新型冠状病毒具有高感染力,其爆发性疫情会短时间内瘫痪局地医疗资源,造成大量患者重症甚至临床死亡.经过前期的精准施策、科学防治,我国疫情已得到良好控制,取得了阶段性的胜利.但新冠病毒的全球传播尚未结束,其高突变性、无症状感染等特征容易造成骤然爆发.因此,新冠疫情仍然是未来相当长时间内需要严肃面对的重大公共卫生安全问题.为了应对新冠疫情,医药院校的研究生免疫学课程可以适当进行调整,适当增加新冠感染及防疫相关的教学内容,选取高品质文献讲读,改进教学方式方法,夯实基础实验技能,并在教学中渗透思政教育.由此,推动研究生教育的发展提升,培养更多德才兼备的医学人才,为抗疫工作提供强大后援.

Human induced pluripotent stem cells (hiPSCs) possess the potential to differentiate toward vascular cells including endothelial cells (ECs), pericytes, and smooth muscle cells. Epigenetic mechanisms including DNA methylation and histone modification play a crucial role in regulating lineage differentiation and specification. Herein, we utilized a three-stage protocol to induce differentiation of mesoderm, vascular progenitors, and ECs from hiPSCs and investigated the regulatory effects of histone acetylation on the differentiation processes. We found that the expression of several histone deacetylases (HDACs), including HDAC1, HDAC5, and HDAC7, were greatly upregulated at the second stage and downregulated at the third stage. Interestingly, although HDAC1 remained in the nucleus during the EC differentiation, HDAC5 and HDAC7 displayed cytosol/nuclear translocation during the differentiation process. Inhibition of HDACs with sodium butyrate (NaBt) or BML210 could hinder the differentiation of vascular progenitors at the second stage and facilitate EC induction at the third stage. Further investigation revealed that HDAC may modulate the stepwise EC differentiation via regulating the expression of endothelial transcription factors ERG, ETS1, and MEF2C. Opposite to the expression of EC markers, the smooth muscle/pericyte marker ACTA2 was upregulated at the second stage and downregulated at the third stage by NaBt. The stage-specific regulation of ACTA2 by HDAC inhibition was likely through regulating the expression of TGFβ2 and PDGFB. This study suggests that HDACs play different roles at different stages of EC induction by promoting the commitment of vascular progenitors and impeding the later stage differentiation of ECs.

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.

目的 分析影响诱导人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)向内皮细胞分化的因素及其机制,探索提高hiPSCs向内皮细胞分化效率的诱导方法.方法 利用3阶段法诱导hiPSCs向内皮细胞定向分化,首先通过激活Wnt信号通路以启动中胚层分化,而后由VEGF、FGF2、BMP4诱导细胞分化为血管前体细胞,最后在VEGF作用下分化为成熟的内皮细胞.期间利用qRT-PCR检测一系列分化相关基因的表达,通过qRT-PCR、细胞免疫荧光、流式细胞术、成管实验验证细胞分化效果.通过VEGF、FGF2、BMP4两两配伍,比较细胞因子对诱导分化的影响;再进一步验证ERK和PI3K通路抑制剂对内皮诱导分化的影响;同时利用细胞免疫荧光分析不同起始细胞密度对诱导分化效果的影响.结果 hiPSCs可诱导分化为内皮细胞;VEGF、FGF2、BMP4三者联合具有高效内皮诱导分化效果;相较于PI3K,抑制ERK信号通路可显著抑制内皮分化效率,促进hiPSCs向平滑肌样细胞分化;低起始细胞密度更有利于提高内皮分化效率.结论 VEGF、FGF2、BMP4联合诱导可取得最佳诱导效果,其中ERK是介导hiPSCs向内皮分化的重要信号通路.

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.