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胚胎干细胞向心肌细胞诱导分化的研究进展 总被引:1,自引:0,他引:1
胚胎干细胞具有多向分化潜能,在器官移植及组织修复治疗方面具有广阔的应用前景。近来研究表明,胚胎干细胞在相关基因、转录因子调控以及体内外细胞因子、激素、微环境等作用下能够诱导分化为心肌细胞。但临床应用胚胎干细胞进行心脏修复治疗还有许多问题需要研究和解决。 相似文献
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胚胎干细胞具有多向分化潜能,在器官移植及组织修复治疗方面具有广阔的应用前景.近来研究表明,胚胎干细胞在相关基因、转录因子调控以及体内外细胞因子、激素、微环境等作用下能够诱导分化为心肌细胞.但临床应用胚胎干细胞进行心脏修复治疗还有许多问题需要研究和解决. 相似文献
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目的 :比较小鼠胚胎干细胞 (ESC)在不同诱导条件下分化为心肌细胞的分化比率。方法 :用鼠胚成纤维细胞(MEF)作为饲养层促进 ESC增殖并抑制其分化 ,用维甲酸 (RA )、二甲基亚砜 (DMSO)、转化生长因子β1 (TGF-β1 )、激活素 - A(activin- A)为分化诱导剂 ,采用三步法诱导 ESC分化为心肌样细胞 ,并比较各组分化比率。结果 :各组实验用的各种诱导剂均能诱导 ESC分化为心肌样细胞 ,尤以 TGF- β1 (2 ng/m l)、activin- A(2 0 ng/ml)及 2 0 %胎牛血清 (FCS)组成的分化培养基可以使其分化比率高达 88% ,显著高于其它各组 (P<0 .0 1)。结论 :以 MEF饲养层培养 ,TGF- β1 、activin- A作分化诱导剂可作为一种比较简便、稳定、高效的 ESC分化条件。 相似文献
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目的:利用不加任何诱导剂的悬浮培养法,体外诱导人胚胎干细胞分化为心肌细胞并检测其分化效率。方法:人胚胎干细胞克隆用200U/mL胶原酶Ⅳ,370C处理10min后,挑起克隆碎片转移到低黏附性培养皿中悬浮培养以形成EB(拟胚体),4d后将EB转移到基质胶处理过的6孔板中贴壁培养(1-3EBs/cm2),显微镜下观察记录出现跳动心肌细胞的时间和跳动频率,并计算跳动克隆百分比,24孔板一组,共记录4组96孔;免疫荧光染色检测心肌细胞特异标志物cTnT;膜片钳实验检测心肌细胞自发性动作电位;跳动心肌细胞经过24h缺氧刺激后,用凋亡试剂盒检测心肌细胞凋亡比例。结果:在悬浮培养14d左右开始出现大量的自发跳动心肌细胞,分化出现自发跳动心肌细胞的平均时间(13.9±0.9)d,百分比为20.8%,平均跳动频率为(63.8±5.6)次/min;跳动心肌细胞cTnT染色阳性;跳动心肌细胞检测到自发性动作电位;跳动心肌细胞缺氧24h的凋亡比例为(8.1±0.4)%。结论:不加诱导剂的悬浮培养可以诱导人胚胎干细胞分化为心肌细胞,分化效率达到20.8%,分化时间14d左右。为进一步的干细胞移植治疗动物心肌梗死模型提供种子细胞。 相似文献
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目的探讨内脏内胚层样END-2细胞体外诱导胚胎干细胞(embryon ic stem cells,ESCs)分化为心肌细胞的特征。方法用鼠胚胎成纤维细胞(mouse embryon ic fibrob lasts,MEF)作为饲养层促进ESCs增殖并抑制其分化,先将ESCs悬浮培养形成23 d的拟胚体(embryoid bod ies,EBs),再和END-2细胞共培养诱导向心肌细胞分化。实验分4组。第1,2组EBs分别和END-2细胞或END-2细胞条件培养液共培养;第3组EBs和表面铺有一层琼脂糖的END-2细胞共培养;第4组自然分化组为对照组。相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T(TnT)的表达;透射电镜观察分化心肌细胞的超微结构。结果各实验组均可见自发节律性收缩的拟胚体。随着培养的延长,自发节律性收缩的拟胚体数目也增加,均表达心肌细胞特异性蛋白TnT,以及观察到心肌样超微结构。在和END-2细胞直接接触的诱导条件下,分化的细胞形态较单一。结论END-2细胞通过分泌可溶性细胞因子可诱导ESCs向心肌细胞分化,直接接触在END-2细胞诱导作用中并不是必要的,但可诱导出较单一的细胞。 相似文献
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胚胎干细胞是具有分化为三个胚层来源的各种类型组织细胞潜能的多能干细胞,可在体外无限增殖。信号分子、化学诱导剂、激素和细胞内转录因子可诱导和调控胚胎干细胞进行心肌细胞分化,为干细胞移植用于心肌再生和改善心功能提供了潜在的细胞来源。 相似文献
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目的 探讨小鼠胚胎干细胞早期分化来源的心肌细胞进行同种异体移植后的电生理特性.方法 首先通过电转染构建a-MHC-EGFP-ESc系[将心肌细胞特异的OL-MHC启动子与表达基因--绿色荧光蛋白(EGFP)基因融合,构建成真核表达载体],悬滴法诱导胚胎干细胞分化,在分化早期(7+4)d利用流式细胞仪筛选带绿色荧光的心肌细胞,将纯化的心肌细胞(5×106个/ml)移植到小鼠心室壁,对照组注入等体积培养基,移植前3 d开始应用环孢素A(静脉注射,5 mg·kg-1·d-1)和泼尼松龙(静脉注射,2.5 mg·kg-1·d-1)抑制免疫排斥反应.移植后2周分离两侧颈迷走神经行电刺激抑制窦房结与房室结,记录刺激前后的体表心电图,然后分别行免疫荧光显像和膜片钳研究.结果 刺激迷走神经前,移植组和对照组均呈正常的窦性心律,两组无室性心律失常的发生;刺激迷走神经后,两组动物均出现异位的心室起搏心律,细胞移植组与对照组心室频率无差异.移植区冰冻切片免疫荧光分析可见EGFP标记的移植细胞具有肌钙蛋白I(cTnI)表达,说明分化细胞移植后仍具有心肌特性,且移植细胞与宿主心肌细胞间有连接蛋白43的表达,表明移植细胞与宿主心肌细胞间形成电偶联通道.膜片钳分析,绿色荧光细胞移植前后其具有起搏细胞动作电位的比例分别为(85.1%vs 1 1.4%,P<0.05),该类细胞在移植前后的起搏电流强度有所增强((11.2±2.4)pA/pF vs(15.5±1.9)pA/pF,P<0.05].移植区分离的绿色荧光细胞中48%具有心室肌动作电位.结论 胚胎干细胞早期分化来源的心肌细胞在体移植后能进一步分化为成熟的心室肌细胞及起搏细胞. 相似文献
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目的:检验催产素能否将去分化脂肪(DFAT)细胞诱导分化成为心肌样细胞。方法: 从脂肪中分离成熟的脂肪细胞,通过天花板培养使成熟脂肪细胞去分化为DFAT细胞。按照不同诱导的时间(1周、2周、3周)及低中高3个不同浓度的(1×10-8 mol/L、1×10-7 mol/L、1×10-6 mol/L)分组对DFAT进行诱导培养。通过免疫荧光检测法、RT-PCR及Western blot分别在基因水平和蛋白水平上检测心肌特异性标记的表达,以人类心肌组织作为阳性对照,未加诱导的DFAT细胞作为阴性对照。结果: 高浓度的催产素(1×10-6 mol/L)造成实验细胞大量死亡,无法进行后续试验。以1×10-8 mol/L及1×10-7 mol/L浓度的催产素诱导3周后,DFAT细胞在基因及蛋白水平上检测到心脏特异性标记GATA4、Nkx2.5及cTnT的表达,但未发现自主搏动现象。结论: 生理浓度和中等浓度的催产素,可以将 DFAT细胞诱导成为心肌样细胞。 相似文献
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Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes 总被引:21,自引:0,他引:21
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Paquin J Danalache BA Jankowski M McCann SM Gutkowska J 《Proceedings of the National Academy of Sciences of the United States of America》2002,99(14):9550-9555
We recently discovered the existence of the oxytocin/oxytocin receptor (OT/OTR) system in the heart. Activation of cardiac OTR stimulates the release of atrial natriuretic peptide (ANP), which is involved in regulation of blood pressure and cell growth. Having observed elevated OT levels in the fetal and newborn heart at a stage of intense cardiomyocyte hyperplasia, we hypothesized a role for OT in cardiomyocyte differentiation. We used mouse P19 embryonic stem cells to substantiate this potential role. P19 cells give rise to the formation of cell derivatives of all germ layers. Treatment of P19 cell aggregates with dimethyl sulfoxide (DMSO) induces differentiation to cardiomyocytes. In this work, P19 cells were allowed to aggregate from day 0 to day 4 in the presence of 0.5% DMSO, 10(-7) M OT and/or 10(-7) M OT antagonist (OTA), and then cultured in the absence of these factors until day 14. OT alone stimulated the production of beating cell colonies in all 24 independently growing cultures by day 8 of the differentiation protocol, whereas the same result was obtained in cells induced by DMSO only after 12 days. Cells induced with OT exhibited increased ANP mRNA, had abundant mitochondria (i.e., they strongly absorbed rhodamine 123), and expressed sarcomeric myosin heavy chain and dihydropyridine receptor-alpha 1, confirming a cardiomyocyte phenotype. In addition, OT as well as DMSO increased OTR protein and OTR mRNA, and OTA completely inhibited the formation of cardiomyocytes in OT- and DMSO-supplemented cultures. These results suggest that the OT/OTR system plays an important role in cardiogenesis by promoting cardiomyocyte differentiation. 相似文献
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小鼠胚胎干细胞体外定向心肌细胞分化的研究 总被引:9,自引:1,他引:9
目的:小鼠胚胎干细胞(embryonic stem cell,ES细胞)体外定向心肌细胞分化模型的建立。方法:通过悬滴培养技术定向诱导ES细胞分化为拟胚体,进而分化为心肌细胞,并用RT-PCR对四种肌肉肌动蛋白在EBs中的表达进行了鉴定。结果:在拟胚体和进一步分化的ES细胞中观察到自发节律性收缩的心肌细胞,α-平滑肌肌动蛋白(血管和胃肠道平滑肌)以及纹状肌肉肌动蛋白(心肌和骨骼肌)在这些心肌细胞样的细胞中均能表达。结论:小鼠胚胎干细胞体外定向心肌细胞分化模型的建立,可用于研究相关基因在心肌发育过程中的直接或间接作用,有助于胚胎干细胞衍生的心肌细胞用于临床治疗作用的研究。 相似文献
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YANG Huang-tian 《岭南心血管病杂志》2011,(Z1):18-19
Rats(Rattus norvegicus) have many advantages over mice in scientific studies,for example, they are more relevant to human in physiological and pharmacological responses.Therefore,rats are broadly used in experimental studies.The recent breakthrough in the generation of rat embryonic stem cells(rESCs) opens the door to application of gene targeting to create models for the study of human diseases.In addition,the in vitro differentiation of rESCs into derivatives of three germ lines will serve as a powerful tool and resource for the investigation of mammalian development,cell function, tissue repair,and drug discovery.However, the distinct culture condition and signal inhibitor-depended maintenance of rESCs stand as a considerable challenge for its in vitro differentiation.To address it,we investigated whether rESCs are capable of forming terminal differentiated cardiomyocytes. We found that the embryoid bodies(EBs)-based method used in mouse ESC(mESC) differentiation failed to work in the cultivation of rESCs.We then modified the differentiation protocol and successfully developed an in vitro differentiation system to differentiate rESCs into three embryonic germ layers.By using this method,the rESCs form spontaneous beating cardiomyocytes with the properties similar to those derived from fetal rat hearts and mESCs.This unique cellular system will provide a new approach to study the early development and cardiac function as well as to perform pharmacological test and cell therapy study(Grants:the State Major Research Program of China(2009ZX09503-024,2010CB945603) and CAS(XDA01030000). 相似文献
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Embryonic stem (ES) cells have been established as permanent lines of undifferentiated pluripotent cells from early mouse embryos. ES cells provide a unique system for the genetic manipulation and the creation of knockout strains of mice through gene targeting. By cultivation in vitro as 3D aggregates called embryoid bodies, ES cells can differentiate into derivatives of all 3 primary germ layers, including cardiomyocytes. Protocols for the in vitro differentiation of ES cells into cardiomyocytes representing all specialized cell types of the heart, such as atrial-like, ventricular-like, sinus nodal-like, and Purkinje-like cells, have been established. During differentiation, cardiac-specific genes as well as proteins, receptors, and ion channels are expressed in a developmental continuum, which closely recapitulates the developmental pattern of early cardiogenesis. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro "gain-of-function" or "loss-of-function" genetic studies. Recently, human ES cell lines have been established that can be used to investigate cardiac development and the function of human heart cells and to determine the basic strategies of regenerative cell therapy. This review summarizes the current state of ES cell-derived cardiogenesis and provides an overview of how genomic strategies coupled with this in vitro differentiation system can be applied to cardiac research. 相似文献
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目的:观察Wnt3a信号对小鼠胚胎干细胞(embryonicstemcell,ESC)心肌细胞分化的影响。方法:用悬滴培养法促进ESCs形成拟胚体(embryoidbodies,EBs)。用免疫荧光染色法检测心肌特异性蛋白cTnT的表达。在不同分化时间加入Wnt3a及Wnt信号通路抑制剂Dkk一1观察对搏动EBs百分率的影响。用RT—PCR检测Nk同源异型盒5(Nkx2.5)、锌指转录因子-4(GATA.4)、B.肌球蛋白重链([~-MHC)及心房钠尿肽(ANP)基因表达水平的变化。用Westernblot检测cTnT表达水平的变化。将不加入Wnt3a,分化第0—5天及5~lO天加入Wnt3a的组分别命名为对照组,D0-5组及D5.10组。加入Dkk.1的组命名为Dkk.1组。结果:通过悬滴培养法形成的EBs能够出现自发性搏动并且有TnT表达。EBs形成过程中即分化第0—5天加入Wnt3a与对照组相比具有更高的搏动EBs百分率,Nkx2.5、GATA4、B.MHC和ANP基因表达的水平,以及cTnT蛋白表达水平,而EBs形成后即分化第5—10天加入Wnt3a的结果相反。分化第5—10天加入Dkk.1与对照组相比具有更高的搏动EBs百分率及cTnT蛋白表达水平,并且在分化第0—5天分别加入Wnt3a及Dkk.1与单独加入Wnt3a及Dkk-1相比,搏动EBs百分率及cTnT蛋白表达水平更高。结论:Wnt3a对ESCs向心肌细胞分化的调控呈时间依耐性,EBs形成过程中激活Wnt3a信号及EBs形成后阻断Wnt3a信号能够获得更多的心肌细胞。 相似文献
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目的:观察Wnt3a信号对小鼠胚胎干细胞(embryonic stem cell,ESC)心肌细胞分化的影响。方法:用悬滴培养法促进ESCs形成拟胚体(embryoid bodies,EBs)。用免疫荧光染色法检测心肌特异性蛋白cTnT的表达。在不同分化时间加入Wnt3a及Wnt信号通路抑制剂Dkk-1观察对搏动EBs百分率的影响。用RT-PCR检测Nk同源异型盒5(Nkx2.5)、锌指转录因子-4(GATA-4)、β-肌球蛋白重链(β-MHC)及心房钠尿肽(ANP)基因表达水平的变化。用Western blot检测cTnT表达水平的变化。将不加入Wnt3a,分化第0~5天及5~10天加入Wnt3a的组分别命名为对照组,D0-5组及D5-10组。加入Dkk-1的组命名为Dkk-1组。结果:通过悬滴培养法形成的EBs能够出现自发性搏动并且有TnT表达。EBs形成过程中即分化第0~5天加入Wnt3a与对照组相比具有更高的搏动EBs百分率,Nkx2.5、GATA4、β-MHC和ANP基因表达的水平,以及cTnT蛋白表达水平,而EBs形成后即分化第5~10天加入Wnt3a的结果相反。分化第5~10天加入Dkk-1与对照组相比具有更高的搏动EBs百分率及cTnT蛋白表达水平,并且在分化第0~5天分别加入Wnt3a及Dkk-1与单独加入Wnt3a及Dkk-1相比,搏动EBs百分率及cTnT蛋白表达水平更高。结论:Wnt3a对ESCs向心肌细胞分化的调控呈时间依耐性,EBs形成过程中激活Wnt3a信号及EBs形成后阻断Wnt3a信号能够获得更多的心肌细胞。 相似文献
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Bcl2 is a potent antiapoptotic gene that can increase resistance of adult bone marrow hematopoietic progenitor cells to lethal irradiation, and thereby preserve their ability to differentiate. However, the effect of Bcl2 on murine embryonic stem (ES) cells induced to undergo hematopoietic differentiation in the absence of a toxic stress is not known. To test this, murine CCE-ES cells that can be induced to undergo hematopoietic differentiation in a two-step process that results in upregulation of Bcl2 were used. Upregulation of Bcl2 precedes formation of hematopoietic embryoid bodies (EB) and their further differentiation into hematopoietic colony-forming units, when plated as single cells in methylcellulose. ES cells stably expressing a Bcl2 siRNA plasmid to "knock-down" endogenous expression or cells expressing wild-type (WT) Bcl2 or phosphomimetic Bcl2 mutants were examined. ES cells expressing the Bcl2 siRNA or those expressing a dominant-negative, nonphosphorylatable Bcl2 display a strikingly reduced capacity to form hematopoietic EBs and colony-forming units compared to cells expressing WT or phosphomimetic Bcl2 that demonstrate an increased capacity. Bcl2's effect on induced-hematopoietic differentiation of ES cells does not result from either decreased apoptosis or a reduced number of cells. Rather, Bcl2-enhances hematopoietic differentiation of ES cells by upregulating p27, which results in retardation of the cell cycle at G1/G 0. Thus siRNA silencing of p27 reverts Bcl2's enhancement phenotype in a manner similar to that of Bcl2 "silencing" or expression of a nonphosphorylable Bcl2. In addition to Bcl2's well-described antiapoptotic and cell-cycle retardant effect on somatic cells, Bcl2 may also function to enhance induced hematopoietic cell differentiation of murine ES cells. These findings may have potential relevance for expanding hematopoietic stem/progenitor cell numbers from an ES cell source for stem cell transplantation applications. 相似文献