基于HCN转染VSELs构建生物起搏信号通路筛选诱导剂

Very small embryonic-like stem cells (VSELs) have successfully been separated, identified and cultivated and their induction procedure has been preliminarily established in our early study subject. We have also found that VSELs with pluripotent characteristics and the stronger ability to rebuild the biological pacemaker in repairing sinus node or the His Bundle conduction tissue. Based on these new problems and recent progress of the above studies, the lentiviral vector with HCN isoforms were transfected to swine VSELs, then cell proliferation, transfection efficiency and paracrine action were compared under the hormones and drugs inducing to them. RNA-sequencing technology will be used to explore the signaling pathway and the key gene inducer in the differentiation process in order to clarify the molecular mechanisms of the VSELs differentiation into pacemaker-like cells; and the expression of pacemaker channel protein in the transfected cells and dynamic characteristics of If current will be detected to get pacemaker-like cells which are stable, long-lasting and high-level expressed of HCN. Further, pacemaker-like cells with advantages of HCN combination will be implanted into His Bundle damage zone in animal cardiac model with permanent complete atrioventricular block under the guideline of three-dimensional mapping system to explore a new method to treat bradyarrhythmia and to provide an experimental basis for building the new generation of stable long-term biological pacemaker.

本课题前期已对极小胚胎样干细胞（VSELs）进行了分离、鉴定和培养并初步建立了诱导分化体系，同时发现VSELs具有多潜能特征,对于重建心脏起搏传导组织可能有更强的修复能力。基于此，本课题通过筛选超极化激活环核苷酸门控(HCN)基因及其两亚型基因组合为心脏生物起搏靶基因，以慢病毒作载体转染猪VSELs，同时采用多种药物诱导分化，比较转染后VSELs诱导效率，旁分泌作用；通过RNA-seq技术，探寻VSELs诱导分化过程中的信号通路，弄清其分化为起搏样细胞分子机制，基于信号通路的关键基因筛选诱导剂；并对起搏通道蛋白表达及If电流动力学特性进行检测，获得高效表达的起搏样细胞。进一步将HCN组合优势的起搏样细胞于三维标测下在体靶向植入完全性永久房室传导阻滞动物模型的希氏束损伤区，为构建稳定长效物心脏起搏器奠定实验基础。

对于房室传导阻滞患者的治疗，目前普遍使用的电子起搏器存在多种固有缺陷，而构建心脏生物起搏器的研究还处于动物实验阶段。为进一步探索干细胞诱导分化为起搏样细胞的分子机制，探寻诱导分化过程中的信号通路，本课题组完成了极小胚胎样干细胞的分离、鉴定和培养，建立了适宜的诱导分化体系，阐明HCN基因调控心脏起搏细胞发生的机制及关键信号通路。.本研究在国际上首次建立并完善了猪骨髓VSELs细胞的分离、纯化及鉴定体系，将红细胞裂解法、免疫磁珠分选法与流式细胞分选术相结合，有效提高猪骨髓中VSELs的提取效率。对所分离和培养的VSELs，透射电镜显示该细胞核大胞浆少，胞核中无明显细胞器，呈现明显幼稚细胞的结构特点，PCR及WB结果显示其表达CD133、SOX-2、OCT-4、NANOG等干细胞特异性基因，上述结果从超微结构、基因水平及蛋白水平等多个角度证明其具有干细胞特性。.为验证其多向分化潜能，本课题组以骨髓间充质干细胞定向诱导分化为参照，摸索并建立干细胞诱导分化体系，参照该体系将所获得的VSELs诱导分化为神经元细胞、心肌细胞和脂肪细胞，定量PCR明确诱导过程中相关基因表达上调，证实其具有具有多向分化潜能。裸鼠成瘤实验中，将VSELs进行注射后连续观察未形成肿瘤包块，组织切片染色未见肿瘤细胞形成，证实VSELs不具备自行成瘤能力。结合既往研究结果，考虑VSELs具有向三胚层细胞分化能力，但不具有恶性增殖成瘤性能，该特点与其表观遗传修饰相关，进一步验证其后续应用中可避免肿瘤形成风险，具有较高的安全性。.此外，本研究将不同HCN基因1、2、4亚型分别构建慢病毒质粒载体，联合转染VSELs，与小分子化合物共同作用促进VSELs向心肌细胞分化。通过免疫共沉淀证明HCN基因相关蛋白参与心肌形成，通过调节信号通路中关键基因的表达发挥调节作用，促进起搏样细胞的形成和发展。