CN112608972B - 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 - Google Patents

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 Download PDF

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CN112608972B
CN112608972B CN202011518860.6A CN202011518860A CN112608972B CN 112608972 B CN112608972 B CN 112608972B CN 202011518860 A CN202011518860 A CN 202011518860A CN 112608972 B CN112608972 B CN 112608972B
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apoptosis
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曾嵘
林彬
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Guangdong Yuanxin Regenerative Medicine Co ltd
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Abstract

本发明属于生物医药技术领域，特别是指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。本发明通过体外构建血管紧张素II诱导的人诱导多能干细胞分化的心肌细胞凋亡模型，首次揭示了转录因子MYOG在抑制心肌细胞凋亡方面的作用，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，是心血管疾病药物研发的新靶点。

Description

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
技术领域
本发明属于生物医药技术领域，具体涉及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。
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。据世界卫生组织(WHO)统计，每年全球超过1700万人死于CVD，占全球死亡总数的31％。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扔呈明显上升趋势。
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。目前临床上抑制心肌细胞凋亡的药物有他汀类、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，使得我使得体外制备及培养人心肌细胞成为可能。利用iPS诱导体外诱导心肌细胞的方法，可以在体外制备足够数量的人心肌细胞，来进行各种功能实验，模拟心血管疾病的发生发展过程，极大推动了针对心血管疾病机制的研究进展。
目前，体外心肌细胞凋亡模型一般是通过过氧化氢(H2O2)或血管紧张素II(Angiotensin II)诱导。血管紧张素II(Angiotensin II)可通过激活血管紧张素II受体引起心肌细胞凋亡，与H2O2诱导相比更贴近正常生理过程，是目前建立心肌细胞凋亡模型的常用诱导剂。
转录因子基因MYOG编码肌细胞生成素(Myogenin)蛋白，是生肌调节因子(MRFs)基因家族的成员之一。MRFs转录因子家族(包括Myod、Myf5、Mrf4和MYOG)在骨骼肌发生的每一个阶段都起着关键作用。该家族所有成员共同含有一个保守的螺旋-环-螺旋(bHLH)基序，可以与下游基因的E盒结合，从而激活下游肌肉特异性基因的表达。研究表明，MYOG通过控制、启动成肌细胞的融合和肌纤维的形成，而在肌肉分化过程中起关键作用。在小鼠中的研究显示，MYOG基因缺失会导致严重的肌肉分化缺陷，从而造成围产期死亡。因此，MYOG是骨骼肌发育过程中必需的调控因子，并且是不可替代的。目前，已有研究证实，MYOG基因在小鼠、鸭、草鱼、京海黄鸡等动物的心脏中都有表达，可能与心肌的生长发育相关。但MYOG基因在人心脏组织中的表达及在心脏发育中的作用尚未见报道。
发明内容
本发明通过体外构建血管紧张素II(Angiotensin II)诱导的hiPSC-CM(人诱导多能干细胞分化的心肌细胞)细胞凋亡模型，首次揭示了转录因子MYOG在抑制心肌细胞凋亡方面的作用，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，是心血管疾病药物研发的新靶点。
本发明通过调控MYOG基因的表达来抑制心肌细胞凋亡。因此，上述药物可以是传统的药物，也可以是基因调控药物，如包装可调控MYOG基因表达的慢病毒等。
附图说明
图1为本发明的技术流程图；
图2为实施例2中MYOG表达水平测定图；
图3为实施例4中血管紧张素Ⅱ处理对心肌细胞活性的影响图；
图4为Annexin V法检测不同浓度血管紧张素Ⅱ处理后的心肌细胞凋亡情况的流式结果图；
图5为Annexin V法检测不同浓度血管紧张素Ⅱ处理后的心肌细胞凋亡情况的统计结果图；
图6为TUNEL法检测不同浓度血管紧张素Ⅱ处理后的心肌细胞凋亡情况的免疫荧光染色结果图；
图7为TUNEL法检测不同浓度血管紧张素Ⅱ处理后的心肌细胞凋亡情况的统计结果图；
图8为PrestoBlue细胞活性检测试剂检测心肌细胞活性结果图；
图9为MYOG抑制血管紧张素II诱导的细胞凋亡的免疫荧光染色结果图；
图10为MYOG抑制血管紧张素II诱导的细胞凋亡的统计结果图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例进行详细描述。
如图1所示，本发明的技术流程如下：
1、构建pCW-MYOG载体。
2、包装慢病毒。
3、转染hiPSC细胞。
4、将hiPSC、pCW-MYOG分化成心肌细胞并进行纯化。
5、用盐酸多西环素(Dox)诱导hiPSC-MYOG细胞表达MYOG基因，Dox浓度为2μg/mL。
6、用血管紧张素II诱导hiPSC-CM、pCW-MYOG-CM凋亡，血管紧张素II浓度为1nM～1mM。
7、测定细胞活力。
8、测定细胞凋亡。
实施例1：获得hIPSC-MYOG细胞株
1.1慢病毒表达载体构建：用常规分子克隆方法将MYOG cDNA和一个嘌呤霉素抗性基因亚克隆到pCW-Cas9-Blast载体中(Addgene，83481)，取代原载体中的Cas9和Blast基因，得到pCW-MYOG。
1.2慢病毒包装
1.2.1接种HEK293T细胞到6孔板中，用D10培养液(DMEM培养液+10％胎牛血清)进行培养，待细胞汇合度达到70％-80％时准备进行转染。
1.2.2在转染前1h，弃去原来培养液，加入2mL/孔预热的无血清OptiMEM培养液。
1.2.3按照说明书用Lipofectamine 2000试剂进行转染。将pCW-MYOG(20μg)、pVSVg(10μg)(Addgene)、psPAX2(15μg)(Addgene)共转染HEK293T细胞。
1.2.4 6h后，将培养液更换成D10培养液(DMEM培养液+10％胎牛血清+1％BSA)。
1.2.5继续培养约60h后，取培养液于3000rpm、4℃离心10min，去除细胞残渣。
1.2.6用0.45μm低蛋白结合滤膜(Millipore Steriflip HV/PVDF)过滤上清液，去除细胞残渣。
1.2.7将含病毒的培养液按照体积比4:1与10％蔗糖缓冲液(50mM Tris-Hcl，pH7.4，100mM NaCl，0.5mM EDTA)，10000g、4℃离心4h。小心弃去上清液，离心管在吸水纸上沥干3min，加入1×PBS重悬，-80℃保存。
1.3转染hiPSC细胞
1.3.1 hiPSC培养：将人诱导多能干细胞(hiPSC)DYR0100(ATCC)接种于Matrigel基质(康宁，354277)包被的平板上，然后用STEMUP(Nissan Chemical Corporation)进行培养。STEMUP培养基每两天更换一次。iPSC每隔3天传代一次，或在细胞培养达到80-90％汇合度时传代。传代过程中用1×DPBS(Gibco，14040133)冲洗1次，然后在室温下用使用1×DPBS(Gibco，14190144)稀释的0.5mM EDTA(Invitrogen，15575020)处理10min。传代比为1:3-1:6。
1.3.2转染：待hiPSC细胞汇合度达到70％-80％时进行转染。感染复数(MOI)约为0.3-0.5。转染24h后，将培养液更换成新鲜STEMUP(含终浓度为2μg/mL的盐酸多四环素(Dox))。2天后，将培养液更换成STEMUP(含Dox 2μg/mL+嘌呤霉素(puromycin)(InvivoGen))进行筛选。筛选2-3天后，能够得到约30％的转化效率。挑取单个的克隆接种到不同的皿中培养，即得hiPSC-MYOG细胞株。
实施例2：Dox诱导MYOG表达
2.1诱导：在STEMUP中加入终浓度为2μg/mL的盐酸多西环素(Dox)(Sigma，D9891)诱导MYOG表达，并以DMSO作为对照。对照组没有DOX只有DMSO，标注为C1，加入到STEMUP；试验组有DOX也有DMSO，DOX先加入到DMSO，标注为C2，再加入到STEMUP；DOX在STEMUP里的终浓度是2μg/mL；C1和C2的加入量相同；
2.2总RNA提取：用UNlQ-10柱式Trizol总RNA抽提试剂盒(Sangon Biotech，B511321-0100)提取细胞总RNA。(样品提前用DNase I(脱氧核糖核酸酶I，Sangon Biotech，B618252)处理30min)；
2.3逆转录：用逆转录试剂盒iScript Reverse Transcription Supermix(Bio-Rad，1708841)对RNA进行逆转录。
2.4 qPCR检测MYOG mRNA表达水平：按照SsoAdvancedTM Universal
Figure BDA0002848864220000051
GreenSupermix(Bio-Rad,1725271)说明书，使用PikoReal Real-Time PCR System(ThermoFisher)系统，以NAPDH为内参，设计引物，对hiPSC、hiPSC-MYOG(Dox诱导组、DMSO对照组)中MYOG表达水平进行检测。引物序列如下：
MYOG-RT-F:GCCCAAGGTGGAGATCCT；
MYOG-RT-R:GGTCAGCCGTGAGCAGAT；
GAPDH-RT-F:TGGGTGTGAACCATGAGAAG；
GAPDH-RT-R:GTGTCGCTGTTGAAGTCAGA.
结果分析：如图2所示，Dox诱导组中MYOG的表达水平高出DMSO对照组100余倍。结果表明，已经成功构建出能够高表达MYOG基因的hiPSC-MYOG细胞株。
实施例3：hiPSC-CM/hiPSC-MYOG-CM的获得
3.1hiPSC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。
3.2hiPSC-MYOG-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4：血管紧张素II诱导hiPSC-CM凋亡模型的建立
2.1血管紧张素II诱导hiPSC-CM细胞凋亡：纯化后的hiPSC-CM细胞，用添加不同量(1nM、10nM、100nM、1μM、10μM、100μM、1mM)血管紧张素Ⅱ的心肌细胞培养液(MedChemExpress，HY-13948)进行血管紧张素Ⅱ处理，每隔2天更换一次血管紧张素Ⅱ培养液。
2.2 hiPSC-CM凋亡验证：
2.2.1分别于24h、48h、6d、10d用PrestoBlue细胞活性检测试剂(Invitrogen，A13261)检测心肌细胞活性，结果如图3所示。
2.2.2应用凋亡检测试剂盒Annexin V，Alexa FluorTM488conjugate(Invitrogen，V13201)检测血管紧张素Ⅱ处理(100μM、1mM)和未处理10d的心肌细胞凋亡情况。心肌细胞用凋亡标记物Annexin V标记后，用FACSAriaTM II流式细胞分析仪(BD)进行分析。结果如图4、图5所示。
2.2.3 TUNEL实验：使用TdT原位凋亡检测试剂盒(TdT In Situ ApoptosisDetection Kit(R&D Systems，4812-30-K))检测血管紧张素Ⅱ处理(1nM、1μM、100μM、1mM)和未处理10d的心肌细胞凋亡情况。将心肌细胞消化后重新接种在玻片上，按照试剂盒说明书进行染色，并用DAPI标记细胞核。
结果分析：PrestoBlue细胞活性检测结果显示(图3)，用高浓度血管紧张素Ⅱ(100μM、1mM)处理长时间(6d、10d)处理hiPSC-CM后，细胞活力显著下降；Annexin V、TUNEL实验结果显示(图4-7)，用高浓度血管紧张素Ⅱ(100μM、1mM)处理长时间(10d)处理hiPSC-CM后，凋亡细胞比例有显著升高。说明使用高浓度血管紧张素Ⅱ(100μM、1mM)长时间处理可以诱导hiPSC-CM凋亡，心肌细胞凋亡模型构建成功。
实施例5：MYOG抑制血管紧张素II诱导的hiPSC-CM凋亡
5.1血管紧张素II诱导hiPSC-MYOG-CM细胞凋亡：纯化后的hiPSC-MYOG-CM细胞，分4组(Ang II(+)&Dox(+)，Ang II(+)&Dox(-)，Ang II(-)&Dox(+)，and Ang II(-)&Dox(-))进行处理，其中Dox浓度为2μg/mL血管紧张素Ⅱ的浓度为1mM，处理时间为6d。
5.2细胞活力检测：用PrestoBlue细胞活性检测试剂(Invitrogen，A13261)检测心肌细胞活性，结果如图8所示。
5.3细胞凋亡检测：TUNEL实验：使用TdT In Situ Apoptosis Detection Kit(R&DSystems,4812-30-K)检测4组(Ang II(+)&Dox(+)，Ang II(+)&Dox(-)，Ang II(-)&Dox(+)，and Ang II(-)&Dox(-))心肌细胞的凋亡情况。将心肌细胞消化后重新接种在玻片上，按照试剂盒说明书进行染色，并用DAPI标记细胞核。
结果分析：Ang II(+)&Dox(+)组与Ang II(+)&Dox(-)相比，细胞活性显著增高(图8)，并且凋亡细胞比例显著降低(分别是对照组的1.49倍和2.01倍)(图9-10)，说明MYOG可以抑制血管紧张素II诱导的hiPSC-CM凋亡。同时，Ang II(-)&Dox(+)组与Ang II(-)&Dox(-)组相比，细胞凋亡比例无显著差异，说明在没有血管紧张素Ⅱ的情况下MYOG对细胞的凋亡无影响。
由此可知：
(1)本发明成功构建了iPSC-MYOG细胞株，经验证，Dox可诱导iPSC-MYOG细胞高表达MYOG基因。
(2)本发明建立了一个血管紧张素II诱导的心肌细胞凋亡模型。
(3)高表达MYOG基因可以抑制由血管紧张素II诱导的心肌细胞凋亡。
本发明首次发现了MYOG在抑制心肌细胞凋亡方面的作用，为心肌细胞凋亡相关的心血管疾病药物的研发和临床治疗提供了一个新思路。
以上所述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来说，在不脱离本发明所述原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

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1.促进MYOG基因高表达的药物在制备抑制血管紧张素II诱导的心肌细胞凋亡的药物中的应用。
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