5-氮胞苷体外诱导骨髓间质干细胞分化为心肌细胞的实验研究

目的观察5-氮胞苷对骨髓间质干细胞(BMMSC)体外向心肌细胞诱导分化的影响.方法分离大鼠BMMSC体外培养,使用终浓度为10 μmol/L的5-氮胞苷(5-aza)对其定向诱导,观察细胞形态学的变化,荧光免疫组织化学方法进行鉴定,电镜观察细胞的超微结构.结果BMMSC体外经5-aza诱导4周,肌钙蛋白及Connexin43表达阳性,电镜下在部分细胞内肌节样结构及细胞间形成缝隙连接.结论BMMSC可以在体外经5-氮胞苷诱导分化为心肌细胞.     

Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation?

OBJECTIVE: The potential use of bone marrow stromal cells (MSCs) as a cellular therapy for chronic cardiac diseases relies on the ability of the cell to replicate extensively in vitro and to give rise to myogenic cells that can replace the damaged cardiomyocytes. For this reason the present study investigated the replication lifespan and chemical-induced cardiomyogenic differentiation of rat MSCs in vitro. METHODS: The primary and the successively passaged Wistar rat MSCs were exposed to different concentrations (3, 5 and 10 microM) of 5-azacytidine using different methods (single- or repeat-treatment). The growth properties and the fate of the cells were compared to their untreated counterparts by cell counting, immunocytochemistry and Western analysis. RESULTS: When seeded at a density of 2845 cells/cm(2) and cultured under common conditions, rat MSCs could be expanded up to 21.94 cell doublings in 30 days of successive subcultures. This was accompanied by a gradual loss of their replication ability with passages. When treated with 5-azacytidine for 24 h at day 3 of primary culture and the first subculture, the growth properties of the MSCs were not obviously affected. Neither the spontaneously beating cells nor the formation of myotubes were found in the primary and first passaged MSCs after a single treatment with 5-azacytidine and in cultures which underwent repeated 5-azacytidine-treatments during continuous subculturing to passage 2. The expressions of cardiac troponin I, cardiac myosin heavy chain and connexin 43 by the 5-azacytidine-treated MSCs were also undetectable at both immunocytochemistry and Western blot levels. The specificity and reliability of the detection methods were technically confirmed with cultured rat cardiomyocytes. CONCLUSIONS: Rat MSCs cannot be extensively expanded in vitro or be induced to differentiate in an expected cardiomyogenic way by 5-azacytidine-treatment, if the cells are not immortalized.     

骨髓间质干细胞心肌分化过程中细胞内钙离子浓度的变化

目的研究骨髓间质干细胞心肌分化前、后钙离子浓度变化。方法用5-杂氮胞苷(5-azacytidine)体外诱导猪骨髓间质干细胞使之向心肌分化；ELASA法测定分化前、后细胞内心肌肌钙蛋白Ⅰ(cTnⅠ)变化；应用离子图像分析系统测定分化前、后细胞内钙离子浓度变化。结果(1)经5-杂氮胞苷诱导后细胞形态发生变化；(2)诱导后第3周起细胞内cTnⅠ明显增高；(3)诱导组细胞内钙离子浓度较对照组高，且诱导前后细胞内钙释放机制不同。结论骨髓间质干细胞体外经5-杂氮胞苷诱导后可具有心肌细胞的某些特性，这一过程与钙信号有关。     

人脐血间充质干细胞诱导分化成心肌细胞的研究

目的探讨体外脐血间充质干细胞诱导分化成心肌细胞的可行性和最佳方法。方法收集获知情同意的健康产妇脐血细胞,分离单个核细胞,从中进一步分离间充质干细胞,传代培养至第3代,应用免疫荧光流式细胞仪标记间充质干细胞特异性抗原CD34、CD44和CD90。5-氮胞苷诱导分化4周后,免疫组织化学染色和RT-PCR法分别检测心肌细胞标记物肌钙蛋白Ⅰ、转录因子GATA 4和β-肌球蛋白重链。结果在第3代细胞中,可检测到CD44、CD90的表达,未检测到CD34的表达。脐血间充质干细胞经5-氮胞苷诱导分化后,呈现成纤维细胞样形态和克隆增殖特点。免疫组织化学染色和RT-PCR可检测到肌钙蛋白Ⅰ,GATA 4和β-肌球蛋白重链的表达。结论脐血间充质干细胞能够被诱导分化成心肌样细胞,可成为干细胞移植的细胞来源。     

Cardiomyogenic differentiation potential of human adipose precursor cells

DNA demethylation agent 5-azacytidine has been widely described in literature as an effective chemical stimulus used to promote cardiomyogenic differentiation in various cell types, ranging from embryonic stem cells, P19 cells, bone marrow-derived mesenchymal stem cells, and recently to adipose-derived stem cells. The purpose of this study was to examine the effects of 5-azacytidine on human adipose precursor cell differentiation along the cardiomyogenic lineage.     

Use of adult marrow mesenchymal stem cells for regeneration of cardiomyocytes

Bone marrow cells have been suggested to have multilineage plasticity including formation of cardiac tissue. We have established a cardiomyogenic (CMG) cell line from mouse bone marrow stromal cells that can be induced to differentiate into cardiomyocytes in vitro by 5-azacytidine treatment. A number of lines of evidence confirm the cardiomyocyte characteristics of CMG cells.     

骨髓干细胞诱导转化成心肌细胞的实验研究

目的探讨骨髓基质干细胞(BMCs)在5-氮杂胞苷(5-aza)的作用下是否能向心肌细胞转化。方法用梯度离心法分离大鼠BMCs;用不同浓度的5-氮胞苷对BMCs进行体外诱导转化,并使用心肌特异性抗体(肌钙蛋白I和肌凝蛋白重链)对诱导后的BMCs进行免疫组织化学染色,光镜和电镜观察。结果分离培养后的BMCs生长密集,形态呈纺锤状,在5μmol/L和10μmol/L 5-aza的诱导下分化成类心肌细胞,前者诱导后生长更好。HE染色细胞质嗜酸性,免疫组织化学染色心肌特异性抗体阳性。电镜发现胞核居细胞中央,肌丝和幼稚肌结形成。结论在5-aza的诱导下,BMCs能转化成心肌细胞,5μmol/L 5-aza的诱导效果更好。     

骨髓间(充)质干细胞体外定向诱导心肌样细胞超微结构特征研究

目的：研究兔骨髓间（充）质干细胞（mesenchymal stem cell，MSC）经5～氮杂胞苷（5-azacytidine，5-aza）诱导在体外定向分化的心肌样细胞超微结构特征。方法：取兔髂骨骨髓，分离并培养骨髓MSC，用5～aza定向诱导向心肌样细胞分化。以相差显微镜、透射电镜观察心肌样钏胞形态学变化及超微结构特征。结果：5-aza诱导后，部分细胞体积增大，呈“捧状”或“珠状”结构，有肌管样结构形成，透射电镜下见有肌丝、心房颗粒及线粒体等心肌样细胞超微等结构。结论：经5-氮杂胞苷诱导分化的骨髓间（充）质干细胞具有心肌样细胞超微结构特征．     

5-氮胞苷体外诱导骨髓间充质干细胞向心肌样细胞的分化

目的: 研究5-氮胞苷（5-Aza）对培养人骨髓间充质干细胞（MSC）的作用,并对分化后的心肌样细胞进行鉴定。方法: 采用密度梯度离心法分离到骨髓单个核细胞（MB-MNC）,用含200 ml/L胎牛血清的低糖型DMEM培养液进行培养。采用差速贴壁法纯化MSC,用流式细胞仪检测细胞表面抗原。以5-Aza诱导第3代MSC 24 h后继续培养。培养4周,用免疫细胞化学染色法检测肌系标记抗原:α-肌动蛋白（α-actin）及心肌细胞特异性标记抗原:肌钙蛋白T（cTnT）;在透射电镜下观察细胞的超微结构。结果: MSC经5-Aza诱导分化后,可表达α-actin和cTnT,未经诱导的同培养天数的MSC中均未见表达。透射电镜可观察到肌丝等心肌细胞的特异性结构。结论: 5-Aza可诱导MSC分化为心肌样细胞。     

骨髓间充质干细胞向心肌分化的实验研究

目的:研究骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)分化为心肌样细胞的能力,用于心肌补片治疗心肌梗死的研究。方法:分离C57/BSL小鼠BMSCs,全培养差速贴壁法,经过贴壁培养至第3代,流式细胞仪鉴定细胞表面标志(CD34、CD45、CD73、CD90),经10μmol/L的5-氮杂胞苷诱导细胞,24 h后更换完全培养基培养,2 w后进行免疫荧光染色,荧光显微镜观察心肌钙蛋白T(cTnT)和连接素蛋白43(CX43)的表达。结果:流式鉴定结果显示CD34、CD45阴性,CD73强阳性,CD90弱阳性。免疫荧光染色显示,诱导后细胞高表达心肌细胞特异性蛋白cTnT,连接素蛋白CX43表达水平明显增加。结论:5-aza可以诱导BMSCs大量表达心肌特异性蛋白cTnT和细胞连接素蛋白(CX43),干细胞分化为心肌样细胞,为干细胞移植治疗小鼠心梗提供种子细胞。     

兔骨髓间充质干细胞移植后移植细胞凋亡的实验研究

目的利用TUNEL法检测5-氮胞苷体外诱导骨髓间充质干细胞（MSCs）移植后的凋亡情况。方法5-氮胞苷体外诱导骨髓间充质干细胞MSCs向肌源性心肌细胞分化,通过免疫组化,鉴定诱导后的MSCs是否向类心肌细胞转化。建立兔心肌梗死模型,将细胞移植于心梗区域。移值2周后,利用TUNEL法检测植入细胞的凋亡率。结果移植2周后,可见DAPI标记带蓝色荧光的供体细胞核,分布比较广泛,形态呈椭圆形类似心肌细胞核,并与心肌纤维排列方向一致,证明移植细胞已存活。移值细胞表达troponinT,证明移植的MSCs分化为类心肌细胞。移植细胞均出现不同程度细胞凋亡。结论移植的MSCs细胞可在缺血的心肌组织存活,并分化为类心肌细胞,但移植细胞均出现不同程度凋亡。     

间接接触共培养下骨髓间充质干细胞分化为心肌样细胞及相关调控基因的时序表达

目的:研究间接接触共培养条件下骨髓间充质干细胞(mesenchymal stem cells,MSCs)向心肌细胞(myocadium-lkce cells,CMs)的分化及相关调控基因的时序表达;筛选MSCs定向分化为CMs的重要调控基因。方法:将MSCs与CMs按1∶5的比例进行间接接触共培养,连续观察两周,在相差显微镜下观察MSCs的形态变化。采用免疫荧光染色法检测心肌特征性肌动蛋白α(α-actin)和心脏肌钙蛋白T(cTnT)的表达。应用半定量RT-PCR分析TGF-β、Nkx-2.5、GATA-4、MEF-2C及TEF-1等相关调控基因在分化过程中的时序表达。结果:共培养后,MSCs的体积增大,其梭形形态逐渐变短变粗近似棒状或椭圆形,细胞之间形成连接,排列方向趋于一致。共培养两周时,α-actin及cTnT阳性细胞的比例分别为29.63%和27.38%。TGF-β、Nkx-2.5、GATA-4和MEF-2C基因在共培养后1 d表达开始增强,诱导后7 d达高峰,以后虽有所下降但仍维持在较高水平;TEF-1基因在诱导过程中表达无明显变化。结论:间接接触共培养条件下,MSCs可分化为心肌细胞。在此过程中,TGF-β、Nkx-2.5、GATA-4和MEF-2C基因可能是调控MSCs定向分化为心肌样细胞的重要调控基因。     

5-氮胞苷对大鼠骨髓基质细胞心钠素及β-肌球蛋白重链表达的影响

目的 观察5-氮胞苷(5-AZ)诱导后体外培养骨髓基质细胞(BMSCs)心钠素(ANP)、β肌球蛋白重链(β-MHC)表达的变化.方法 分离培养SD大鼠BMSCs及新生乳鼠心室肌细胞.BMSCs的诱导分化采用第8代BMSCs,于传代后第3 d分为4组:正常对照组、上清液组、5-AZ组、5-AZ+上清液组.反转录聚合酶链反应法测定心肌特异性蛋白ANP、β-MHC基因表达水平的变化.结果 正常培养及心肌细胞上清培养液诱导的BMSCs不表达心肌特异性ANP、β-MHC;5-AZ诱导后的BMSCs表达ANP、β-MHC,分别31.5±5.6、32.1±8.3和33.7±5.6、46.6±8.3.心肌细胞上清培养液增加5-AZ诱导的BMSCs中β-MHC的表达水平,而对ANP表达无影响.结论 体外培养大鼠成体BMSCs在5-AZ诱导下可表达心肌特异性ANP、β-MHC.心肌细胞上清培养液可增加β-MHC表达水平.     

多种细胞移植治疗大鼠心肌梗死后功能性室壁瘤的疗效比较

目的:观察比较骨髓间充质干细胞(MSCs)、5-氮杂胞苷(5-Aza)诱导分化的心肌样细胞及2种细胞联合移植治疗大鼠心肌梗死后功能性室壁瘤的疗效.方法:体外培养大鼠的MSCs,及用5-Aza诱导成的心肌样细胞.结扎大鼠冠状动脉左前降支,形成心肌梗死,4周后用超声心动图检测并筛选形成功能性室壁瘤者,分4组:A组(n=10),在室壁瘤瘤部及周边部点状注射MSCs(106～107个);B组(n=10),注射心肌样细胞(106～107个);C组(n=10),联合移植MSCs和心肌样细胞(106～107个);D组(n=10)为对照组,注射0.9%氯化钠.术后4周用超声心动图和血流动力学方法测定大鼠的心功能.另外,用组织学方法评价细胞移植后毛细血管密度,Masson氏三色染色法测量室壁瘤范围.结果:心脏彩超结果显示,与D组相比,A组、B组和C组的左室舒张内径、左室收缩内径均明显缩小(P<0.05),短轴缩短率及左室射血分数明显增大(P<0.05),且C组优于A组和B组(P<0.05),A组和B组比较,差异无统计学意义(P>0.05).血流动力学检测结果显示,与D组相比,A组、B组和C组的左室舒缩压差和左室正负最大变化速率均明显增高(P<0.05),其中C组增高最明显(P<0.05),A组和B组比较,差异无统计学意义(P>0.05).苏木精-伊红染色血管计数结果显示,A组(3.452±0.168/高倍视野)和C组(3.383±0.129/高倍视野)的毛细血管密度高于D组(1.827±0.052/高倍视野)(P<0.05),B组(1.917±0.038/高倍视野)与D组比较,差异无统计学意义(P>0.05).Masson氏三色染色法评价室壁瘤范围(%)结果显示,A组[(21.32±0.90)%]、B组[(22.14±0.74) %]和C组[(21.98±0.51) %]的室壁瘤范围小于D组[(25.70±1.71) %],差异有统计学意义(P<0.05).结论:MSCs移植,心肌样细胞移植,及二者联合移植对大鼠室壁瘤均有修复作用,联合MSCs和心肌样细胞移植改善心功能及心室重构方面效果优于单项细胞移植.     

Differentiation, engraftment and functional effects of pre-treated mesenchymal stem cells in a rat myocardial infarct model

BACKGROUND: Mesenchymal stem cells (MSCs) offer a novel therapeutic option in the treatment of acute myocardial infarction. MSCs are able to differentiate into myogenic cells after 5-azacytitdine treatment. However, 5-azacytidine might have genotoxic effects. Recently, it was reported that combined treatment with bone morphogenetic protein-2(BMP-2) and fibroblast growth factor-4(FGF-4) caused cardiac differentiation in non-precardiac mesoderm explants. Therefore, we investigated whether MSCs treated with combined BMP-2 and FGF-4 showed evidence of myogenic differentiation in vitro, and whether these cells resulted in sustained engraftment, myogenic differentiation, and improved cardiac function after implantation in infarcted myocardium. METHODS AND RESULTS: In vitro study: MSCs were treated with BMP-2 + FGF-4 (GF-MSCs) and myogenic phenotype was evaluated immunohistochemically. Cell growth curve was used to compare MSC proliferative capacity between the growth factors and 5-azacytidine treatments. In vivo study: two weeks after coronary artery occlusion, GF-MSCs (n=15), MSCs (n=5) labelled with PKH26 were injected into infarcted myocardium. Control animals (n=5) received a culture medium into the infarcted myocardium.Two weeks after implantation, some engrafted GF-MSCs or MSCs expressed sarcomeric-alpha-actinin and cardiac myosin heavy chain, as was observed in culture. Echocardiography showed that the GF-MSC group had a better (p < 0.05) left ventricular performance than the other groups. CONCLUSION: GF-MSCs induced myogenic differentiation in vitro. Moreover, GF-MSCs engrafted into the infarcted myocardium increased myogenic differentiation, prevented dilation of the infarcted region, and eventually improved heart function.     

Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP-cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression. CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.     

心肌细胞裂解液对骨髓间充质干细胞向心肌细胞分化诱导作用的研究

目的通过向骨髓间充质干细胞(MSCs)培养体系中添加心肌细胞裂解液的方法,体外模拟心肌微环境,观察MSCs向心肌细胞分化的诱导作用,并与诱导分化剂5-氮杂胞苷(5-aza)比较。方法分离新生乳鼠的心肌细胞并制成心肌细胞裂解液,自成年大鼠骨髓中分离MSCs,用含有心肌细胞裂解液的培养基(A组)、含有5-aza的培养基(B组)、含有5-aza和心肌细胞裂解液的培养基(c组)以及普通培养基(对照组)培养。观察细胞形态的改变,并通过免疫组化分析分化后细胞表达α-肌动蛋白、心脏特异性肌钙蛋白T(cTnT)、连接蛋白43及CD31的情况。结果A、B组的MSCs在培养1周后均形成肌细胞形态,并且均表达α-肌动蛋白和cTnT;A组MSCs分化的肌样细胞所含的肌纤维较B组更丰实,细胞生长趋势也优于B组,并且可以表达CD31;B组MSCs分化的肌样细胞不表达CD31;对照组细胞仅表达α-肌动蛋白。结论心肌细胞裂解液是体外诱导MSCs分化为心肌样细胞的理想条件,优于传统的5-aza,在心肌细胞移植技术中可以用于体外模拟心肌细胞微环境。     

Bone marrow Oct3/4+ cells differentiate into cardiac myocytes via age-dependent paracrine mechanisms

The mechanisms that govern the capacity of the bone marrow stem cells to generate cardiac myocytes are still unknown. Herein we demonstrate that the cardiomyogenic potential of bone marrow-derived Oct3/4(+)/cKit(+/-)/CXCR4(+/-)/CD34(-)/Sca1(-) cells is governed by age-dependent paracrine/juxtacrine platelet-derived growth factor (PDGF) pathways. Specifically, bone marrow cell cultures from both 3- and 18-month-old mice formed aggregates of Oct3/4(+) cells circumscribed by PDGFRalpha(+)/Oct3/4(-)/Sca1(+) cells. In young (3-month) bone marrow cell cultures, induction of PDGF-AB preceded the induction of cardiac genes and was required for the generation of cardiomyogenesis. Indeed, in old (18-month) cultures, diminished PDGF-B induction was associated with impaired cardiomyogenic potential, despite having Oct3/4 levels similar to those in the young cells. Importantly, supplementation with PDGF-AB specifically restored the cardiac differentiation capacity of the old bone marrow cells. Together these results demonstrate that, regardless of age, the bone marrow niche contains Oct3/4 stem cells that are capable of differentiating into cardiac myocytes. Moreover, this differentiation is governed by age-dependent PDGF-AB-mediated paracrine/juxtacrine pathways that may be essential in the translation of bone marrow cell-mediated cardiomyogenesis.     

大鼠骨髓间充质干细胞体外定向诱导分化为心肌样细胞的实验研究

目的研究大鼠骨髓间充质干细胞的体外培养及向心肌样细胞转化的条件。方法获取成年大鼠胫骨干骨髓,采用贴壁法进行间充质干细胞的培养、传代,观察经5-氮胞苷诱导后骨髓间充质干细胞的生长和分化。结果大鼠骨髓基质细胞贴壁呈集落生长,5-氮胞苷诱导骨髓基质细胞转化为心肌样细胞。结论骨髓基质细胞能够在体外被诱导分化为心肌样细胞,为自体心肌细胞移植提供了一种良好的来源。