骨髓多能成体祖细胞向起搏细胞的诱导分化及在心脏生物起搏器中的应用

Cardiac biological pacemaker is a hot issue in recent cardiac conduction study. Pacemaker cell transplantation is a major way to construct cardiac biological pacemakers and stem cells are main source of seed cells. There has been substantial progression in differentiation of adult stem cells to pacemaker cells induced by embryonic stem cells, induced pluripotent stem cells and gene transfection cells. Supported by two National Natural Science Foundations, we make a great step in this field. All those mentioned methods, however, remain to be further studied for ethics, reject reaction,clinical reliability,and so on. For the sake of clinical application, researchers have attempted to use non-genetic modification to induce and differentiate adult stem cells towards pacemaker cells. Regrettably, there is very little published information regarding the differentiation of adult stem cells into pacemaker cells by non-genetic modification. It is one of keys to seek ideal seeding cells and induce them into pacemaker cells for the development of cardiac biological pacemaker. In this study, bone marrow multipotent adult progenitor cells were used as seed cells. Conditioned culture and co-culture with sinus node cells was used to find a way to induce and differentiate pacemaker cells. We studied its mechanism and explored the feasibility of the inducer cells in constructing a cardiac biological pacemaker in vivo and in vitro, which can lay a clinical and experimental foundation of cardiac biological pacemaker.

心脏生物起搏器（CBP）是近年来心传导领域研究的热点，起搏细胞移植是构建CBP的主要方法，干细胞是主要的种子细胞来源。近年来，胚胎源性干细胞、诱导性多能干细胞（iPS），和通过基因转染的方法诱导成体干细胞向起搏细胞分化，取得了长足的进展。我们在二项国家自然科学基金的资助下，也取得了可喜的成绩。然而，胚胎源性干细胞、iPS细胞、和基因转染的细胞，由于其伦理、排斥反应和安全性等问题尚待深入研究，难以近期应用于临床。为此，国内外积极探索了用非基因改造的方法诱导干细胞向起搏细胞分化，但，目前尚无成体干细胞诱导成功的报道。寻找适宜的种子细胞和诱导方法，是CBP研究的关键问题之一。本研究拟以骨髓多能成体祖细胞为种子细胞，利用条件培养、与窦房结细胞共培养等方法，寻找将其诱导分化为起搏细胞的方法；研究其诱导分化机制；并探讨该诱导细胞在体内外构建CBP的可行性，旨在为CBP的临床应用奠定理论和实验基础。

心脏生物起搏器是近年来心传导领域研究的热点问题，起搏细胞、组织移植是构建心脏生物起搏器的主要方法，干细胞是主要的种子细胞来源。但胚胎源性干细胞、iPS细胞、和基因转染的细胞，由于其排斥反应和安全性问题，尚难以近期应用于临床。本研究以骨髓成体多能祖细胞(MAPCs)为种子细胞，探讨其诱导分化为起搏细胞的非基因干涉的方法及构建心脏生物起搏器的可行性。课题研究证实，通过非基因改造的办法，可以将MAPCs诱导为具有起搏离子通道的起搏样细胞，诱导后细胞能够驱动静息心肌细胞搏动，利用干细胞源性起搏细胞可以体外构建组织工程化起搏组织，体内移植能够建立异位起搏点。应用基因芯片和蛋白组学的方法初步分析了干细胞诱导分化为起搏细胞的机制，发现Bmp2/Alk3/Smad信号通路、 MAPK信号通路、及多组“关键分子-信号转导通路-lncRNA形成的调控网络”可能参与了这一过程。此外，为了探索更好的诱导分化效果，课题组尝试从骨髓中分离出比MAPCs更幼稚的胚胎样干细胞（ELSCs），作为诱导为起搏细胞的种子细胞；初步研究证实，ELSCs较MAPCs、MSCs更易于诱导分化为起搏样细胞。上述研究，为成体干细胞源性起搏细胞性生物心脏起搏器的临床应用，奠定了实验基础。