【作者】 程颖；

【导师】 唐明；

【作者基本信息】 华中科技大学 ， 生理学， 2011， 博士

【摘要】 第一部分葛根素促进小鼠胚胎干细胞向心肌细胞分化及调控机制目的：干细胞源心肌细胞是再生医学中治疗心力衰竭等心脏疾病的很有前景的细胞来源。如何进一步提高干细胞源心肌细胞的分化率以确保充足的细胞来源是目前急需解决的首要问题。传统中药的活性成分葛根素已经广泛运用于临床治疗各种心血管疾病。本课题旨在研究葛根素对离体培养的小鼠胚胎干细胞(murine embryonic stem cells, mES细胞)向心肌细胞分化的作用及其调控机制。方法：首先采用MTT法检测不同浓度的葛根素对mES细胞增殖能力的影响,筛选适合用于诱导分化的葛根素浓度。采用经典诱导心肌分化的三步法,即“悬滴-悬浮-贴壁”,持续用1μmol/L、10μmol/L、100μmol/L葛根素诱导mES细胞分化。倒置显微镜下连续观测胚体(embryoid body, EB)大小和跳动EB(含跳动区域的EB)的比例。运用免疫荧光染色法检测心肌特异性蛋白α-肌小节辅肌动蛋白(α-sarcomeric-actinin)的表达。运用反转录PCR检测不同胚层标志物的表达。用western blot检测葛根素诱导分化后丝氨酸-苏氨酸激酶(serine-threonine kinase,Akt)和磷酸化丝氨酸-苏氨酸激酶(phosphorylation of the serine-threonine kinase,P-Akt)的表达。结果：葛根素促进mES细胞增殖,增加分化过程中跳动EB比例。100μmol/L葛根素为最佳实验浓度。免疫荧光结果显示葛根素明显增多分化第12天α-sarcomeric-actinin阳性细胞。半定量RT-PCR分析表明葛根素明显上调分化早期内胚层、中胚层标志性基因表达,但显著下调外胚层标志性基因。葛根素组EB的直径显著增大,P-Akt的表达上调。结论：葛根素促进mES细胞增殖,影响mES细胞分化方向。其作用可能与磷脂酰肌醇(-3)激酶／丝氨酸-苏氨酸激酶(phosphatidylinositol 3-kinase/serine-threonine kinase, PI3K/Akt)途径有关。第二部分葛根素诱导小鼠胚胎干细胞向心肌分化并促进心室样心肌细胞的发育目的：多能干细胞分化出的心肌样细胞包括起搏样心肌细胞,心房样心肌细胞,心室样心肌细胞。心肌梗死部位好发于心室,用心室样心肌细胞修复心肌梗死部位明显更具优势。干细胞来源的心肌细胞含有多种类型,如何在提高心肌分化率的同时,促进心室样心肌细胞的特化的研究仍然甚少。已有关于植物雌激素能够调节心肌发育调控基因生长因子β(TGF-β)、骨形成蛋白(BMP)和Wnt的报道,而且第一部分的研究也显示植物雌激素葛根素能促进鼠胚胎干细胞(mES cells)向心肌分化,所以此部分的课题主要研究葛根素对mES细胞向心肌分化的影响,检测向心室样心肌细胞分化的效率和机制。方法：采用经典的“悬滴-悬浮-贴壁”三步法,持续用100μmol/L葛根素诱导mESCs分化。用流式细胞术检测心肌肌小节特异性α-肌小节辅肌动蛋白(α-sarcomeric-actinin)阳性细胞的含量。RT-PCR检测心肌特异性标志物和心肌发育调控基因。采用膜片钳技术分别测定起搏样细胞,心房样心肌细胞和心室样心肌细胞的含量。结果：葛根素能提高跳动胚体的百分比,能提高α-sarcomeric actinin阳性细胞的百分比。在分化早期葛根素处理组T-box5 (Tbx5),心肌细胞增强因子2c (myocyte enhancer factor 2c, MEF2c)和心肌肌球蛋白重链(α-myosin heavy chain,α-MHC)的表达较阴性对照组明显提高。在进一步的分化过程中葛根素能提高心室肌特异性基因MLC2的表达,同时降低起搏样细胞的特异性基因HCN4的表达。膜片钳结果也显示葛根素诱导mES细胞分化出的心室样心肌细胞含量较阴性对照组高。在分化早期葛根素能明显上调TGF-β1,2,3,和Wnt11的表达,而对Wnt3a, BMP2和BMP 4的表达无影响。结论：葛根素能促进mES细胞向心肌分化,尤其是向心室样心肌细胞分化。TGF-β和Wnt信号途径可能参与介导葛根素的早期诱导效应。更多还原

【Abstract】 Aims:Currently CMs derived from stem (ES) cells represent the most promising CMs for cardiac regenerative medicine.lt is important to screen and identify chemical compounds that induce high efficiency cardiac differentiation.Phytoestrogen puerarin is a traditional Chinese herbal medicine to treat many cardiac diseases. The aim of this study was to investigate the possible inducible effects of puerarin on embryonic stem cells differentiation and regulatory mechanism in vitro.Methods:Effect of puerarin on proliferation of murine ES cells were analyzed via MTT assay, then were differentiated using The classic "hanging drop-Suspension-adherent" three step differentiation protocol with or without 1μmol/L、10μmol/L、100μmol/L puerarin. Differentiating ES cells were analyzed via morphological analysis for diameter of embryonic bodys (EBs) and percentage of beating EBs, immunofluorescence staining forα-sarcomeric-actinin, RT-PCR for specific genes of different mesodern, western blot for serine-threonine kinase (Akt) and phosphorylation of the serine-threonine kinase (P-Akt).Results:Puerarin (100μmol/L) resulted in significantly increased number of cardiac cells and percentage of beating EBs,100μmol/L puerarin was the optimal concentration Puerarin resulted in a significantly increased the number ofα-sarcomeric-actinin positive cells on differentiation day 12. RT-PCR results showed expressions of endoderm, cardiac (mesoderm) and endothelial cells specific gene and decreased expressions of neuronal cells (ectoderm) specific gene at early differentiation stages. Furthermore, we confirmed that embryoid body (EB)-size increased by puerarin might affect the direction of differentiation. Puerarin also facilitated phosphorylation of the serine-threonine kinase (Akt). Conclusions:Taken together, our data suggest that PI3K/Akt pathway might mediate effects of puerarin on proliferation and differentiation of mES cells. Aims:Ventricular cells are obviously the best candidate for reconstructing injured ventricles. However, Cardiomyocytes derived from embryonic stem cells(ES cells) are the mixture of pacemaker-, ventricular-, atrial-like cells, which can be distinguished based on their electrophysiological properties. Therefore, it is important to screen and identify chemical compounds that induce high efficiency cardiac differentiation and specialization of ESCs. Phytoestrogen puerarin has protective effect against myocardial reperfusion injury, has been used clinically for treatment of myocardial infarction. In the present work we aimed to investigate the effects of puerarin on cardiac differentiation of ESCs and its ventricular specialization.Methods:Murine ES cells were differentiated using standard embryoid body-based differentiation protocol with or without 100μmol/L puerarin. Flow cytometry, semi-quantitative RT-PCR and patch clamp were employed to assess differentiating ES cells.Results:Puerarin resulted in a significantly increased percentage of ES cells-derived cardiomyocytes (ES-CMs), the up-regulated transcript levels of transforming growth factor-beta (TGF-β)1,2,3, and Wnt11, but not Wnt3a, bone morphogenetic protein (BMP)2 and 4. The expressions of T-box5, myocyte enhancer factor 2c, and a-myosin heavy chain are significantly increased in early differentiation stage. In advanced differentiation stage, puerarin enhanced expressions of ventricular-specific myosin light chain 2 ventricular transcrip. Patch-clamp analysis confirmed that puerarin doubled percentage of ventricular-like cells.Conclusions:Our results suggest that puerarin promotes cardiac differentiation and enhances specialization of mES cells into ventricular-like cells through regulating expression of multiple genes involved in cardiac development and ventricle differentiation.更多还原