目的:评价小分子化合物雷帕霉素、羟氯喹和MC1568对心肌分化效率和心肌分化转录因子Mef2c表达的影响.方法:利用DMSO诱导P19CL6小鼠畸胎瘤干细胞分化为自发跳动的心肌细胞,检测自噬诱导剂雷帕霉素对小鼠P19CL6畸胎瘤细胞向心肌分化的影响,研究相关机制.结果:自噬诱导剂雷帕霉素能够有效促进心肌分化,增强心肌标志性基因表达;自噬抑制剂羟氯喹能抑制DMSO诱导的P19CL6细胞心肌分化.雷帕霉素促进心肌分化转录因子Mef2c表达;采用Mef2c的小分子抑制剂MC1568,则能抑制雷帕霉素诱导的心肌早期和晚期分化标志基因表达.结论:雷帕霉素诱导的Mef2c表达是增强心肌分化能力所必需的.

上皮间质转化(epithelial-mesenchymal transition,EMT)是指上皮细胞失去连接和极性转变为间质细胞的过程,这一现象普遍存在于胚胎发育、创伤愈合、器官纤维化以及肿瘤转移.在胚胎早期发育和晚期发育过程,例如着床、原肠运动、心血管发育等事件中有EMT和间质上皮转化(mesenchymal-epithelial transition,MET)的参与.EMT和MET参与调控干细胞表型变化、细胞迁移运动,是细胞差异分化和三维组织构建的重要机制.EMT的重要标志是细胞黏附分子表达由E-钙黏着蛋白(E-cadherin)向N-钙黏着蛋白(N-cadherin)转换.E-钙黏着蛋白通过与β-联蛋白、p120-联蛋白、a-联蛋白联合,影响Wnt、小GTP酶超家族等信号通路活化,调控细胞骨架运动.TGFβ、Notch、Wnt、BMP、FGF等信号通路,Snail、Twist、Zeb等转录因子,联合表观修饰酶,协同参与EMT的启动和调控.体外研究模型表明,E-钙黏着蛋白参与干细胞自我更新;而体细胞重编程可视为MET,重编程因子辅助体细胞获得E-钙黏着蛋白表达.体外研究发现,EMT及相关分子(例如E-钙黏着蛋白、Snail、Twist、Zeb等)参与了早期三胚层分化及晚期特定细胞类型的形成.对EMT机制的研究有助于理解和改善干细胞体外诱导分化效率,促进类器官的构建和诱导.

微生物与免疫学绪论可以结合马克思主义唯物辩证法,开展多种形式的教学探索.微生物与免疫学绪论是人类与各种病原微生物进行不懈斗争的历史浓缩.在人类对感染性疾病的认识当中,贯穿着世界观的自然演进过程,从唯心主义到朴素唯物主义、机械唯物主义,直到辩证唯物主义,同时伴随着不同医学模式的转换.因此,在教学过程中结合唯物辩证法哲学和医学模式转换进行授课,可以起到较好的思政教育效果.

目的 雷帕霉素是小分子mTOR抑制剂,可激活细胞自噬.本研究检测了雷帕霉素对胚胎干细胞(ES细胞)向心肌分化的影响.方法 采用拟胚体(embryoid body,EB)加抗坏血酸诱导ES细胞向心肌分化.悬浮诱导阶段添加自噬激动剂雷帕霉素或抑制剂羟氯喹,通过免疫印迹检测LC3蛋白剪切以监测细胞自噬水平,通过检测EB球的心肌搏动百分比以及心肌分化标志物troponin-T和α-actinin的表达来评估细胞分化效率.采用real-time PCR检测心肌分化转录因子Mef2c和Isl1 mRNA表达,免疫印迹法检测Oct4表达,进一步阐释雷帕霉素的效应机制.结果 雷帕霉素能够诱导自噬,促进LC3剪切,促进EB球产生自发搏动,增强心肌分化标志基因表达.机制研究发现,雷帕霉素促进心肌转录因子Mef2c和Isl1 mRNA的表达和Oct4在分化细胞的下调.而自噬抑制剂羟氯喹能拮抗雷帕霉素的上述效应.结论 雷帕霉素诱导自噬可以促进心肌转录因子表达和促进干性因子Oct4水平下调,从而促进心肌分化.

4-phenylbutyrate (4-PBA), a terminal aromatic substituted fatty acid, is used widely to specifically attenuate endoplasmic reticulum (ER) stress and inhibit histone deacetylases (HDACs). In this study, we investigated the effect of 4-PBA on cardiac differentiation of mouse embryonic stem (ES) cells. Herein, we found that 4-PBA regulated cardiac differentiation in a stage-specific manner just like trichostatin A (TSA), a well-known HDAC inhibitor. 4-PBA and TSA favored the early-stage differentiation, but inhibited the late-stage cardiac differentiation via acetylation. Mechanistic studies suggested that HDACs exhibited a temporal expression profiling during cardiomyogenesis. Hdac1 expression underwent a decrease at the early stage, while was upregulated at the late stage of cardiac induction. During the early stage of cardiac differentiation, acetylation favored the induction of Isl1 and Nkx2.5, two transcription factors of cardiac progenitors. During the late stage, histone acetylation induced by 4-PBA or TSA interrupted the gene silence of Oct4, a key determinant of self-renewal and pluripotency. Thereby, 4-PBA and TSA at the late stage hindered the exit from pluripotency, and attenuated the expression of cardiac-specific contractile proteins. Overexpression of HDAC1 and p300 exerted different effects at the distinct stages of cardiac induction. Collectively, our study shows that timely manipulation of HDACs exhibits distinct effects on cardiac differentiation. And the context-dependent effects of HDAC inhibitors depend on cell differentiation states marked by the temporal expression of pluripotency-associated genes.

Pathological cardiac hypertrophy, characterized by enlarged cell size and fetal gene reactivation, ultimately leads to cardiac dysfunction and heart failure. The expression of transforming growth factor beta 1 (TGFβ1) is often elevated in experimental models of cardiac hypertrophy. In the present study, we observed the activation of Wnt/β-catenin signaling in TGFβ1-induced cardiac hypertrophy. TGFβ1 stimulation decreased the phosphorylation levels of β-catenin and triggered the nuclear accumulation of β-catenin. In turn, TGFβ1 enhanced the expression of c-Myc, which is a transcriptional target of canonical Wnt/β-catenin pathway. Knockdown of β-catenin completely blocked TGFβ1-induced c-Myc upregulation. Wnt3a is an important Wnt ligand associated with cardiac fibrosis and hypertrophy. Further investigation revealed that TGFβ1 can upregulate Wnt3a expression in an ALK5-Smad2/3-dependent manner. A consensus Smad binding sequence is located within the Wnt3a promoter, and TGFβ1 stimulation enhanced recruitment of Smad2/3 onto the Wnt3a promoter. Meanwhile, Wnt3a overexpression also stimulated TGFβ1 expression. Chemical inhibition of Wnt/β-catenin signaling partially attenuated TGFβ1-induced hypertrophic responses. These findings suggest crosstalk between TGFβ1 and canonical Wnt/β-catenin pathways in cardiac hypertrophy.

表观遗传调控,如组蛋白乙酰化修饰,是决定干细胞分化方向的重要机制.组蛋白去乙酰化酶抑制剂(HDACi)通过影响不同亚类的组蛋白去乙酰化酶(HDAC)活性,提高组蛋白乙酰化水平,调控基因表达,从而影响胚胎干细胞自我更新,以及沿神经元、心肌和造血等细胞谱系的定向分化.HDACi类小分子化合物在体细胞重编程中也有广泛的应用,可替代致癌因子c-Myc和Klf4,促进体细胞克隆.研究显示,HDACi的效应与药物剂量、细胞类型和细胞分化状态密切相关.本文主要阐述了HDACi在干细胞分化和体细胞重编程中的应用进展,并对所涉及的分子通路进行讨论,有助于揭示干细胞定向分化的关键分子机制,优化干细胞定向分化诱导策略,对干细胞诱导分化具有重要的理论和实用价值.