论文题名(中文): | 成年心脏成纤维细胞调控幼稚心肌细胞 向成熟心肌细胞转化机制研究 |
姓名: | |
论文语种: | chi |
学位: | 博士 |
学位类型: | 学术学位 |
学校: | 北京协和医学院 |
院系: | |
专业: | |
指导教师姓名: | |
论文完成日期: | 2019-04-01 |
论文题名(外文): | Adult cardiac fibroblasts regulate naive cardiomyocytes transformation into mature cardiomyocytes |
关键词(中文): | |
关键词(外文): | Cardiac Maturation Cardiomyocyte Fibroblast Single-Cell RNA-Sequencing Microenvironment |
论文文摘(中文): |
研究背景与目的 急性心肌梗死是由冠状动脉粥样硬化引起血栓形成、冠状动脉的分支堵塞,使一部分心肌失去血液供应而坏死的病症,多发生于中年以后。近年来,随着再生医学与干细胞研究的兴起,细胞移植是一种较具前景的治疗方式。在研究使用过程中经常观察到诸如心律失常和畸胎瘤形成,造成这种影响多能干细胞来源的心肌细胞的原因是由于其不成熟的特性。心脏成熟奠定了出生后心脏发育和疾病的基础,但很少了解心肌细胞微环境的贡献及其作用这个过程中的动态。因此,我们亟待鉴定非心肌细胞类型和心肌细胞成熟的重要信号通路。 研究方法 首先,我们对出生后P1、P4、P7、P14以及P56小鼠心脏细胞进行单细胞测序检测,经生物信息学分析确定细胞分群类型、细胞各亚群基因变化以及非心肌细胞的信号通路变化。其次,我们鉴定对心肌细胞成熟起重要作用的成纤维细胞,并将其与P1心肌细胞共培养3.5天,经real-time PCR技术检测心肌细胞成熟基因以及增殖基因相对表达量;免疫荧光共染检测共培养体系心肌细胞增殖标志物AURKB、MKI67和pH3表达变化,缝隙连接蛋白GJA1表达变化,T-tubule检测共培养体系表达变化以及鬼笔环肽检测细胞分级;膜片钳技术检测共培养体系心肌细胞动作电位、离子通道(钠、钾、钙)变化以及fluo4,AM检测心肌细胞瞬时钙变化。再次,我们对共培养体系分别进行RNA测序和单细胞测序,以检测其与成熟心肌细胞的差异变化以及信号通路差异变化。紧接着,我们采用趋化因子受体抑制剂Plerixafor,细胞因子抑制剂Linifanib和ECM受体抑制剂 BP-1-102对共培养体系给药处理,免疫荧光检测其增殖蛋白AURKB、MKI67和pH3变化以及鬼笔环肽检测心肌细胞分级;三种抑制剂分别对出生后乳鼠,成年小鼠以及心梗模型小鼠给药处理14天和21天,超声检测其心脏功能变化,免疫荧光共染检测增殖蛋白AURKB、MKI67和pH3变化以及GJA1表达变化。最后,我们将人胚胎干细胞来源的心肌细胞与成人心脏成纤维细胞共培养7天,免疫荧光共染检测共培养体系心肌细胞增殖蛋白AURKB、MKI67和pH3变化以及GJA1表达变化以及鬼笔环肽检测心肌细胞分级;膜片钳技术检测共培养体系心肌细胞动作电位和钙通道变化。 研究结果 研究结果表明,出生后心肌细胞随着时间的延长逐渐成熟,心脏成纤维细胞参与的细胞因子、趋化因子受体以及ECM受体等信号通路在心肌细胞成熟过程中起着重要的调控作用,尤其以ENTPD2+为标志物的成纤维细胞在该过程中发挥着主要促进作用(p<0.05)。在共培养体系中,AF诱导乳鼠心肌细胞增殖基因降低,成熟基因表达升高,增殖标志物AURKB、MKI67和pH3表达量显著降低,且细胞分级主要处于CⅠ、Ⅱ,而CⅢ、Ⅳ 显著下降(p<0.05);在电生理实验中,共培养体系促进心肌细胞APD50和APD90显著升高,且钠、钾、钙离子通道以及瞬时钙也呈现出显著变化,而成熟心肌细胞标志之一的t-tubule呈阳性,GJA1表达显著增加(p<0.05)。RNA测序热图分析显示,共培养体系中升高基因与成熟心肌细胞中相似,而下降基因也与成熟心肌细胞相似;GSEA结果显示,共培养体系上升的基因主要集中于成熟心肌细胞P56一侧,而下降基因主要集中于乳鼠心肌细胞P1一侧。单细胞测序结果显示,共培养体系中存在从幼稚形态向成熟细胞形态的演进;而共培养体系与在体分析显示,共有9条相同信号通路参与,其中包括细胞因子信号通路、趋化因子受体信号通路以及ECM受体相互作用信号通路;这三条信号通路的抑制剂Plerixafor,Linifanib和BP-1-102对共培养体系给药处理后,增殖标志物AURKB、MKI67和pH3表达无显著性变化(p>0.05),而细胞形态发生显著性变化,给药组心肌细胞主要处于CⅢ、Ⅳ(p<0.05)。在乳鼠心脏中,Linifanib组乳鼠死亡,Plerixafor组和BP-1-102组给药14天后,心脏心肌细胞增殖蛋白AURKB、MKI67和pH3表达显著升高(p<0.05),而给药21天却无显著变化(p>0.05);Plerixafor和BP-1-102分别对成年小鼠的抑制结果显示,心脏心肌细胞增殖蛋白AURKB、MKI67和pH3表达无显著变化(p>0.05);Plerixafor和BP-1-102分别对心梗模型给药处理结果显示,给药14天后,在心梗周围区域尚有增殖的心肌细胞存在,增殖蛋白AURKB、MKI67和pH3表达显著升高(p<0.05),给药21天尚无变化(p>0.05)。成人心脏AF促进人胚胎干细胞来源的心肌细胞增殖标志物AURKB、MKI67和pH3表达量显著降低,且细胞分级主要处于CⅠ、Ⅱ,而CⅢ、Ⅳ 显著下降(p<0.05);在电生理实验中,共培养体系促进心肌细胞APD50和APD90显著升高,且钙离子通道也呈现出显著变化(p<0.05)。场电位和阻抗实验结果显示,AF 促进了ESC-CM的复极时间延长,而nifidipine(0.3 μM)加大了复极时间(p<0.05),而对照组无明显变化。 研究结论 我们的研究揭示了心脏成纤维细胞作为心肌细胞微环境的主要成分驱动心脏成熟,且参与心肌细胞的配体-受体相互作用的信号通路在心脏疾病和心脏再生中可能调节心肌细胞的成熟状态。 |
论文文摘(外文): |
background and ive acute myocardial infarction is most commonly caused by coronary artery atherosclerosis. atherosclerotic plaque rupture can lead to blocking of the left anterior descending artery, causing reduced blood supply, and therefore necrosis. in recent years, stem cell therapy has become a promising option for cardiac regenerative medicine. however, due to the immature nature of pluripotent stem cell-derived cardiomyocytes, they often produce serious side effects, such as arrhythmia and teratoma formation. hence, the study of cardiac maturation not only forms the core of understanding postnatal heart development and certain cardiac diseases, but also provides important clues to enhance cardiomyocyte maturity in vitro. although the role of the cardiomyocyte microenvironment has been extensively studied, little is known regarding its dynamics, and contributions to heart maturation. thus, the goal of this study is to elucidate regulatory roles of non-cardiomyocytes in cardiac maturation to serve a therapeutic purpose.
methods firstly, we performed single-cell rna sequencing on heart cells isolated from p1, p4, p7, p14 and p56 postnatal mice. bioinformatic analysis identified several known cell types, and further clustered them into subtypes. changes in cell subtype composition, corresponding gene expression, and putative signaling pathways revealed cardiac fibroblasts as a major player in cardiac maturation. to this end, we performed co-cultures of adult (p56) cardiac fibroblasts with neonatal (p1) cardiomyocytes for 3.5 days to observe the effect of the former on the maturation status of the latter. relative expression of cardiac maturation genes and proliferation genes were assessed by real-time pcr. expression of proliferation markers aurkb, mki67 and ph3 were detected by immunofluorescence. structural features of mature cardiomyocytes, such as gap junctions (gja1) and t-tubules, were measured by staining. further, maturity grading of cardiomyocytes was based on phalloidin staining of actin. patch clamping was used to detect action potentials and ion channel currents (sodium, potassium, calcium) of cardiomyocytes in co-culture, whereas changes in calcium transients were detected by fluo-4 am. in addition, we performed rna sequencing and single-cell rna sequencing on co-cultured cells to identify pathways critical for fibroblast-induced cardiomyocyte maturation. based on the preceding information, we used chemokine receptor inhibitor plerixafor, cytokine inhibitor linifanib, and ecm receptor inhibitor bp-1-102, to individually inhibit pathways predicted to be important to cardiomyocyte maturation. proliferation and maturation status of cardiomyocytes were determined via staining. to assess their effects in vivo, we applied them separately to neonatal, adult and infarcted mice for 14 days and 21 days, and cardiac function was detected by cardiac ultrasonography. proliferation and formation of gap junctions were determined by immunofluorescence. finally, we co-cultured human embryonic stem cell-derived cardiomyocytes with adult cardiac fibroblasts for 7 days, and examined proliferation and maturation status via various techniques.
results these results illustrated the maturation of postnatal cardiomyocytes, in which cardiac fibroblasts played a central regulatory role, mainly through inter-cell type crosstalk via cytokines, chemokines and ecm receptor signaling pathways. in particular, a subset of cardiac fibroblasts marked with entpd2+ played a significant role in this process (p<0.05). in the co-culture system, adult fibroblasts (afs) induced a series of changes in cardiomyocytes, including decreased proliferation and increased maturation grading (p<0.05). in electrophysiological experiments, af co-culture promoted a significant increase in apd50 and apd90 in cardiomyocytes. changes in sodium, potassium, calcium channels and calcium transients all exhibited signs of cardiac maturation. markers of mature cardiomyocytes, the number of t-tubules and gja1 expression, both significantly increased (p<0.05). further analysis showed that genes elevated in the co-culture system were similar to high-expressing genes in mature cardiomyocytes, while those decreased were similar to low-expressing ones. gsea showed that upregulated genes in the co-culture system were highly enriched on the p56 side (mature) of cardiomyocytes, while downregulated genes were significantly enriched on the p1 side (neonatal) of cardiomyocytes. single-cell sequencing results validated the evolution from naive to mature gene changes of cardiomyocytes in the co-culture system. kegg analysis of both in vitro and in vivo maturating cardiomyocytes showed 9 overlapping signaling pathways, including cytokine, chemokine receptor and ecm receptor interaction signaling pathways. interestingly, application of individual inhibitors to those pathways suppressed morphological maturation of cardiomyocytes in co-culture (p<0.05), but did not promote proliferation (p>0.05). drug administration to neonatal mice retarded cardiomyocyte proliferation, evidenced by increased expression of proliferation markers at 14, but not 21, days after treatment with plerixafor or bp-1-102. such changes were absent in adult mice. administration of plerixafor and bp-1-102 to the myocardial infarction model gave rise to proliferating cardiomyocytes in the area around the infarct after 14 days (but not 21 days), marked by significant increases in the expression of proliferating proteins aurkb, mki67 and ph3 (p<0.05). importantly, co-culture with adult cardiac afs decreased the expression of aurkb, mki67 and ph3 in human embryonic stem cell-derived cardiomyocytes, with the majority graded as ci, ii (mature). at the electrophysiological level, afs promoted significant increases in apd50 and apd90 of cardiomyocytes (p<0.05). similar changes towards maturation upon co-culture with afs were also manifested by changes in calcium transients, efp and impedance.
conclusions our study revealed that cardiac fibroblasts act as a major component of cardiac microenvironment to drive cardiac maturation, and that signaling pathways involved in ligand-receptor interactions with cardiomyocytes may regulate cardiomyocyte maturation in heart disease and cardiac regeneration. |
开放日期: | 2019-05-24 |