CN107460165A - 一种心脏干细胞及其用途 - Google Patents
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Abstract
本发明提供了一种心脏干细胞及其用途，所述心脏干细胞表达TNFR2。本发明所述心脏干细胞能够分化为心肌细胞，为治疗人类缺血性心脏病提供了一种新的治疗策略。
Description
技术领域
本发明涉及一种心脏干细胞及其用途，具体是一种表达TNFR2的心脏干细胞及其在制备治疗人类缺血性心脏病的药物中的用途。
背景技术
心血管疾病是世界范围内威胁人类生命的最主要疾病之一，其中急性心肌梗塞是冠心病中最严重的疾病，具有很高的发病率和死亡率。心肌梗死会造成不可逆性功能心肌细胞数量的减少，导致心肌收缩功能降低。目前心梗传统的治疗方法主要包括药物治疗、介入治疗和手术治疗，虽能改善心肌缺血症状，但都无法挽救已坏死的心肌细胞。
干细胞是一类具有自我更新和多向分化潜能的细胞，可以替代损伤的心肌细胞，构建新的血管，修复心脏泵血功能，为心梗的治疗提供了一种新的选择。越来越多的证据支持成年心脏干细胞(Cardiac stem cells,CSCs)的存在，并在缺血状态下被激活，在心脏功能修复中发挥重要作用。
发明内容
本发明的目的在于提供一种心脏干细胞，本发明还提供了所述心脏干细胞的用途，所述心脏干细胞为治疗人类缺血性心脏病提供了新的治疗策略。
为实现上述目的，本发明采取的技术方案为：鉴定一种心脏干细胞，所述心脏干细胞表达TNFR2。
同时，本发明还提供一种所述心脏干细胞在制备心肌细胞中的用途。
另外，本发明还提供一种所述心脏干细胞在制备治疗心肌梗塞的药物中的用途。
此外，本发明还提供一种所述心脏干细胞在制备治疗人类缺血性心脏病中的用途。
本发明的有益效果在于：本发明提供了一种心脏干细胞及其用途，所述心脏干细胞能够表达TNFR2，所述心脏干细胞能够分化为心肌细胞，为治疗人类缺血性心脏病提供了一种新的治疗策略，具有重大研究意义。
附图说明.
图1为本发明的实验技术路线图；
图2为利用GiWi-心脏干细胞分化的方法获取iPSC-CSC；A.GiWi-心脏干细胞分化方法的流程图；B.QPCR检测TNFR2,GATA4以及ISL1在已分化的 iPSC-CSC中的表达结果，纵坐标表示靶基因相对于内参(GAPDH)表达的百分比，横坐标表示hESCs细胞系和分化的CPC细胞；C.iPSC-CSC中TNFR2, NKX2.5以及GATA4的免疫荧光染色图像(10×)；D.iPSC-CSC中TNFR2以及原肌球蛋白的免疫荧光染色(63×)；E.iPSC-CSC中NKX2.5以及原肌球蛋白的免疫荧光染色(63×)；F.iPSC-CSC中NKX2.5,TNFR2以及原肌球蛋白的免疫荧光染色(63×)；G.iPSC-CSC中Ki67,TNFR2以及原肌球蛋白的免疫荧光染色(63×)；
图3为在标准规范条件下将CSC以及成熟CM进行分化；分别利用qRT-PCR 和IB的方法在基因水平(A)和蛋白水平(B)进行检测，与对照组比起来，OCT4 只在人的胚胎干细胞中表达，而α-SA只在成熟心肌细胞中表达；
图4为本发明实施例2中TNFR2中和抗体抑制心肌细胞分化。A－B： NKx2.5和cTnT阳性细胞染色及定量。C：NKx2.5mRNA表达定量；
图5为本发明实施例2中R2-TNF促进心肌细胞分化。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例及附图对本发明作进一步说明。
实验技术路线如附图1所示。
实验方法具体为：
用四环素诱导的慢病毒载体转染H1hESCs细胞系(US National Institutes ofHealth-registered as WA01)或，表达对照shRNA,TNFR2或Bmx shRNA，同时共表达GFP。采用诱导的shRNA系统拮抗各阶段已知的因子，例如Nkx2.5/ Gata4作为对照。相反，构建过表达的TNFR2或Bmx。诱导的慢病毒shRNA 系统可以在分化的特定阶段下调目标基因。应用GFP共表达检测转染效率，并特异性追踪转染细胞。采用qRT-PCR，IB，FACS检测hESCs中的下调或上调的TNFR2。
应用已确立的方法诱导hESCs分化。将标准分化步骤用两种方法调整：1) 在培养液中加入TNFR2中和抗体或TNFR2激活剂R2-TNF，应用生理盐水、WT-TNF、R1-TNF作为对照。2)应用缺氧小室(5％CO2，0.5-1％O2)模拟缺血环境，检测心肌分化情况。细胞每3天更换新鲜的分化培养液，每三天收集一次，直到第30天心肌细胞完全成熟。分别检测蛋白表达和TNFR2信号，CSC 标志物(Nkx2.5/Gata4)，分化标志物(α-SA/cTnT)。
透射电镜术检测细胞肌原纤维组织，培养细胞的跳动区域被分割、固定、并用电镜树脂包埋。用超薄切片荧光素醋酸盐和柠檬酸盐双染。检测心肌活化药物异丙肾上腺素的(β1-肾上腺素能受体激动剂)，Bay k8644(Ca2+通道激活剂)，苯肾上腺素(α1-肾上腺素能受体激动剂)，碳酰胆碱(毒蕈碱受体激动剂) 的心脏变时效应评价心肌细胞成熟功能。随机区域切割单细胞检测成熟心肌细胞电生理特性，随后用全细胞膜片钳检测检测动作电位和钙内流。心肌细胞终末分化的特性是细胞停止增殖，跳出细胞周期，因此也可用BrdU标记检测细胞增殖。选用新生乳鼠细胞作为染色和收缩检测实验的对照。
实施例1：hESCs和iPS分化为CSC和CMs细胞
我们采用Dr.Kamp’s课题组建立的基质胶三明治法，培养、分化细胞，从而增加细胞产出(图2)。其具体实验方法为：hESC或iPS在Matrigel上单层培养，细胞外基质准备，然后覆盖基质胶。该法如同原肠胚形成一样，产生N-cadherin- 阳性间充质细胞，促进EMT发生。将一些生长因子(Activin A,BMP4,FGF)联合基质胶应用，可以从多种细胞得到高纯度(高至98％)和高数量(高至11CMs/hESC) 的心肌细胞。心肌细胞在培养液中30天后逐渐成熟，具备肌丝表达和有丝分裂活性。
实验结果如附图2所示：如附图2中的A所示，观察到细胞分化第14天，细胞开始表现心肌细胞特征，包括自发收缩及心脏相关基因蛋白表达；如附图2中的 B所示，我们通过qPCR检测了心脏标记物GATA4和Isl1，分化后表达均显著增高，同时如附图2中的C-G所示，免疫染色显示这些细胞为TNFR2、NKX2.5阳性， TNFR2/NKX2.5及TNFR2/Ki67共阳性。
此外，我们采用相似的实验方法，观察了CSC产生和分化中的TNFR2表达和信号。实验结果如附图3所示，用qRT-PCR和IB的方法在基因水平(图3 中A)和蛋白水平(图3中B)进行检测，与对照组比起来，OCT4只在人的胚胎干细胞中表达，而α-SA只在成熟心肌细胞中表达.
实施例2：检测TNFR2-Bmx信号对hESC/hiPSC来源的心肌细胞分化的作用
本发明的最终目标是应用TNFR2激活策略治疗人类缺血性心脏病，但是显然我们无法应用人体心脏明确TNFR2-Bmx信号通路在eCSC中的作用，因此我们采用TNFR2阳性外源心脏干细胞—hESC/iPSC来源的CSC进行研究，检测 TNFR2-Bmx信号对hESC/hiPSC来源的心肌细胞分化的作用。
实验方法具体为：
应用已确立的方法诱导hESCs分化。将标准分化步骤用两种方法调整：1) 在培养液中加入TNFR2中和抗体或TNFR2激活剂R2-TNF，应用生理盐水、 WT-TNF、R1-TNF作为对照。2)应用缺氧小室(5％CO2，0.5-1％O2)模拟缺血环境，检测心肌分化情况。细胞每3天更换新鲜的分化培养液，每三天收集一次，直到第30天心肌细胞完全成熟。分别检测蛋白表达和TNFR2信号，CSC 标志物(Nkx2.5/Gata4)，分化标志物(α-SA/cTnT)。其结果如图4和图5所示，从图4可以看出，TNFR2中和抗体抑制心肌细胞分化；从图5可以看出，R2-TNF 促进心肌细胞分化。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (4)
1.一种心脏干细胞，其特征在于，所述心脏干细胞表达TNFR2。
2.如权利要求1所述的心脏干细胞在制备心肌细胞中的用途。
3.如权利要求1所述的心脏干细胞在制备治疗心肌梗塞的药物中的用途。
4.如权利要求1所述的心脏干细胞在制备治疗人类缺血性心脏病中的用途。
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| CN101437938A (zh) * | 2004-11-08 | 2009-05-20 | 约翰霍普金斯大学 | 心脏干细胞 |
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