黄芩苷促进小鼠胚胎干细胞向功能性心肌细胞分化及其调控机制
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摘要
背景：
胚胎干细胞(embryonic stem cells, ES细胞)是一类可进行自我复制和更新的细胞,在一定条件下能够向骨、软骨、脂肪、神经、肌肉等多个胚层的组织细胞分化。多项研究表明,ES细胞移植入缺血心脏后可以分化形成新生心肌细胞、血管内皮细胞等,还可以通过分泌多种生长因子,促进微循环的建立,增加血流灌注,提高梗死后心脏功能。将ES细胞的多向分化特性用于临床治疗,可能为心肌梗死后的慢性心力衰竭患者带来新的希望。
ES细胞在体外培养中可以被诱导分化为心肌样细胞,具备心肌细胞的部分表型,表达心肌特异性标志物,甚至出现自发跳动。ES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。但是ES细胞在体外自然分化为心肌样细胞的效率很低,研究者已经研究了用很多诱导因子在体外诱导ES细胞向心肌细胞分化,尽管取得了不少成就,但还是未能达到临床移植治疗心肌疾病所需要的细胞的数量和纯度。如何进一步提高ES细胞源心肌细胞的分化率以确保充足的心肌细胞来源是目前亟需解决的首要问题。寻找新的更有效、更安全、更经济的诱导剂仍是研究的热点问题。传统中药的活性成分黄芩苷(baicalin)具有抗氧化、抗炎、保护缺血后受损的心肌细胞的作用,已经广泛运用于临床治疗各种心血管疾病。本课题旨在研究黄芩苷对离体培养的小鼠ES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。
胚胎干细胞分化是外界刺激和细胞内特异性信号转导调控的共同作用结果。ES细胞分化为心肌细胞的过程类似胚胎时期的表现,心肌发育相关调控基因在这一过程中会被激活,但其表达规律尚未被阐明。结合现有的中药诱导ES细胞向心肌细胞分化的研究现状,我们希望通过使用黄芩苷诱导ES细胞向心肌细胞分化的实验,试图在以下几个方面有所发现：(1)黄芩苷能否诱导mES细胞向心肌细胞分化?(2)在诱导后不同时间点心肌细胞的标志物表达有何差异?(3)黄芩苷在影响ES细胞向心肌细胞分化的过程中具体影响到了哪些调控心肌分化的信号通路?希望通过上述问题的研究阐明ES细胞向心肌细胞分化的影响因素,为提高心肌细胞的分化比率提供理论依据,加快ES细胞源心肌细胞在临床上的应用进程。
目的：
1.观察黄芩苷对mES细胞增殖的影响；
2.观察经黄芩苷诱导分化后不同时间点胚体大小的变化；
3.分别研究经黄芩苷处理后不同分化时间点心肌特异性标志物在RNA水平和蛋白水平的表达变化；
4.研究调控心肌分化的各信号通路的相关基因在黄芩苷处理后不同分化阶段的表达变化,揭示黄芩苷影响心肌分化的可能作用机制。
方法：
1.采用MTT(四甲基偶氮苯唑盐)法检测不同浓度的黄芩苷对mES细胞增殖能力的影响,筛选最佳浓度用于诱导mES细胞向心肌细胞分化。2.mES细胞的体外诱导分化培养：
采用经典的“悬滴-悬浮-贴壁”三步法,持续用终浓度为10μmol/L和50μmol/L黄芩苷诱导mES细胞分化。每2天更换一次培养基,并保持分化过程中培养基始终维持相同的药物浓度。
3.形态学观察：
镜下观察mES细胞克隆的形态。诱导分化后,用倒置显微镜连续观测胚体(embryoid bodies, EBs)的直径和含跳动区域的EBs数占总EBs数的百分比。
4.流式细胞检测：
应用Phycoerythrin(PE)标记的抗体分别检测分化第10、16、20天的EBs的心肌细胞占总细胞数的百分率,即a-sarcomeric-actinin标记阳性细胞的百分率。
5.免疫荧光检测：
分化第16天,用免疫荧光染色法检测分化出的心肌样细胞的心肌特异性标志物a-肌小节辅肌动蛋白(α-sarcomeric-actinin)和心室肌特异性肌球蛋白轻链2 (Myosin light chain-2, ventricular, MLC-2v)的表达。
6.逆转录聚合酶链式反应(RT-PCR)和实时荧光定量聚合酶链式反应(real-timePCR)检测：
分别在分化后第2、4、6、8、10、12、14、16、20天时提取细胞总RNA,经逆转录反应(RT)得到cDNA,以3-磷酸甘油醛脱氢酶(GAPDH)作为内参基因,通过RT-PCR和real-time PCR检测三个胚层标志物如Soxl7、PEC AM和NCAM,心肌转录因子如Nkx2.5、MEF2c、TBX5、GATA4,心肌特异性基因如α-心肌肌球蛋白重链(Myosin heavy chain,α-MHC)、MLC-2v、心房钠尿肽(atrial natriuretic peptide, ANP)和HCN4 (hyperpolarization-activated cyclic nucleotide-gated channel 4),及心肌发育调控信号通路相关基因如TGF-β1、TGF-β2、TGF-β3、BMP2、BMP4、Wnt11、Wnt3a的mRNA表达水平。
7.免疫印迹(western blot)检测：
分别取分化第10、16、20天的细胞,提取细胞的总蛋白,经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印迹、ECL显色曝光等步骤,检测心肌特异性标志蛋白α-actinin、MLC-2v的表达。
8.膜片钳记录：
利用膜片钳技术检测干细胞来源的心肌细胞的电生理学特性和激素调节反应性。
结果：
不同浓度的黄芩苷对mES细胞的增殖均有抑制作用,此抑制作用呈浓度依赖性。在分化的过程中,黄芩苷处理组的EB直径显著小于对照组。但是50μmol/L黄芩苷明显地提高了mES细胞向心肌细胞的分化比例,表现在：(1)黄芩苷提高了中晚期跳动胚体的比例。(2)黄芩苷提高了α-actinin阳性细胞的比例。(3)分化中后期,心肌特异性标志物α-MHC、MLC-2v及ANP的mRNA水平在黄芩苷组的表达明显高于对照组。免疫荧光结果显示在对照组和黄芩苷处理组得到的mmES细胞来源的心肌细胞中都有心肌特异性的蛋白α-actinin和MLC-2v的表达。膜片钳记录结果显示分化出来的心肌细胞中存在着起搏样细胞、心房样细胞、心室样细胞。黄芩苷处理组中的心房及心室样心肌细胞的比例高于空白对照组,起搏样细胞的比例低于对照组。除了起搏样细胞和心室样细胞的跳动频率在黄芩苷组高于对照组外,其他的动作电位参数在两组间没有明显的差异。黄芩苷诱导出的心肌细胞具有了完整的激素调节功能。检测的心肌转录因子中,仅有Nkx2.5在分化晚期被黄芩苷上调,其他转录因子的表达未明显受到黄芩苷处理的影响。黄芩苷在分化中期上调Wnt3a的表达,然而在分化晚期,黄芩苷则明显地抑制了Wnt3a的表达,黄芩苷对检测的其他调控心肌发育的基因mRNA水平没有产生明显的影响。
结论：
1.黄芩苷抑制mES细胞的增殖,此抑制作用具有浓度依赖性。
2.黄芩苷(50μmol/L)可以促进mES细胞分化为心肌细胞,此促进作用主要表现在分化晚期。
3.黄芩苷促进mES细胞向心房肌和心室肌细胞分化,黄芩苷诱导得到的心肌细胞具有完整的激素调节功能。
4.黄芩苷促进mES细胞向心肌细胞分化可能是通过调控经典的Wnt/β-catenin信号通路实现的。
Background:
Embryonic stem cells (ES cells) are capable of self-replication and renewal. Under the certain condition, they will differentiate into multiple lineages cells, including osteoblast, chondrocyte, adipocyte, neurocyte, muscle cells and so on. A big number of evidence have indicated that ES cells transplanted into the ischemic heart will regenerate the necrotic cardiomyocytes and vascular endothelial cells, can also secrete various growth factors, promote the establishment of a micro-circulation, increase perfusion and improve heart function after infarction. The clinical therapy based on the utilizing of pluripotential ES cells has become the new hope for numerous chronic heart failure patients after myocardial infarction.
ES cells can be induced to differentiate into cardiomyocyte-like cells in vitro, these cardiomyocytes express part of the phenotype of myocardial cells, cardiac-specific markers, or even show spontaneous beating. Cardiomyocytes derived from the stem cells are promising candidate cell sources used for regenerative medicine in the treatment of heart failure disease. But the in vitro spontaneously cardiomyocytes differentiation from ES cells is low efficiency. Researchers have studied many inducible factors to induce cardiac differentiation from ES cells in vitro. Although the effects of these chemicals on cardiac differentiation are impressive, it still does not meet the request of quantity and purity of cells used in clinical transplantation. How to further enhance the efficiency of cardiac differentiation of ES cells is the key problem to be solved. Looking for a new and more effective, safer, more economical inducer is still a hot research topic. Baicalin is one of the active ingredients of traditional Chinese medicine, it possesses anti-oxidant and anti-inflammatory activities, and thus can protect cardiomyocytes exposed to ischemia/reperfusion. Baicalin has been widely used in clinical treatment of various cardiovascular diseases. The purpose of this project is to study the effect of baicalin on cardiac differentiation of mouse embryonic stem cells (mES cell) and its underlying mechanism.
The differentiation of ES cells draws assistance from the regulation of stimulus signal coming from outside accompany with intercellular specific transcription course. It has been approved that ES cells differentiation into cardiomyocytes-like cells is partly similar to embryonic development. Some regulation factors can be reactivated during the cell differentiation. However, the expression pattern has not yet been elucidated. Based on the existing status of traditional Chinese medicine-induced cardiac differentiation of ES cells, we try to use baicalin as an inducer to study the cardiac differentiation of ES cells to make discoveries in the following areas:(1) Whether baicalin can induce cardiac differentiation from mouse embryonic stem cells? (2) Whether the expression of myocardial cell marker after induction is different at every differentiation time points? (3) Which underlying signaling pathways is affected by baicalin during cardiac differentiation from ES cells? Clarifying the above issues can provide a theoretical basis to promote the efficiency of myocardial cell differentiation and speed up the process of clinical use of ES cells derived cardiomyocytes.
Aims:
(1) To observe the effect of baicalin on the proliferation of mouse embryonic stem cells;
(2) To observe the size of embryoid body (EB) after induction by baicalin at different development time points;
(3) To detect the expression of cardiac-specific markers at the mRNA and protein levels;
(4) To study the expression of genes related to cardiac differentiation during different differentiation stages after baicalin treatment, revealing the possible mechanism of baicalin affecting myocardial differentiation.
Methods:
1.MTT (3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide the tetramethyl azo oxacillin salt) assay was used to detect the effect of different concentrations of baicalin on the proliferation capacity of mES cells, screening the best concentration used to induce cardiac differentiation of embryonic stem cells.
2. Differentiation of mouse embryonic stem cells:
The classic "hanging drop-suspension culture-adherent culture" three-steps method was used to initiate cardiac differentiation. A final concentration of 10μmol/L or 50μmol/L baicalin was maintained in the differentiation medium during all the stages of differentiation. The medium was changed every 2 days.
3. Morphological observation:
Observe the morphology of embryonic stem cells before differentiation. After differentiation, We use an inverted microscope to observe the size of the embryo (embryoid body, EB) and the percentage of beating EBs (EBs with beating area).
4. Flow cytometry analysis:
We evaluated the percentage of cardiomyocytes at 10,16,20 days of EB by PE labeledα- sarcomeric actinin positive cells.
5. Immunofluorescence analysis:
At day 16 of differentiation, the expression of cardiac-specific markerα-sarcomeric actinin and ventricular muscle-specific myosin light chain 2 (myosin light chain-2, ventricular, MLC-2v) was detected using immunofluorescence staining.
6. Reverse transcriptase polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative polymerase chain reaction (real-time PCR):
The total cellular RNA was extracted from the EBs of days 2,4,6,8,10,12,14,16,20, cDNA was obtained by reverse transcriptase reaction (RT), housekeeping gene 3-phosphate dehydrogenase (GAPDH) was used as internal control, the mRNA levels of three germ layer marker and cardiac-specific genesα-cardiac myosin heavy chain (α-MHC), the MLC-2v, atrial natriuretic peptide(ANP), hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and myocardial developmentally regulated signaling pathway genes such as TGF-β1, TGF-β2, TGF-β3, BMP2, BMP4, Wnt11, Wnt3a and cardiac transcription factor such as Nkx2.5, MEF2c, TBX5, of GATA4 were detected by RT-PCR and real-time PCR.
7. Western blot:
The total protein was extracted from EBs at day10,16,20. After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to a PVDF membrane. After western blotting, ECL color development, exposure, the a-actinin and MLC-2v was detected.
8. Patch clamp analysis:
Electrophysiological characteristics and hormonal regulation of the cardiomyocytes derived from ES cells were detected using patch clamp technique.
Results:Baicalin inhibited the proliferation of mES cells in a concentration dependent manner. During all the differentiation stages, the size of EBs was significantly decreased in baicalin treatment groups than controls.50μmol/L baicalin successfully promoted the late stage cardiac differentiation in vitro, as demonstrated by increases in the percentage of beating EBs,α-actinin+ cells, and expression of cardiac specific markers. Typical pacemaker-like, atrial-like and ventricular-like action potentials (APs) were recorded by patch-clamp. Baicalin enhanced the percentage of working cardiomyocytes while decreased the percentage of pacemaker-like cells.β-adrenergic and muscarinic signaling cascades are present and functional in baicalin-induced ES-CMs. Further analysis revealed that baicalin strikingly up-regulated Wnt3a which is an activator of Wnt/β-catenin signaling pathway at the intermediate stage while dramatically down-regulated it at the late stage, but the expressions of TGF-β1/β2/β3, BMP2/4 and Wnt11 were not affected by baicalin. Downstream cardiac transcription factor Nkx2.5 was elevated by baicalin at the late stage, while Mef2c, Tbx5 and Gata4 were not significantly affected by baicalin.
Conclusion:
1. Baicalin inhibited the proliferation of ES cells in a concentration dependent manner.
2.50μmol/L of baicalin successfully promoted cardiac differentiation of mES cells in vitro at the late stage.
3. Baicalin enhanced the percentage of working ceardiomyocytes. The cardiomyocytes derived from ES cells had intact hormonal regulation.
4. Canonical Wnt/β-catenin signaling pathway might play an important role in this effect of baicalin.
胚胎干细胞(embryonic stem cells, ES细胞)是一类可进行自我复制和更新的细胞,在一定条件下能够向骨、软骨、脂肪、神经、肌肉等多个胚层的组织细胞分化。多项研究表明,ES细胞移植入缺血心脏后可以分化形成新生心肌细胞、血管内皮细胞等,还可以通过分泌多种生长因子,促进微循环的建立,增加血流灌注,提高梗死后心脏功能。将ES细胞的多向分化特性用于临床治疗,可能为心肌梗死后的慢性心力衰竭患者带来新的希望。
ES细胞在体外培养中可以被诱导分化为心肌样细胞,具备心肌细胞的部分表型,表达心肌特异性标志物,甚至出现自发跳动。ES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。但是ES细胞在体外自然分化为心肌样细胞的效率很低,研究者已经研究了用很多诱导因子在体外诱导ES细胞向心肌细胞分化,尽管取得了不少成就,但还是未能达到临床移植治疗心肌疾病所需要的细胞的数量和纯度。如何进一步提高ES细胞源心肌细胞的分化率以确保充足的心肌细胞来源是目前亟需解决的首要问题。寻找新的更有效、更安全、更经济的诱导剂仍是研究的热点问题。传统中药的活性成分黄芩苷(baicalin)具有抗氧化、抗炎、保护缺血后受损的心肌细胞的作用,已经广泛运用于临床治疗各种心血管疾病。本课题旨在研究黄芩苷对离体培养的小鼠ES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。
胚胎干细胞分化是外界刺激和细胞内特异性信号转导调控的共同作用结果。ES细胞分化为心肌细胞的过程类似胚胎时期的表现,心肌发育相关调控基因在这一过程中会被激活,但其表达规律尚未被阐明。结合现有的中药诱导ES细胞向心肌细胞分化的研究现状,我们希望通过使用黄芩苷诱导ES细胞向心肌细胞分化的实验,试图在以下几个方面有所发现：(1)黄芩苷能否诱导mES细胞向心肌细胞分化?(2)在诱导后不同时间点心肌细胞的标志物表达有何差异?(3)黄芩苷在影响ES细胞向心肌细胞分化的过程中具体影响到了哪些调控心肌分化的信号通路?希望通过上述问题的研究阐明ES细胞向心肌细胞分化的影响因素,为提高心肌细胞的分化比率提供理论依据,加快ES细胞源心肌细胞在临床上的应用进程。
目的：
1.观察黄芩苷对mES细胞增殖的影响；
2.观察经黄芩苷诱导分化后不同时间点胚体大小的变化；
3.分别研究经黄芩苷处理后不同分化时间点心肌特异性标志物在RNA水平和蛋白水平的表达变化；
4.研究调控心肌分化的各信号通路的相关基因在黄芩苷处理后不同分化阶段的表达变化,揭示黄芩苷影响心肌分化的可能作用机制。
方法：
1.采用MTT(四甲基偶氮苯唑盐)法检测不同浓度的黄芩苷对mES细胞增殖能力的影响,筛选最佳浓度用于诱导mES细胞向心肌细胞分化。2.mES细胞的体外诱导分化培养：
采用经典的“悬滴-悬浮-贴壁”三步法,持续用终浓度为10μmol/L和50μmol/L黄芩苷诱导mES细胞分化。每2天更换一次培养基,并保持分化过程中培养基始终维持相同的药物浓度。
3.形态学观察：
镜下观察mES细胞克隆的形态。诱导分化后,用倒置显微镜连续观测胚体(embryoid bodies, EBs)的直径和含跳动区域的EBs数占总EBs数的百分比。
4.流式细胞检测：
应用Phycoerythrin(PE)标记的抗体分别检测分化第10、16、20天的EBs的心肌细胞占总细胞数的百分率,即a-sarcomeric-actinin标记阳性细胞的百分率。
5.免疫荧光检测：
分化第16天,用免疫荧光染色法检测分化出的心肌样细胞的心肌特异性标志物a-肌小节辅肌动蛋白(α-sarcomeric-actinin)和心室肌特异性肌球蛋白轻链2 (Myosin light chain-2, ventricular, MLC-2v)的表达。
6.逆转录聚合酶链式反应(RT-PCR)和实时荧光定量聚合酶链式反应(real-timePCR)检测：
分别在分化后第2、4、6、8、10、12、14、16、20天时提取细胞总RNA,经逆转录反应(RT)得到cDNA,以3-磷酸甘油醛脱氢酶(GAPDH)作为内参基因,通过RT-PCR和real-time PCR检测三个胚层标志物如Soxl7、PEC AM和NCAM,心肌转录因子如Nkx2.5、MEF2c、TBX5、GATA4,心肌特异性基因如α-心肌肌球蛋白重链(Myosin heavy chain,α-MHC)、MLC-2v、心房钠尿肽(atrial natriuretic peptide, ANP)和HCN4 (hyperpolarization-activated cyclic nucleotide-gated channel 4),及心肌发育调控信号通路相关基因如TGF-β1、TGF-β2、TGF-β3、BMP2、BMP4、Wnt11、Wnt3a的mRNA表达水平。
7.免疫印迹(western blot)检测：
分别取分化第10、16、20天的细胞,提取细胞的总蛋白,经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印迹、ECL显色曝光等步骤,检测心肌特异性标志蛋白α-actinin、MLC-2v的表达。
8.膜片钳记录：
利用膜片钳技术检测干细胞来源的心肌细胞的电生理学特性和激素调节反应性。
结果：
不同浓度的黄芩苷对mES细胞的增殖均有抑制作用,此抑制作用呈浓度依赖性。在分化的过程中,黄芩苷处理组的EB直径显著小于对照组。但是50μmol/L黄芩苷明显地提高了mES细胞向心肌细胞的分化比例,表现在：(1)黄芩苷提高了中晚期跳动胚体的比例。(2)黄芩苷提高了α-actinin阳性细胞的比例。(3)分化中后期,心肌特异性标志物α-MHC、MLC-2v及ANP的mRNA水平在黄芩苷组的表达明显高于对照组。免疫荧光结果显示在对照组和黄芩苷处理组得到的mmES细胞来源的心肌细胞中都有心肌特异性的蛋白α-actinin和MLC-2v的表达。膜片钳记录结果显示分化出来的心肌细胞中存在着起搏样细胞、心房样细胞、心室样细胞。黄芩苷处理组中的心房及心室样心肌细胞的比例高于空白对照组,起搏样细胞的比例低于对照组。除了起搏样细胞和心室样细胞的跳动频率在黄芩苷组高于对照组外,其他的动作电位参数在两组间没有明显的差异。黄芩苷诱导出的心肌细胞具有了完整的激素调节功能。检测的心肌转录因子中,仅有Nkx2.5在分化晚期被黄芩苷上调,其他转录因子的表达未明显受到黄芩苷处理的影响。黄芩苷在分化中期上调Wnt3a的表达,然而在分化晚期,黄芩苷则明显地抑制了Wnt3a的表达,黄芩苷对检测的其他调控心肌发育的基因mRNA水平没有产生明显的影响。
结论：
1.黄芩苷抑制mES细胞的增殖,此抑制作用具有浓度依赖性。
2.黄芩苷(50μmol/L)可以促进mES细胞分化为心肌细胞,此促进作用主要表现在分化晚期。
3.黄芩苷促进mES细胞向心房肌和心室肌细胞分化,黄芩苷诱导得到的心肌细胞具有完整的激素调节功能。
4.黄芩苷促进mES细胞向心肌细胞分化可能是通过调控经典的Wnt/β-catenin信号通路实现的。
Background:
Embryonic stem cells (ES cells) are capable of self-replication and renewal. Under the certain condition, they will differentiate into multiple lineages cells, including osteoblast, chondrocyte, adipocyte, neurocyte, muscle cells and so on. A big number of evidence have indicated that ES cells transplanted into the ischemic heart will regenerate the necrotic cardiomyocytes and vascular endothelial cells, can also secrete various growth factors, promote the establishment of a micro-circulation, increase perfusion and improve heart function after infarction. The clinical therapy based on the utilizing of pluripotential ES cells has become the new hope for numerous chronic heart failure patients after myocardial infarction.
ES cells can be induced to differentiate into cardiomyocyte-like cells in vitro, these cardiomyocytes express part of the phenotype of myocardial cells, cardiac-specific markers, or even show spontaneous beating. Cardiomyocytes derived from the stem cells are promising candidate cell sources used for regenerative medicine in the treatment of heart failure disease. But the in vitro spontaneously cardiomyocytes differentiation from ES cells is low efficiency. Researchers have studied many inducible factors to induce cardiac differentiation from ES cells in vitro. Although the effects of these chemicals on cardiac differentiation are impressive, it still does not meet the request of quantity and purity of cells used in clinical transplantation. How to further enhance the efficiency of cardiac differentiation of ES cells is the key problem to be solved. Looking for a new and more effective, safer, more economical inducer is still a hot research topic. Baicalin is one of the active ingredients of traditional Chinese medicine, it possesses anti-oxidant and anti-inflammatory activities, and thus can protect cardiomyocytes exposed to ischemia/reperfusion. Baicalin has been widely used in clinical treatment of various cardiovascular diseases. The purpose of this project is to study the effect of baicalin on cardiac differentiation of mouse embryonic stem cells (mES cell) and its underlying mechanism.
The differentiation of ES cells draws assistance from the regulation of stimulus signal coming from outside accompany with intercellular specific transcription course. It has been approved that ES cells differentiation into cardiomyocytes-like cells is partly similar to embryonic development. Some regulation factors can be reactivated during the cell differentiation. However, the expression pattern has not yet been elucidated. Based on the existing status of traditional Chinese medicine-induced cardiac differentiation of ES cells, we try to use baicalin as an inducer to study the cardiac differentiation of ES cells to make discoveries in the following areas:(1) Whether baicalin can induce cardiac differentiation from mouse embryonic stem cells? (2) Whether the expression of myocardial cell marker after induction is different at every differentiation time points? (3) Which underlying signaling pathways is affected by baicalin during cardiac differentiation from ES cells? Clarifying the above issues can provide a theoretical basis to promote the efficiency of myocardial cell differentiation and speed up the process of clinical use of ES cells derived cardiomyocytes.
Aims:
(1) To observe the effect of baicalin on the proliferation of mouse embryonic stem cells;
(2) To observe the size of embryoid body (EB) after induction by baicalin at different development time points;
(3) To detect the expression of cardiac-specific markers at the mRNA and protein levels;
(4) To study the expression of genes related to cardiac differentiation during different differentiation stages after baicalin treatment, revealing the possible mechanism of baicalin affecting myocardial differentiation.
Methods:
1.MTT (3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide the tetramethyl azo oxacillin salt) assay was used to detect the effect of different concentrations of baicalin on the proliferation capacity of mES cells, screening the best concentration used to induce cardiac differentiation of embryonic stem cells.
2. Differentiation of mouse embryonic stem cells:
The classic "hanging drop-suspension culture-adherent culture" three-steps method was used to initiate cardiac differentiation. A final concentration of 10μmol/L or 50μmol/L baicalin was maintained in the differentiation medium during all the stages of differentiation. The medium was changed every 2 days.
3. Morphological observation:
Observe the morphology of embryonic stem cells before differentiation. After differentiation, We use an inverted microscope to observe the size of the embryo (embryoid body, EB) and the percentage of beating EBs (EBs with beating area).
4. Flow cytometry analysis:
We evaluated the percentage of cardiomyocytes at 10,16,20 days of EB by PE labeledα- sarcomeric actinin positive cells.
5. Immunofluorescence analysis:
At day 16 of differentiation, the expression of cardiac-specific markerα-sarcomeric actinin and ventricular muscle-specific myosin light chain 2 (myosin light chain-2, ventricular, MLC-2v) was detected using immunofluorescence staining.
6. Reverse transcriptase polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative polymerase chain reaction (real-time PCR):
The total cellular RNA was extracted from the EBs of days 2,4,6,8,10,12,14,16,20, cDNA was obtained by reverse transcriptase reaction (RT), housekeeping gene 3-phosphate dehydrogenase (GAPDH) was used as internal control, the mRNA levels of three germ layer marker and cardiac-specific genesα-cardiac myosin heavy chain (α-MHC), the MLC-2v, atrial natriuretic peptide(ANP), hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and myocardial developmentally regulated signaling pathway genes such as TGF-β1, TGF-β2, TGF-β3, BMP2, BMP4, Wnt11, Wnt3a and cardiac transcription factor such as Nkx2.5, MEF2c, TBX5, of GATA4 were detected by RT-PCR and real-time PCR.
7. Western blot:
The total protein was extracted from EBs at day10,16,20. After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to a PVDF membrane. After western blotting, ECL color development, exposure, the a-actinin and MLC-2v was detected.
8. Patch clamp analysis:
Electrophysiological characteristics and hormonal regulation of the cardiomyocytes derived from ES cells were detected using patch clamp technique.
Results:Baicalin inhibited the proliferation of mES cells in a concentration dependent manner. During all the differentiation stages, the size of EBs was significantly decreased in baicalin treatment groups than controls.50μmol/L baicalin successfully promoted the late stage cardiac differentiation in vitro, as demonstrated by increases in the percentage of beating EBs,α-actinin+ cells, and expression of cardiac specific markers. Typical pacemaker-like, atrial-like and ventricular-like action potentials (APs) were recorded by patch-clamp. Baicalin enhanced the percentage of working cardiomyocytes while decreased the percentage of pacemaker-like cells.β-adrenergic and muscarinic signaling cascades are present and functional in baicalin-induced ES-CMs. Further analysis revealed that baicalin strikingly up-regulated Wnt3a which is an activator of Wnt/β-catenin signaling pathway at the intermediate stage while dramatically down-regulated it at the late stage, but the expressions of TGF-β1/β2/β3, BMP2/4 and Wnt11 were not affected by baicalin. Downstream cardiac transcription factor Nkx2.5 was elevated by baicalin at the late stage, while Mef2c, Tbx5 and Gata4 were not significantly affected by baicalin.
Conclusion:
1. Baicalin inhibited the proliferation of ES cells in a concentration dependent manner.
2.50μmol/L of baicalin successfully promoted cardiac differentiation of mES cells in vitro at the late stage.
3. Baicalin enhanced the percentage of working ceardiomyocytes. The cardiomyocytes derived from ES cells had intact hormonal regulation.
4. Canonical Wnt/β-catenin signaling pathway might play an important role in this effect of baicalin.
引文
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