CN107449913A - 一种心脏干细胞标志物及其用途 - Google Patents
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Abstract
本发明提供了一种心脏干细胞标志物及其用途，所述心脏干细胞标志物包含TNFR2。本发明中发现TNFR2阳性的心脏干细胞在小鼠心肌梗塞模型的缺血心肌中被诱导产生，以及TNFR2‑Bmx通路在小鼠心肌梗塞模型中具有激活缺血诱导的内源性心脏干细胞的作用，并且TNFR2在缺血心脏中与Nkx2.5+/Gata4+CSC标志物共表达。另外，本发明还提供了一种筛选心脏干细胞的组合物和试剂盒，所述组合物和试剂盒包含检测TNFR2的试剂。由此，TNFR2作为心脏干细胞的一种新的生物标志物，为分离内源心脏干细胞提供新的分子标记。
Description
技术领域
本发明涉及一种生物标志物及其用途，尤其是一种与心脏干细胞相关的生物标志物及其用途。
背景技术
生物标志物(Biomarker)是指可以标记系统、器官、组织、细胞及亚细胞结构或功能的改变或可能发生的改变的生化分子指标，具有非常广泛的用途。生物标志物可用于疾病诊断、判断疾病分期或者用来评价新药或新疗法在目标人群中的安全性及有效性。
对于疾病研究，生物标志物一般是指可供客观测定和评价的一个普通生理或病理或治疗过程中的某种特征性的生化分子指标，通过对它的测定可以获知机体当前所处的生物学过程中的进程。检查一种疾病特异性的生物标志物，对于疾病的鉴定、早期诊断及预防、治疗过程中的监控可能起到帮助作用，因此寻找和发现有价值的生物标志物已经成为目前研究的一个重要热点。
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。
但是，目前尚无一个明确的心脏干细胞表面分子标记，因此鉴定出一种心脏干细胞的生物标志物，将给分离内源心脏干细胞提供新的分子标记，具有重大研究意义。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种心脏干细胞的生物标志物，所述生物标志物阳性的细胞代表了一类心脏干细胞亚群；同时，本发明还提供了所述与心脏干细胞相关的生物标志物的用途，所述生物标志物将给分离内源心脏干细胞提供新的分子标记。
为实现上述目的，本发明采取的技术方案为：一种心脏干细胞标志物，所述心脏干细胞标志物包含TNFR2。
同时，本发明还提供一种所述心脏干细胞标志物在筛选心脏干细胞中的用途。
另外，本发明还提供一种筛选心脏干细胞的组合物，所述组合物包含检测TNFR2的试剂。
同时，本发明还提供一种筛选心脏干细胞的试剂盒，所述试剂盒包含检测TNFR2的试剂。
本发明的有益效果在于：本发明提供了一种心脏干细胞标志物，TNFR2作为心脏干细胞的一种新的生物标志物，为分离内源心脏干细胞提供新的分子标记。
附图说明
图1为本发明的整体实验技术路线图；
图2为本发明实施例2中TNF受体分子和下游效应分子在人正常心脏和缺血心脏的表达分布图；其中A为切片免疫荧光染色，分别为TNFR1/α-SA，TNFR2/α-SA和Bmx/α-SA三组(CM-心肌细胞，VEC-血管内皮细胞，IC-间质细胞)；B为正常心脏组织和缺血心脏组织western blot检测相应蛋白表达情况；C为磷酸化组蛋白3(pH3s10)与α-SA免疫荧光染色；D为pH3阳性细胞统计图，纵坐标表示高倍视野pH3阳性细胞数，横坐标表示三组细胞(CM-心肌细胞，VEC-血管内皮细胞，IC-间质细胞)，**，p<0.01，n＝5张切片；E：组织切片免疫荧光染色，显示pH3s10与TNFR2存在共定位，其中比例尺为50μm；
图3为本发明实施例3中人正常心脏和缺血性心脏中心脏前体细胞分布情况；其中A为人正常心脏和缺血心脏组织切片中Gata4，NKx2.5和α-SA免疫荧光染色，箭头指出Gata4在心肌细胞细胞核表达；B为Gata4+和NKx2.5+细胞统计，纵坐标表示高倍视野阳性细胞数，横坐标表示c-Kit、Gata4和NKx2.5染色组别，*p<0.01，n＝5张切片；C为人正常心脏和缺血心脏中的心脏前体细胞，用NKx2.5，Gata4和TNFR2进行标记，箭头指向双阳性的细胞，图片放大倍数为40，比例尺为50μm；
图4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5为hiPSC-CM中TNFR2与心脏祖细胞标记物存在共定位现象。
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。
实施例1:建立心梗模型
本实施例中所有动物实验均得到动物伦理委员会批准。
实验方法：
1)冠状动脉结扎：小鼠麻醉后从口腔导引一软管进入气管外接呼吸机，在左侧胸部从右下至左上做一斜行切口，在第4肋间以手术刀沿下位肋骨上缘切开肋间肌进入胸腔，将心脏轻轻挤出后，在左心耳与肺动脉圆锥之间平左心耳下缘迅速缝扎左前降支近端，同时观察心电图变化，术后立即关胸并用注射器抽出胸腔内气体，以恢复胸腔负压。心脏超声检测射血分数<40％小鼠纳入实验。
2)弥漫性心肌损伤模型：近年报道弥漫性心肌损伤模型更适合心肌修复实验。野生型、TNFR2-KO和Bmx-KO小鼠分别接受在颈部疏松皮肤处接受5mg kg-1异丙肾上腺素单次皮下注射。ISO将发挥类似Takotsubo的心脏病理过程，导致弥漫性心内膜下和心尖心肌坏死，急性损伤将损坏8-10％左心室心肌，导致急性心力衰竭。和传统心梗模型相比，这种心肌损伤和心力衰竭通过增加BrdU-阳性eCSC在28天后形态上和功能上自发恢复eCSC在心肌再生和功能修复中发挥关键作用。
实施例2:TNFR2-Bmx在人体缺血心脏中表达增加
本发明的整体实验技术路线如附图1所示。本实施例的实验方法为运用anti-TNFR2和anti-Bmx抗体进行IB或免疫组化检测TNF受体分子和下游效应分子的表达。实验样品为我们从英国剑桥大学Dr.Bradley获得的心脏移植术中的石蜡包埋组织切片。
其具体的实验方法为:
1)免疫荧光：组织切片固定，Triton-X100通透处理后，用1％BSA(牛血清蛋白)封闭30分钟，之后PBS(磷酸盐缓冲液)漂洗，相应抗体孵育2小时，PBS漂洗，对应荧光二抗避光孵育1小时，PBS漂洗后甘油封片，荧光显微镜下观察拍照。
2)Western blot：提取心脏组织蛋白定量后，进行SDS-PAGE凝胶电泳，之后PVDF膜进行转膜，之后PVDF膜用5％脱脂奶粉进行封闭1小时，相应一抗4℃孵育过夜，PBS漂洗，对应二抗室温孵育1小时，PBS漂洗，化学发光检测蛋白表达水平。
实验结果如附图2所示，切片显示正常(NM；n＝20)与缺血性心脏病(IHD,n＝58)组织形态并无异常。TNFR1在正常心脏的CMs(心肌细胞)，VECs(血管内皮细胞)和ICs(间质细胞)中均强表达(α-SA)。然而，TNFR1和AIP1在缺血心脏移植物中下调。TNFR2和Bmx激酶只在正常人类心脏(normal human)NM中弱表达，而在缺血心脏中上调。
以上结果表明，TNFR2-Bmx在人体缺血心脏中表达增加。
实施例3:TNFR2在缺血心脏中与Nkx2.5+/Gata4+CSC标志物共表达
将野生型、TNFR2-KO和Bmx-KO小鼠分别接受在颈部疏松皮肤处接受5mg kg-1异丙肾上腺素单次皮下注射，分别于1,3,6,14或28天处死小鼠(每个时间点每组n＝10)。处死前行心脏超声检测心功能。运用anti-TNFR2和anti-Bmx抗体进行IB或免疫组化检测TNFR2-Bmx表达。Bmx活化情况通过anti-phospho-Bmx进行检测。小鼠eCSC利用anti-Gata4、anti-Nkx2.5进行评估。同时检测TNFR2和Bmx与eCSC标志物的表达。免疫双标法检测CSC标志物(Gata4，NKx2.5和α-SA)可能的心脏前体细胞。
实验结果如附图3中的A所示，在正常心脏只有部分游离细胞Nkx2.5和Gata4阳性。而在缺血心脏的缺血区域，如附图3中的A和B所示，三种标志物(Gata4，NKx2.5和α-SA)均阳性。之后我们评估它们的免疫表型，运用免疫荧光双染明确它们和TNFR2的关系，结果如图3B中的NM所示，正常心肌细胞中Nxk2.5+TNFR2+或Gata4+TNFR2+表达很低，然而在缺血心脏Nkx2.5+或Gata4+前体细胞中TNFR2也呈阳性，如附图3的图C中的IHD所示。
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。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4所示：如附图4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4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4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。图5用三染色显示hiPSC-CM中TNFR2与心脏祖细胞标记物存在共定位现象。
以上结果表明，TNFR2在缺血心脏中与Nkx2.5+/Gata4+CSC标志物共表达。
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)
1.一种心脏干细胞标志物，其特征在于，所述心脏干细胞标志物包含TNFR2。
2.如权利要求1所述的心脏干细胞标志物在筛选心脏干细胞中的用途。
3.一种筛选心脏干细胞的组合物，其特征在于，所述组合物包含检测TNFR2的试剂。
4.一种筛选心脏干细胞的试剂盒，其特征在于，所述试剂盒包含检测TNFR2的试剂。
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| MARIA MIROTSOU ET AL: "Paracrine mechanisms of stem cell reparative and regenerative actions in the heart", 《JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY》 * |
| RAFIA S.AL–LAMKI, ET AL: "TNF, Acting Through Inducibly Expressed TNFR2, Drives Activation and Cell Cycle Entry of c-Kit+ Cardiac Stem Cells in Ischemic Heart Disease", 《STEM CELLS》 * |
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