CN117018164A - Cpon的应用 - Google Patents
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- CN117018164A CN117018164A CN202310727048.1A CN202310727048A CN117018164A CN 117018164 A CN117018164 A CN 117018164A CN 202310727048 A CN202310727048 A CN 202310727048A CN 117018164 A CN117018164 A CN 117018164A
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Abstract
本发明提出了CPON的应用,属于生物医药技术领域。本发明发现神经肽Y的C端肽段‑CPON在常氧、缺氧和活性氧(ROS)环境中具有抑制心肌细胞凋亡的作用及其在心肌细胞冷冻保存中的应用;本发明通过在常氧、缺氧和活性氧(ROS)环境检测人心肌细胞的凋亡情况,首次发现CPON在以上环境中能抑制心肌细胞凋亡,具有心肌保护作用,并利用该作用开发了一种新型的针对人诱导多能干细胞衍生的心肌细胞(hiPSC‑CM)的冻存方法。
Description
技术领域
本发明属于生物医药技术领域,具体地说,本发明涉及神经肽Y的C端肽段-CPON在常氧、缺氧和活性氧(ROS)环境中具有抑制心肌细胞凋亡的作用及其在心肌细胞冷冻保存中的应用。
背景技术
心血管疾病(cardiovascular disease,CVD)是目前人类健康的头号杀手,全球每年死于心血管疾病的人数多于其它任何原因。随着人民生活水平的提高和饮食结构的变化,心血管疾病的患病率和死亡率仍呈明显上升趋势。
心肌细胞凋亡参与多种心血管疾病病理生理过程,包括原发性高血压、缺血性心脏病和再灌注损伤、心肌炎、心肌病、心律失常、心脏衰竭、先天性心脏病等。心肌细胞凋亡机能异常是发生多种重症心血管病的重要机制。通过干预细胞凋亡程序的进行,抑制心肌细胞凋亡的发生,挽救心脏和心功能,已成为心血管疾病药物研究的重要方向之一。目前临床上的相关药物有他汀类、β-受体阻断剂、血管紧张素转换酶抑制剂等,但存在不同程度的副作用。
由于人心肌细胞样本获取困难、原代细胞在体外培养的存活时间较短、在研究过程中存在伦理问题等原因,直接使用患者的心肌细胞进行心血管疾病方面的研究几乎不可能实现。近年来,随着iPS(诱导多能干细胞)技术的发展、心肌细胞分化及纯化方法的建立,体外制备及培养人心肌细胞技术逐渐完善。利用iPS体外诱导分化心肌细胞的方法,可以在体外制备足够数量的人心肌细胞进行各种功能实验,模拟心血管疾病的发生发展过程,并有开发成为新型细胞药物的巨大潜力,极大推动了针对心血管疾病的机制研究及治疗应用的进展。
冷冻保存是将细胞等生物样品置于超低温环境中,减少细胞代谢以实现长期储存,并能在解冻后恢复生物功能的技术。现有的心肌细胞保存方法存在复苏后细胞凋亡比例上升,活性下降的问题,这极大限制了利用心肌细胞进行心血管疾病等方面的研究和未来临床应用(如通用型细胞药物均需低温保存,使用前必须恢复到常温,巨大的温差会导致细胞活性下降)。
神经肽Y(neuropeptide Y,NPY)的C端肽段(the C-terminal flanking peptideof neuropeptide Y,CPON)是NPY成熟过程中的副产物。成熟的NPY(36个氨基酸残基)由NPY前体(Pre-Pro NPY,97个氨基酸残基)通过一系列的肽酶剪切和羧基末端酰胺化反应而产生,其中Pre-Pro NPY的C端的30个氨基酸残基被肽酶剪切后形成的多肽即为CPON。至1982年首次从猪的下丘脑组织中被分离以来,NPY已经被研究了40年,作为人体中含量最丰富的神经肽之一,其生理功能及相关机制已经得到基本阐明,PubMed数据库中的相关文献已超过15000篇。然而,作为经常在组织中与NPY共表达的CPON,其生物学功能一直未能得到证明。目前没有CPON具有生物活性的报道,唯一一项关于CPON在癫痫发作方面的功能研究也以阴性结果告终。
发明内容
本发明发现神经肽Y的C端肽段-CPON在常氧、缺氧和活性氧(ROS)环境中具有抑制心肌细胞凋亡的作用及其在心肌细胞冷冻保存中的应用;本发明通过在常氧、缺氧和活性氧(ROS)环境检测人心肌细胞的凋亡情况,首次发现CPON在以上环境中能抑制心肌细胞凋亡,具有心肌保护作用,并利用该作用开发了一种新型的针对人诱导多能干细胞衍生的心肌细胞(hiPSC-CM)的冻存方法。
附图说明
图1为实施例2中荧光显微镜观察CPON在心肌细胞的定位情况;
图2为实施例3、4中流式细胞仪采集细胞凋亡结果与统计分析结果;
图3为实施例5中DEG(NCN)与DEG(Ctr)相关性分析结果图;
图4为实施例6中流式检测心肌细胞凋亡情况的统计结果图。
具体实施方式
为让本领域的技术人员更加清晰直观的了解本发明,下面将结合附图,对本发明作进一步的说明。
实施例1:hiPSC-CM的获得
1.1hiPSC的分化:在RPMI-BSA培养基[RPMI1640培养基(HyClone,SH30027.01)+213μg/mL AA2P(l-抗坏血酸2-磷酸镁)(Sigma,A8960)和0.1%牛血清白蛋白(Sigma,A1470)]中添加小分子CHIR99021(Tocris,4423,终浓度为10mM)]处理hiPSC 24小时,然后换RPMI-BSA培养基孵育48h。在分化第4天,在RPMI-BSA培养基中加入小分子IWP2(Tocris,3533,终浓度5μM)处理细胞。48h后换用RPMI-BSA培养基。在此阶段,就从hiPSC分化为hiPSC-CM。在随后的实验中,用RPMI1640培养基加3%的血清替代物(Gibco,10828-028)培养心肌细胞。
1.2hiPSC-CM的纯化:使用代谢选择方法纯化hiPSC-CM。代谢选择培养基为添加0.1%牛血清白蛋白(Sigma,A1470)和1×亚油酸-油酸-白蛋白(Sigma,L9655)的DMEM培养基(无糖)(Gibco,11966-025)。用代谢选择培养基处理细胞3-6天。培养液每2天更换一次。用此方法纯化得到的心肌细胞纯度可高达99%。
实施例2:CPON在心肌细胞膜上定位
2.1人工合成CPON多肽并在其C端连接FITC基团(CPON-FITC),由生工生物工程(上海)股份有限公司提供。
2.2CPON细胞膜定位:将心肌细胞消化后重新接种在玻片上,与5μM CPON-FITC 37℃孵育1小时后,用DPBS(GIBCO,C14190500CP)洗涤细胞2次,在荧光显微镜下观察CPON-FITC在心肌细胞的定位情况。
结果分析:如图1所示,心肌细胞膜上有明显的绿色荧光,细胞内无荧光。心肌细胞,本身不发绿色荧光。FITC基团,发绿色荧光。表示CPON-FITC成功在心肌细胞膜上定位。
实施例3:CPON在常氧、缺氧和活性氧(ROS)的环境中能抑制心肌细胞凋亡
3.1人工合成CPON多肽,由江苏金斯瑞生物科技有限公司提供。常氧环境使用37℃5%CO2,95%空气的CO2细胞培养箱,缺氧环境使用三气培养箱(ESCO,CLL-170T-8)设置95%N2,1%O2,4%CO2模拟。
3.2实验分组:纯化后的hiPSC-CM细胞按2×105/孔接种至六孔板,培养24h后更换为RPMI1640培养基加3%血清替代物的培养基,恢复培养2天。按处理方式将心肌细胞分为6组:常氧对照组(+PBS),常氧+CPON,缺氧对照组(+PBS),缺氧+CPON,缺氧+CPON+H2O2,缺氧+H2O2。CPON终浓度为5μM,孵育时间为48小时;PBS加入量与CPON相等;H2O2终浓度为1mM,处理时间为4小时。
3.3细胞凋亡检测:按分组加入多肽或PBS后进行常氧或缺氧培养2天。使用APCAnnexin V检测试剂盒(Biolegend,640920)检测以上6组心肌细胞的凋亡情况。细胞按分组处理,消化后按试剂盒说明用凋亡早期标记物Annexin V标记后,用NovoCyte D2040R流式细胞分析仪(安捷伦)进行分析。
结果分析:各分组使用流式细胞仪采集细胞凋亡结果如图2-a,c。在常氧环境下,常氧+CPON组的凋亡细胞比例是对照组(常氧+PBS)的凋亡细胞比例的68.7%(图2-b)(常氧+CPON组:16.99%,对照组:24.71%;n=3;*,p<0.01)。在缺氧环境下,缺氧+CPON组的凋亡细胞比例是对照组(缺氧+PBS)的凋亡细胞比例的75.3%(图2-d)(缺氧+CPON:33.40%,对照组:44.38%;n=3;**,p<0.001);缺氧+CPON+H2O2组的凋亡细胞比例比缺氧+H2O2组的凋亡细胞比例降低了9.08%(图2-d)(缺氧+CPON+H2O2:84.91%,缺氧+H2O2:93.99%;n=3;**,p<0.001)。说明在常氧、缺氧和活性氧(ROS)的环境中CPON能抑制心肌细胞凋亡。
实施例4:CPON-pT具有更强的抑制心肌细胞凋亡的作用
4.1人工合成CPON磷酸肽在第16位的苏氨酸被磷酸化(Thr16)并在磷酸肽C端连接FITC基团(CPON-pT-FITC)由生工生物工程(上海)股份有限公司提供。
4.2实验分组:纯化后的hiPSC-CM细胞按2*105/孔接种至六孔板,培养24h后更换为RPMI1640培养基加3%血清替代物的培养基,恢复培养2天。按处理方式将心肌细胞分为2组:缺氧+CPON-FITC,缺氧+CPON-pT-FITC。缺氧培养4天,CPON-FITC和CPON-pT-FITC的终浓度为5μM,每2天更换新的含多肽培养基。
4.3细胞凋亡检测:使用APC Annexin V检测试剂盒(Biolegend,640920)检测以上2组心肌细胞的凋亡情况。细胞按分组处理,消化后按试剂盒说明用凋亡早期标记物Annexin V标记后,用NovoCyte D2040R流式细胞分析仪(安捷伦)进行分析。
结果分析:各分组使用流式细胞仪采集细胞凋亡结果如图2-e。CPON-pT-FITC组的凋亡细胞比例比CPON-FITC组的凋亡细胞比例降低了9.36%(图2-f)(CPON-pT-FITC:53.40%,CPON-FITC组:62.76%;n=3;**,p<0.001),提示在缺氧环境下,CPON-pT较CPON具有更强的抑制心肌细胞凋亡的作用。
实施例5:RNA-seq分析
5.1实验分组:纯化后的hiPSC-CM细胞按2×105/孔接种至六孔板,培养24h后更换为RPMI1640培养基加3%血清替代物的培养基,恢复培养2天。按处理方式将心肌细胞分为8组,常氧对照组,常氧+CPON,常氧+CPON-FITC,常氧+CPON-pT-FITC,缺氧对照组,缺氧+CPON,缺氧+CPON-FITC,缺氧+CPON-pT-FITC。CPON终浓度为5μM,孵育时间为48小时。
5.2总RNA提取:用EZ-10总RNA小量提取试剂盒(Sangon Biotech,B618583-0050)提取细胞总RNA。(样品使用RNase-Free DNA清除试剂盒(Sangon Biotech,B618253-0050)清除残留基因组DNA)。
5.3RNA-seq由Novogene公司完成,收集以上8个分组的样品,获得转录组数据。
结果分析:利用hisat2,htseq,DESeq2及gencode v32等软件进行分析和注释,识别不同条件下的差异表达基因(DEG),结果见表1。
表1不同条件下的差异表达基因(DEG)
筛选常氧+CPON与常氧对照组比较(DEG(NCN))Log2(Fold Change)>1和Log2(FoldChange)<-1前十名的基因(见表2),在上调的基因中,有文章报道IFI27L2、DHRSX是细胞对外界刺激(病毒感染、温度、饥饿等)的应激相关基因,TMEM50B、DENND11、GBA等基因与神经系统相关疾病有关。
表2DEG(NCN)上调和下调前十名的基因
比较DEG(NCN)与DEG(Ctr),发现在p<0.05的基因中,DEG(NCN)与DEG(Ctr)呈正相关,R2=0.3324(图3)。这提示CPON对hiPSC-CM的保护作用可能是由于CPON对hiPSC-CM造成了一定程度的缺氧预处理,激发了hiPSC-CM内在的抗凋亡机制,从而降低其在不同刺激下的凋亡比例。
实施例6:利用CPON的心肌保护作用开发hiPSC-CM的新型冻存方法
6.1实验分组:实验分组共分为12组。各组的处理方式如下表3。
表3各组实验分组处理情况
处理1具体操作:心肌细胞冻存前更换为含CPON(5μM)的培养基孵育24h。
处理2具体操作:使用MEFLC细胞(见专利202210907968.7)培养上清重悬细胞再加入等体积2×DMSO-血清替代物(血清替代物体积比为20%)作为冻存液。
处理3具体操作:使用MEFLC细胞上清重悬细胞,加入等体积2×DMSO-血清替代物(血清替代物体积比为20%)再加入终浓度5μM的CPON作为冻存液。
处理4具体操作:使用MEFLC细胞上清重悬细胞再加入等体积2×DMSO-FBS(FBS体积比为20%)作为冻存液。
处理5具体操作:使用MEFLC细胞上清重悬细胞,加入等体积2×DMSO-FBS(FBS体积比为20%)再加入终浓度5μM的CPON作为冻存液。
处理6具体操作:使用RPMI1640培养基加3%的血清替代物重悬细胞,再加入等体积2x DMSO-FBS(FBS体积比为20%)作为冻存液。
处理7具体操作:使用RPMI1640培养基加3%的血清替代物重悬细胞,再加入等体积2×DMSO-血清替代物(血清替代物体积比为20%)作为冻存液。
6.2实验方案:纯化后的hiPSC-CM细胞使用TrypLETM Express酶消化按2.5×105/孔接种至六孔板,培养24h后更换为RPMI1640培养基加3%血清替代物的培养基,恢复培养2天。按照实验分组冻存细胞,使用程序降温盒(Corning,432001)在-80℃冰箱保存1天后,将细胞转移至液氮保存1周。
6.3细胞复苏方案:从液氮中取出细胞迅速放入37℃水浴,小心摇晃使细胞融化至仅剩一小块冰晶。轻柔吹打2次,将细胞悬液转移至15mL离心管中。吸取4mL完全培养基(15%FBS+1%NEAA+1%P/S+0.1%BSA+DMEM+2μM TZV)缓慢加至细胞中(第1mL,滴1滴轻柔转两下,第2mL,滴2滴轻柔转两下,第3、4mL,滴4滴轻柔转两下),200g离心5分钟。去除上清加入2.5mL完全培养基,轻柔吹打2次,将细胞悬液接种于明胶(sigma,V900863)包被的六孔板中培养3天。
6.4细胞凋亡检测:使用APC Annexin V检测试剂盒(Biolegend,640920)检测以上12组心肌细胞的凋亡情况。细胞使用TrypLETM Express酶消化后按试剂盒说明用凋亡早期标记物Annexin V标记后,用NovoCyte D2040R流式细胞分析仪(安捷伦)进行分析。
结果分析:各分组使用流式细胞仪采集细胞凋亡结果如图4。其中组4的心肌细胞凋亡比例最低,是对照组12凋亡比例的61.0%(组4:27.48%,组12:45.06%;n=3;**,p<0.001)。由此,组4的处理方法及冻存液配方可以进一步优化开发成为新型的hiPSC-CM的冻存方法及试剂。
由此可知:
(1)在常氧、缺氧和活性氧(ROS)的环境中CPON能抑制心肌细胞凋亡。
(2)在缺氧环境下,CPON-pT较CPON具有更强的抑制心肌细胞凋亡的作用。
(3)本发明开发了一种新型hiPSC-CM的冻存方法。
本发明首次发现了CPON的心肌保护功能,即CPON具有抑制心肌细胞凋亡和降低心肌细胞冻存后细胞凋亡的作用。因此,CPON可作为缓解或治疗心肌细胞凋亡相关的心血管疾病的药物,和心肌细胞冻存的试剂,为心肌细胞凋亡相关的心血管疾病的药物研发提供了理论基础和科学依据,为心肌细胞临床应用提供支持。
上述对实施例的描述是为便于该技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于这里的实施例,本领域技术人员根据本发明的揭示,对于本发明做出的改进和修改都应该在本发明的保护范围之内。
Claims (9)
1.CPON或CPON-pT在制备心肌保护产品中的应用。
2.CPON在制备抑制心肌细胞凋亡产品中的应用。
3.CPON在制备缓解或治疗心肌细胞凋亡相关的心血管疾病的药物中的应用。
4.CPON在冻存心肌细胞时或在心肌细胞处于常氧/缺氧/活性氧的环境中作为降低细胞凋亡比例的添加剂的应用。
5.CPON在制备心肌细胞冻存液中的应用。
6.CPON-pT在制备抑制缺氧环境中心肌细胞凋亡产品中的应用。
7.一种心肌细胞冻存试剂,其特征在于,冻存试剂中含有CPON。
8.如权利要求7所述的冻存试剂,其特征在于,所述冻存试剂中CPON的终浓度为5μM。
9.一种心肌细胞的冻存方法,其特征在于,包括如下步骤:
心肌细胞冻存前更换为含CPON的培养基孵育;
使用MEFLC细胞上清重悬细胞,加入DMSO-血清替代物和CPON作为冻存试剂冻存心肌细胞。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080286324A1 (en) * | 2007-05-14 | 2008-11-20 | Cardiac Pacemakers, Inc. | Media and devices for cold storage of therapeutic cells |
CN105473614A (zh) * | 2013-08-20 | 2016-04-06 | 斯洛文尼亚莱柯制药股份有限公司 | 用于控制多肽的α-酰胺化和/或C-末端氨基酸分裂的细胞培养基和方法 |
US20190078053A1 (en) * | 2016-03-15 | 2019-03-14 | Keio University | Method for producing cardiac precursor cell and myocardial cell from pluripotent stem cell |
CN112608972A (zh) * | 2020-12-21 | 2021-04-06 | 广东源心再生医学有限公司 | Myog基因作为靶点在制备治疗心肌细胞凋亡相关的心血管疾病的药物中的应用 |
US20210360913A1 (en) * | 2018-02-09 | 2021-11-25 | Yale University | Methods, systems and compositions for normothermic ex vivo preservation of intact organs |
CN114762496A (zh) * | 2021-01-11 | 2022-07-19 | 南京艾尔普再生医学科技有限公司 | 心肌细胞冻存液及其制备方法 |
-
2023
- 2023-06-19 CN CN202310727048.1A patent/CN117018164A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080286324A1 (en) * | 2007-05-14 | 2008-11-20 | Cardiac Pacemakers, Inc. | Media and devices for cold storage of therapeutic cells |
CN105473614A (zh) * | 2013-08-20 | 2016-04-06 | 斯洛文尼亚莱柯制药股份有限公司 | 用于控制多肽的α-酰胺化和/或C-末端氨基酸分裂的细胞培养基和方法 |
US20190078053A1 (en) * | 2016-03-15 | 2019-03-14 | Keio University | Method for producing cardiac precursor cell and myocardial cell from pluripotent stem cell |
US20210360913A1 (en) * | 2018-02-09 | 2021-11-25 | Yale University | Methods, systems and compositions for normothermic ex vivo preservation of intact organs |
CN112608972A (zh) * | 2020-12-21 | 2021-04-06 | 广东源心再生医学有限公司 | Myog基因作为靶点在制备治疗心肌细胞凋亡相关的心血管疾病的药物中的应用 |
US20230071726A1 (en) * | 2020-12-21 | 2023-03-09 | Guangdong Beating Origin Regenerative Medicine Co., Ltd. | Use of myog gene as target in preparation of drug for treating cardiomyocyte apoptosis-associated cardiovascular disease |
CN114762496A (zh) * | 2021-01-11 | 2022-07-19 | 南京艾尔普再生医学科技有限公司 | 心肌细胞冻存液及其制备方法 |
Non-Patent Citations (1)
Title |
---|
王萍等: "血清NPY和IGF-1在成人OSAHS中的表达及与患者认知功能关系", 《西部医学》, vol. 32, no. 3, 31 March 2020 (2020-03-31), pages 426 - 437 * |
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