTCCTATCCTCTGG | GCCATTTCCTCCGACTTTTCTC |
CollagenⅠ | ACTGCAACATGGAGACAGGTCAGA | ATCGGTCATGCTCTCTCCAAACCA |
CollagenⅢ | GGAACCTGGTTTCTTCTCACC | TAGGACTGACCAAGGTGGCT |
GAPDH | ACTCCACTCACGGCAAATTC | TCTCCATGGTGGTGAAGACA |
(2)解冻cDNA、Mix和引物,打开离心机4℃预冷。
(3)配制混合液:
SYBR Green I Master 10.0μL×3.2×n.5;
PCR水 8.0μL×3.2×n.5;
引物(上+下) 1.0μL×3.2×n.5。
n为样本数量,3.2表示3个副孔。
(4)充分混匀后,将混合液均分至新的600μL EP管中,60.8μL/管。然后每个新的EP管中加入对应cDNA模板3.2μL,充分混匀。
(5)先行布板,然后点样,20μL/孔,共3个副孔。加样完成后,封板膜封板,配套离心机离心,3000rpm/min、4℃离心2min。
(6)Roche高通量RT-PCR仪扩增,程序为:预变性(95℃5min)→扩增(95℃10s→60℃ 10s→72℃30s,40个循环)→熔解曲线。
(7)内参GAPDH,2-ΔΔCT法计算结果。
2.2 Western blot检测纤维化
2.2.1组织蛋白提取
(1)剪取适量左心室梗死交界区组织(约30mg)放入对应的EP管中,称量并记录每个样本的重量,同时打开离心机将温度预冷至4℃。
(2)按照按100mg/1mL计算所需裂解液总量。其中1mL裂解液包含720μL RIPA、100μL Phosstop(磷酸酶抑制剂)、100μL Complete(蛋白酶抑制剂)、50μL NaF、10μL Na3VO4和20μL PMSF(丝氨酸/半胱氨酸蛋白酶抑制剂,最后加入)。将裂解液分别加入到EP管中。
(3)提前预冷研磨适配器,将装有样品的EP管对称放进研磨适配器中,并将研磨适配器安装在研磨仪上,研磨仪的程序设置为30Hz/s,90s。
(4)研磨结束后取出EP管,稍离心后将溶液转移至新的1.5mL EP管中,静置10min。
(5)超声裂解仪裂解样品,每次1-2s,中间间隔1s,重复10-20次,频率设置为5KHz。
(6)将样品放入离心机中,4℃条件下12000g离心30min。离心后取出样品,吸取上清液并转移至新的EP管中,记录上清液体积。
2.2.2组织蛋白定量(BCA法)
(1)配制工作液(按A液:B液50:1的比例)。
(2)配制标准品:准备6个200μL的小EP管,依次标记为A、B、C、D、E和F。A管标准品的浓度1000μg/mL,B管标准品的浓度500μg/mL,C管标准品的浓度250μg/mL,D管标准品的浓度125μg/mL,E管标准品的浓度25μg/mL,F管浓度为0μg/mL。
(3)吸取4μL待测样品并分别加入装有116μL去离子水的EP管中(稀释30倍),混匀后备用。
(4)按25μL/孔吸取标准品或待测样品,加入到96孔板中,同时做2个复孔。每孔均加入200μL的工作液,37℃恒温箱中孵育30min。
(5)96孔板置于酶标仪中,测量所有样品在562nm波长处的吸光度值,计算标准曲线和待测样品的总蛋白浓度。
(6)根据待测样品的总蛋白浓度进行定量处理,计算待测样品的终体积。加入相应量的5×蛋白上样缓冲液和去离子水。
(7)将样品混匀后分装,放入72℃水浴锅中水浴10min以促进蛋白变性(每2-3min翻动一次),结束后置于-80℃冰箱中备用。
2.2.3制备SDS-PAGE凝胶电泳
(1)清洗制胶架和玻璃板,将玻璃板安装在制胶架上,其中厚玻璃板朝内,薄玻璃板朝外。加满去离子水测试渗漏情况,若无渗漏,则缓慢倒出去离子水,滤纸吸尽水滴后备用。
(2)准备2个50mL的离心管,分别用记号笔写上分离胶和浓缩胶。根据目的蛋白的分子量配制适宜浓度的分离胶(按5mL/玻璃板配制),依次向标有分离胶的离心管中加入去离子水、30%丙烯酰胺、1.5M三羟甲基氨基甲烷-盐酸(Tris-HCl)、10%十二烷基硫酸钠(SDS)、过硫酸铵(AP)和四甲基乙二胺(TEMED),最后加入)。加入TEMED后漩涡混匀器混匀分离胶,将分离胶缓慢加入玻璃板中。缓慢注入无水乙醇封顶(注意不要把胶冲散),待分离胶凝结后倒出乙醇,残留乙醇可用滤纸吸尽。
(3)按照说明书制备5%浓缩胶(按1.25mL/玻璃板配制):依次向标有浓缩胶的离心管中加入去离子水、30%丙烯酰胺、1M Tris-HCl、10%SDS、AP和TEMED(促凝剂,最后加入),加入TEMED后立即用漩涡混匀器混匀浓缩胶,将浓缩胶缓慢加入玻璃板中,将齿梳插入浓缩胶中,同时避免气泡产生,待浓缩胶凝固后取下玻璃板,置于4℃冰箱备用。
(4)上样跑胶:于4℃冰箱取出玻璃板,拔出齿梳,将厚玻璃板侧朝外,固定在电泳架上。将电泳架放进电泳槽中,向内槽中缓慢加入1×电泳内液(约150mL),向外槽倒入缓慢电泳外液。
(5)按10μL/孔的量将蛋白依次加入到SDS-PAGE凝胶加样孔,其中第一个孔先加入5μL 的蛋白Marker,再加入5μL的1×蛋白上样缓冲液。初始75V恒压电泳,待蛋白到达分离胶时加压至120V,继续电泳。
(6)提前配制转膜液,置于4℃冰箱中预冷。同时根据所需大小裁剪PVDF膜。
(7)电泳结束后取出凝胶,裁掉多余部分并将凝胶放入转膜液中。将夹板左右摊开,两边各铺上海绵和滤纸,铺平后润湿,夹板的黑色面(负极)朝右。将凝胶平铺在黑色面的滤纸上,大分子量侧朝内。将PVDF膜做好标记后,在甲醇中浸泡1min并覆盖在凝胶上,注意各层之间不留气泡,合上夹板。
(8)将夹板固定在转膜槽中,缓慢倒入转膜液。将转膜槽置于盛有碎冰的泡沫盒中, 0.2A恒流转膜约1.5h(可根据蛋白分子量调整转膜时间)。
(9)转膜结束后,取出PVDF膜,TBST液洗膜3次,每次5min。之后将PVDF膜置于提前配制好的封闭液中,在脱色摇床上缓慢摇动,室温封闭1h。
(10)封闭结束后,TBST液洗膜3次,每次5min。将PVDF膜放入孵育盒中,加入适量的一抗(CollagenⅠ,CollagenⅢ,α-SMA,GAPDH)。将孵育盒置于摇床上缓慢摇动,4℃条件下孵育过夜。
(11)一抗孵育后,TBST液洗膜3次,每次5min,同时回收一抗。将PVDF膜放入孵育盒中,加入适量的二抗,室温条件下避光孵育1h。
(12)二抗孵育后,TBST液洗膜3次,每次5min,同时回收二抗。使用Odyssy双色红外成像系统扫描PVDF膜,分析目的条带的表达情况。
心脏梗死面积和心肌重构结果如图2所示。Neo1中和抗体未能明显升高心重/体重和心重 /胫骨长度(图2A-B),但能够增加梗死面积(图2C-D),加重心肌梗死后心肌重构(图2E-F) 和心肌纤维化(图2G-L)。
实验例3Neo1中和抗体加重心肌梗死后心肌细胞凋亡
3.1 Western Blot监测凋亡相关蛋白
蛋白提取,电泳凝胶同上,使用一抗为Bax、Bcl-2和GAPDH。
3.2免疫荧光
3.2.1制备石蜡标本及切片
(1)将小鼠心脏组织从10%福尔马林溶液取出,在通风橱内修剪心脏组织,并将修剪好的心脏组织和相对应的标签置于脱水盒中。
(2)脱水浸蜡:将脱水盒放进脱水机中,进行脱水浸蜡处理。75%乙醇(4h)→85%乙醇(2h)→90%乙醇(2h)→95%乙醇(1h)→无水乙醇I(30min)→无水乙醇II(30min) →醇苯(5-10min)→二甲苯I(5-10min)→二甲苯II(5-10min)→65℃融化石蜡I(1h)→65℃融化石蜡II(1h)→65℃融化石蜡III(1h)。
(3)心脏组织包埋:先将融化的石蜡注入进包埋框中,趁石蜡未凝固时,从脱水盒内将心脏组织取出,心底部朝下放入包埋框中,并贴上相对应的标签,水平置于-20℃冻台上冷却。待石蜡凝固后,从包埋框中将蜡块取出。
(4)心脏组织切片:将蜡块进行修整并固定在石蜡切片机上,切片厚度为4-5μm。转动切片机手柄连续切片,使切片漂浮于温水中并将切片展平。用载玻片将切片捞起,使切片位于载玻片的正中位置,置于60℃烘箱内烘烤,保存备用。
3.2.2石蜡切片依次经过以下步骤,完成免疫荧光检测与分析。
(1)脱蜡至水:依次将心脏组织切片放入一下试剂:二甲苯Ⅰ-二甲苯Ⅱ-无水乙醇Ⅰ- 无水乙醇Ⅱ-85%乙醇-75%乙醇,分别需要20分钟,20分钟,5分钟,5分钟,5分钟,5分钟。最后纯水清洗。
(2)抗原修复:滴蛋白酶K,37℃25分钟,PBS洗3次。
(3)血清封闭:山羊血清封闭切片30分钟。
(4)一抗孵育:PBS洗3次,滴加一抗(c-caspase3),4℃孵育过夜。注意参考说明书确定抗体最佳稀释比例。
(5)二抗孵育。
(6)DAPI复染细胞核。
(7)封片。
(8)使用奥林巴斯荧光显微镜观察切片。
3.3 TUNEL测定心肌细胞凋亡
(1)石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ20min-二甲苯Ⅱ20min-无水乙醇Ⅰ 5min-无水乙醇Ⅱ5min-85%酒精5min-75%酒精5min-蒸馏水洗。
(2)修复:切片稍甩干后用组化笔在组织周围画圈(防止液体流走),在圈内滴加蛋白酶K工作液覆盖组织,37℃温箱孵育25min。将玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次,每次5min。
(3)破膜:切片稍甩干后在圈内滴加破膜工作液覆盖组织,常温下孵育20min,将玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次,每次5min。
(4)加试剂1、2:按片子数量和组织大小取TUNEL试剂盒内适量试剂1(TdT)和试剂2(dUTP)按1:9混合,加到圈内覆盖组织,切片平放于湿盒内,37℃温箱孵育2小时,湿盒内加少量水保持湿度。
(5)血清封闭:将玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次,每次5min。切片稍甩干后,在圈内滴加山羊血清室温孵育30min。山羊血清可结合组织中的非特异性结合位点,防止对POD的吸附作用,以及非特异性产生。
(6)阻断内源性过氧化物酶:将玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次,每次5min。切片放入3%双氧水溶液,室温避光孵育15min,将玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次,每次5min。
(7)加试剂3:切片稍甩干后,每张切片加适量试剂3(converter-POD)覆盖组织,切片平放于湿盒内,37℃温箱孵育30min。将玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤 3次,每次5min。
(8)DAB显色:切片稍甩干后在圈内滴加新鲜配制的DAB显色液,显微镜下控制显色时间,阳性为细胞核呈棕黄色,自来水冲洗切片终止显色。
(9)复染细胞核:苏木素复染3min左右,自来水洗,苏木素分化液分化数秒,自来水冲洗,苏木素返蓝液返蓝,流水冲洗。
(10)脱水封片:将切片依次放入75%酒精5min-85%酒精5min--无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-二甲苯Ⅰ5min中脱水透明,将切片从二甲苯拿出来稍晾干,中性树胶封片。
(11)荧光镜下观察,拍照。若需保存,于暗湿盒中4℃保存。在荧光显微镜下观察,拍照,计数凋亡神经元细胞。
心脏组织梗死交界区心肌细胞凋亡情况测定结果如图3所示。研究发现Neo1中和抗体加重心肌梗死后心肌细胞凋亡,表现在促凋亡蛋白Bax表达增加、抑制凋亡蛋白Bcl-2表达减少(图3A-D)、促凋亡分子c-caspase3表达增加(图3E)、TUNEL+阳性细胞数量增加(图3F)。
研究结果表明,在急性心肌梗死模型中,Neo1中和抗体能够明显增加梗死面积,加重病理性心肌重构,恶化心脏功能。
实验例4 Neo1过表达改善心肌梗死后心脏功能
1、Neo1腺病毒构建
Neo1过表达腺病毒(Ad-Neo1)和对照病毒(Ad-NC)由汉恒生物科技有限公司构建和检测,病毒滴度1.26×1010pfu/mL。
2、心肌梗死模型获得及腺病毒心肌注射
(1)3%戊巴比妥钠90mg/kg腹腔注射麻醉,用小鼠剃毛器剃除小鼠胸部及腋下毛发(充分暴露手术区),用碘酒和75%乙醇手术区消毒。
(2)气管插管:麻醉后,夹趾检测无反应即可进行MI手术。打开外置光源、显微镜开关,打开呼吸机,设置好各参数(呼吸频率110bpm),将气管插管沿声门插入气管,取下小鼠接上呼吸机,观察小鼠呼吸状况,胸廓起伏与呼吸机频率一致表示插管成功,即可进行 MI手术。
(3)小鼠采用右侧卧位,用眼科剪在左前肢腋下,用显微剪于三、四肋间打开胸腔充分暴露心脏,显微直镊轻轻夹起少量心包并于左心耳下撕开少许心包,充分暴露左冠状动脉前降支(LAD)或所在区域。
(4)结扎冠状动脉:于显微镜下找到LAD走向或可能所在位置,持针器持取7-0带针缝合线,于左心耳下缘2mm处进针,缝线穿过LAD,以完全阻断LAD血流,可见血管远端心肌缺血。
(5)使用胰岛素注射针于缺血心肌(4个位点)和梗死交界区(2个位点)注射Neo1过表达腺病毒或对照病毒,总体积18μL。
(5)关胸:结扎完成后,6-0缝线完全缝合胸腔开口(保证无缝隙、无错位)关闭胸腔,由内向外逐层缝合各层肌肉和皮肤。
(6)术后管理:术后密切关注小鼠状态,有无呼吸异常等。待小鼠自然苏醒后将小鼠从呼吸机上取下并取下气管插管,正常饲养。于心肌梗死术后14天取材。
(7)分组:实验遵循随机、盲法原则分为4组:未手术(sham)+Ad-NC、sham+Ad-Neo1、心肌梗死组(MI)+Ad-NC、MI+Ad-Neo1。
3、心脏功能评估
(1)心肌梗死术后14天,小鼠处死前,用剃毛刀剔除胸前鼠毛。
(2)1.5%异氟烷麻醉,使用超声仪(VINNO 6,飞依诺科技有限公司,苏州,中国)行小鼠超声心脏检查;短轴测心率(HR)、左室舒张末径(LVEDD)、左室收缩末径(LVESD)、左室射血分数(LVEF)左室射短分数(LVFS)。
心脏超声结果如图4所示。Neo1过表达改善心肌梗死后心脏功能(图4A-E)。
实验例5Neo1过表达减小梗死面积,改善心肌梗死后心肌重构
1、苏木精-伊红染色(HE)
将石蜡标本块切为5μm厚的切片,依次进行以下操作:
(1)二甲苯(Ⅰ)15min;
(2)二甲苯(Ⅱ)15min;
(3)100%乙醇(Ⅰ)5min;
(4)100%乙醇(Ⅱ)5min;
(5)95%酒精5min
(6)90%酒精5min
(7)80%乙醇5min;
(8)蒸馏水5min;
(9)苏木精液染色5min;
(10)流水稍洗去苏木精液1-3s;
(11)1%盐酸乙醇1-3s;
(12)稍水洗10-30s;
(13)蒸馏水过洗1-2s;
(14)0.5%伊红液染色1-3min;
(15)蒸馏水稍洗1-2s;
(16)80%乙醇稍洗1-2s;
(17)95%乙醇(Ⅰ)2-3s;
(18)95%乙醇(Ⅱ)3-5s;
(19)无水乙醇(I)5-10min;
(20)无水乙醇(II)5-10min;
(21)二甲苯(Ⅰ)2min;
(22)二甲苯(Ⅱ)2min;
(23)二甲苯(Ⅲ)2min;
(24)中性树胶封固;
待玻片晾干后,使用光学显微镜拍照记录,并用Image-Pro Plus进行计算心肌梗死面积。
2、天狼猩红(PSR)染色
(1)将石蜡切片置于二甲苯溶液中5min,同样重复3次。
(2)相继经过100%、90%、70%的乙醇1次1min。
(3)用流水冲洗标本10min。
(4)冲洗完毕将标本置入纯水中清洗1min,再置于0.2%磷钼酸中1-5min。
(5)将切片摆放于潮湿的染色盒中,滴入数滴0.1%天狼猩红苦味酸溶液使其充分与标本接触,染色90min。
(6)充分甩去天狼猩红苦味酸溶液,再浸入纯水中数次。
(7)最后经过70%、95%乙醇各30秒,100%乙醇30秒3次。
(8)二甲苯2min重复3次,封片。
(9)染色后切片用光学显微镜拍照记录,并用Image-Pro Plus进行计算心肌纤维化面积。
心肌梗死面积和纤维化面积如图5所示,Neo1过表达减小心肌梗死面积,减轻梗死后心肌纤维化,表明Neo1过表达减轻心肌梗死后病理性心肌重构。
上述结果表明,在急性心肌梗死模型中,Neo1过表达能够明显减小梗死面积,减轻病理性心肌重构,改善心脏功能。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 武汉大学
<120> Neogenin在制备预防、缓解和/或治疗心肌梗死及其相关疾病药物中的应用
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Claims (4)
1.以Neo1为靶点在制备预防、缓解和/或治疗心肌梗死及其相关疾病的药物中的应用。
2.Neo1在制备预防、缓解和/或治疗心肌梗死及其相关疾病的药物中的应用。
3.根据权利要求1或2所述的应用,其特征在于:包括Neo1作为药物、Neo1激活剂作为药物、促进Neo1表达的物质作为药物。
4.根据权利要求1-3任一项所述的应用,其特征在于:所述的心肌梗死及其相关疾病包括:急性ST段抬高型心肌梗死,急性非ST段抬高型心肌梗死,缺血性心肌病,心力衰竭。
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