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。     

心肌细胞裂解成分诱导骨髓间充质干细胞分化的超微结构观察

目的:观察骨髓间充质干细胞(MSCs)在心肌细胞裂解成分作用下超微结构改变,了解心肌细胞裂解成分对MSCs分化的诱导作用。 方法:分离并裂解新生大鼠的心肌细胞;自成年大鼠骨髓中分离MSCs;将分离的MSCs分3组培养:仅用普通培养基培养(对照组);5-氮杂胞苷(5-aza)诱导后用普通培养基培养(5-aza诱导组);含有心肌细胞裂解成分的培养基培养(心肌细胞裂解成分培养组)。培养1周,观察细胞形态及超微结构的改变,对培养的细胞进行抗心脏特异性肌钙蛋白T(cTnT)及抗分化决定簇31(CD31)细胞免疫化学染色,并分析各组细胞的增殖情况。 结果:对照组MSCs无明显的肌样细胞或内皮样细胞形成,抗cTnT和抗CD31染色阴性。5-aza诱导组部分MSCs分化为肌样细胞,电镜下可见大量细胞器空化,抗cTnT染色阳性,但细胞增殖缓慢。心肌细胞裂解成分培养组的MSCs分化为肌样细胞,电镜下可见肌丝样结构,抗cTnT染色阳性,细胞增殖旺盛,另见部分MSCs分化为内皮样细胞,形成内皮细胞特有的胞质突起和质膜小泡等超微结构,且抗CD31染色阳性。 结论:含有心肌细胞裂解成分的培养基可以诱导MSCs向心肌样细胞和内皮样细胞方向分化。     

心肌组织裂解液诱导骨髓间充质干细胞向心肌样细胞分化的研究

目的探讨心肌组织裂解液定向诱导骨髓间充质干细胞(BMSCs)分化为心肌样细胞的可行性。方法体外分离、培养及鉴定大鼠BMSCs,实验分为3组:空白对照组、正常心肌组织裂解液诱导组和梗死心肌组织裂解液诱导组。分别对第3代BMSCs进行定向诱导,倒置相差显微镜观察诱导后细胞形态变化。诱导4周后,Western-blot检测心肌肌钙蛋白T(cTnT)、心肌连接蛋白43(Cx43)的表达。结果 P3代BMSCs CD34染色呈阴性,而CD105、CD106染色呈阳性。两个诱导组cTnT、Cx43蛋白表达量均高于空白对照组,且梗死心肌裂解液诱导组的蛋白表达量高于正常心肌组织裂解液诱导组,差异有统计学意义(P0.05)。结论正常心肌组织裂解液及梗死心肌组织裂解液均可诱导BMSCs分化为心肌样细胞,且梗死心肌组织裂解液组诱导分化效率更高。     

不同浓度的窦房结细胞裂解液对兔骨髓间充质干细胞的诱导分化作用

目的 探索不同浓度的窦房结细胞裂解液对兔骨髓间充质干细胞的诱导分化作用。方法 体外分离兔窦房结细胞并制备窦房结细胞裂解液。全骨髓贴壁法分离纯化兔骨髓间充质干细胞,分别加入含1、5、10倍浓度的窦房结细胞裂解液作为诱导培养基,普通培养基培养作对照,观察细胞形态变化,通过免疫荧光染色法检测超极化激活的环核苷酸门控的离子通道蛋白HCN4的表达,免疫细胞化学染色检测心肌特异性肌钙蛋白T(cTnT)的表达。结果 经1、5、10倍浓度窦房结细胞裂解液诱导后的细胞均表达cTnT和HCN4;4组间HCN4阳性细胞率比较,差异有统计学意义(P<0.05)。随着细胞裂解液浓度的提高,所诱导成的阳性细胞数再逐步增加,但增加至5倍浓度以上,效果并不明显。结论 在一定范围内,不同浓度的窦房结细胞裂解液能够体外诱导骨髓间充质干细胞分化为起搏细胞,但并不遵从剂量效应关系。     

同种异体骨髓间充质干细胞在大鼠心脏的迁移及分化特点

目的探讨同种异体骨髓间充质干细胞(MSCs)在大鼠心脏定居存活情况、同种异体移植是否可行?正常心脏微环境与梗死心脏微环境对骨髓间充质干细胞(MSCs)迁移、分化的影响.方法将体重200～250g雌性大鼠35只随机分为4组①正常心脏+rMSCs移植组(Control+rMSCs,n=10)正常大鼠接受同种异体rMSCs移植治疗(5×106MSCs/100 ul,分4点注入);②急性心肌梗死对照组(AMI,n=10)大鼠心肌梗死后注射等量培养基;③心肌梗死+rMSCs移植组(AMI+rMSCs,n=10)大鼠心肌梗死后1～3h接受同种异体rMSCs移植治疗(5×106MSCs/100ul,分4点注入);④单个核细胞治疗组(n=5)大鼠心肌梗死后1-3h接受同种异体单个核细胞移植治疗(5×106MSCs/100μl,分4点注入).结扎左冠状动脉前降支造成心肌梗死模型.用比重1.073的Percoll分离液分离来自雄性Wistar大鼠骨髓MSCs,经贴壁、及时传代纯化MSCs;用FITC或PE标记的表面分子CD34、CDlla、CD71、CD29抗体对二代MSC进行流式细胞术鉴定;采用01ivette方法结扎冠状动脉建立急性心肌梗死模型,经DAPI标记移植细胞(MSCs、单个核细胞),分别置入到各组大鼠心脏组织,模拟同种异体细胞移置治疗.于细胞移植后10周取心脏,荧光显微镜下直接观察植入细胞迁移分布;免疫荧光检测心肌特异蛋白(肌钙蛋白、转录因子4和连接蛋白43)抗原表达.结果(1)采用密度为1.073的percoll分离液分离MSCs,形态呈较均一的长梭形,由原代的(5.0±0.21)×105个MSCs,经扩增16代后可获得(4.0±0.36)×1012个细胞,扩增约(6.8±0.45)×106倍,形态均一并保持原有特性.(2)流式细胞术鉴定,扩增二代的MSCs强表达CD90(动物干细胞表面标志),其阳性达97.27%,不表达CD45(白细胞分化抗原)和CD31(内皮细胞表面标志),CD44(黏附分子)仅有微弱表达(2,72%),这表明MSCs是骨髓中区别于造血干细胞的一群处于未分化状态的非定向干细胞.(3)经纯化的大鼠MSCs在同种异体大鼠心脏可定居、生存,而未经纯化的单个核细胞移植在同种异体大鼠心脏未见存活;(4)在正常心脏微环境中,带蓝色荧光的供体MSCs细胞核呈小岛屿状分布,与宿主心肌纤维排列无关;而在心肌梗死模型大鼠心脏微环境中,蓝色细胞核分布广泛,形态呈椭圆形类似心肌细胞核,并与宿主心肌纤维排列方向一致,免疫组化检测胞浆心肌特异蛋白染色阳性.结论及时传代可避免大鼠MSCs自身分化,也是纯化大鼠MSCs的重要因素之一;纯化的MSCs具有免疫耐受特性,无需加用免疫抑制剂,可进行同种异体移植;同种异体MSCs在正常心脏微环境中不发生迁移、分化;而在急性梗死大鼠心脏中具有向缺血梗死区迁移并分化为心肌样细胞的特点.针对慢性心肌梗死瘢痕的治疗,Bittira发现移植诱导后的MSCs较未诱导的MSCs更利于瘢痕局部心肌分化,今后需要阐明MSCs定向分化的分子机制,还需要在增加移植细胞存活率、心肌细胞分化率及功能化研究等方面取得突破,推动MSCs治疗心肌梗死、慢性心功能不全的研究工作,对未来MSCs移植应用于临床具有十分重要的意义.     

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。     

人骨髓间充质干细胞在心肌损伤裸鼠体内的归巢与分化

目的: 探讨在心肌细胞移植中机体内环境因素对人骨髓间充质干细胞（MSC）向心肌细胞（CM）定向分化的影响。方法: 取人骨髓血,用Percoll（1 073 g/L）进行密度梯度离心及贴壁筛选结合的方法,体外培养扩增人骨髓MSC,以流式细胞仪鉴定其纯度,并进行免疫细胞化学染色。随后将人骨髓MSC植入异丙肾上腺素所致急性心肌损伤的裸鼠体内。3周后,取心脏组织块进行免疫组织化学染色检测。 结果: 体外分离纯化培养扩增的人骨髓MSC,可高表达CD44,CD105和波形蛋白,不表达CD31,CD34,CD45,肌钙蛋白I和结蛋白。细胞移植3周后,人骨髓MSC在心肌损伤区域均能分化为表达肌钙蛋白I,结蛋白和波形蛋白的细胞。 结论: 体外分离培养扩增的人骨髓MSC可归巢于受损心脏,并参与体内心肌损伤的修复,具有向CM分化的潜能。     

三七皂甙诱导骨髓基质细胞分化为心肌样细胞的实验研究

目的探讨三七皂甙对体外定向诱导猪骨髓基质细胞(MSCs)分化为心肌样细胞的影响。方法用穿刺方法抽取猪骨髓,分离并培养骨髓基质细胞,中药三七皂甙定向诱导分化为心肌样细胞。倒置显微镜下观察细胞形态,免疫细胞化学法鉴定心肌细胞特异性抗原标志物肌钙蛋白T,用RT-PCR对心肌肌球蛋白重链(β-MHC)进行鉴定。取培养的猪心肌细胞作为阳性对照,未经三七皂甙诱导正常培养的骨髓基质细胞作为阴性对照。结果猪MSCs经三七皂甙诱导2h后,大部分MSCs可分化为心肌样细胞,免疫细胞化学检测细胞中的肌钙蛋白T呈阳性,RT-PCR显示能表达β-MHC。结论MSCs是骨髓来源的具有多向分化潜能的干细胞,在三七皂甙的定向诱导下,可以向心肌样细胞分化,有望成为心衰及心肌梗死等心肌损伤时干细胞移植治疗的理想细胞材料。     

二种不同的诱导方法诱导人骨髓间充质干细胞分化的心肌样细胞L型钙电流的变化

目的利用膜片钳技术检测体外两种不同诱导方法诱导人骨髓间充质干细胞(hMSCs)分化为心肌样细胞的L型钙电流(ICa-L)。方法体外培养扩增hMSCs,采用①5-氮胞苷(5-Aza,10μmol/L)化学诱导4周;②原代分离培养SD乳鼠搏动心肌细胞(CMs)制备的心肌微环境诱导10天直至CMs停止搏动。利用膜片钳技术检测两种不同方法诱导分化的心肌样细胞、hMSCs及原代CMs的ICa-L。结果 5-Aza与心肌微环境诱导分化的心肌样细胞较hMSCs的ICa-L增强(112.60±8.26,120.50±9.35pAvs96.67±13.50pA,P均<0.05);低于CMs组(263.86±39.01pA,P<0.01)。结论两种诱导方法诱导的心肌样细胞ICa-L均增强。     

复方丹参滴丸含药血清诱导大鼠骨髓间充质干细胞分化为心肌样细胞

目的观察复方丹参滴丸含药血清体外诱导大鼠骨髓间充质干细胞(MSCs)分化为心肌样细胞的能力。方法采用全骨髓直接贴壁的方法分离培养大鼠MSCs,流式细胞仪鉴定细胞表面标记;采用复方丹参滴丸的含药血清体外定向诱导第四代MSCs,应用免疫细胞化学法、原位杂交组织化学法、透射电子显微镜鉴定心肌样细胞。结果大鼠MSCs细胞表面抗原CD90、CD106阳性,CD34、CD45、CD31阴性。经复方丹参滴丸含药血清诱导向心肌样细胞分化后,免疫细胞化学法显示α辅肌动蛋白(α-Actinin)、结蛋白(desmin)强阳性,原位杂交组织化学法显示肌球蛋白重链(MHC)强阳性表达。结论复方丹参滴丸含药血清能促使MSCs分化为心肌样细胞,为中药干预MSCs治疗缺血性心脏病提供干细胞实验依据。     

微环境中人骨髓间充质干细胞向心肌细胞表型分化的实验研究

目的 探讨人骨髓间充质干细胞(hBMSC)在非接触共培养的微环境中向心肌细胞分化的能力.方法 密度梯度离心法分离培养hBMSC,用流式细胞仪鉴别其表面标记特征.采用transwell按1:10的比例共同培养hBMSC和SD乳鼠心室肌细胞.用聚合酶链反应检测心肌转录因子GATA4的基因表达水平.免疫细胞化学、免疫荧光检测横纹肌α-辅肌动蛋白、结蛋白、肌钙蛋白(cTn)T和cTnI等心肌细胞标志蛋白的表达.结果 hBMSC的标志物主要为CD29(98.64%±0.80%)和CD44(96.70%±1.50%),而极少表达CD105(2.21%±0.60%)、CD11b(0.80%±0.05%)、CD45(1.39%±0.13%)和CD34(1.73%±0.08%).与心肌细胞共培养后第7天可在hBMSC细胞中观察到少量搏动的细胞,随着诱导时间的增加,细胞的搏动更规则、有力.与心肌发育有关的转录因子GATA4基因在诱导后1、2、3周均有表达.共培养后2周,检测的心肌标志蛋白均有阳性表达,分别是结蛋白,α-辅肌动蛋白,cTnI和cTnT.荧光标记也检测到cTnT和cTnI的阳性表达.结论 hBMSC具有向心肌细胞分化的潜能,在共培养的微环境中,可以分化成心肌细胞表型,其机制是通过心肌细胞的旁分泌作用,而不需要hBMSC与心肌细胞的直接接触.     

Bone marrow mesenchymal stem cells differentiate into functional cardiac phenotypes by cardiac microenvironment

Heart attacks and congestive heart failure remain among the world's most prominent health challenges despite the many breakthroughs. Bone marrow mesenchymal stem cells (BMSCs) have the potential to transdifferentiate into myocytes if an appropriate cardiac environment is provided. This study is meant to investigate the ability of BMSCs to differentiate into cardiomyocytes in a conditioned medium. BMSCs were isolated from rat femurs and tibias using Percoll gradient centrifugation method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer. BMSCs were cocultured with neonatal rat ventricular myocytes in a rate of 1:10 separated by semipermeable membrane. BMSCs marker of CD29 were highly expressed (98.89+/-1.2%); however, CD34 could hardly be identified (5.61+/-0.1%). After coculturing with myocytes, some of BMSCs showed contraction which became more regular and more vigorous. As assessed by RT-PCR, SERCA2 and RyR(2) were expressed by newly formed cells from 1 to 3 weeks. Immunostaining of newly differentiated BMSCs revealed positivity for cTnT. Some of these cells were positive for sarcomeric alpha-actinin, desmin, cTnT, and cTnI. Western blotting showed that cTnI protein expression was upregulated in these cells from 1 to 3 weeks. Newly formed BMSCs exhibited ultrastructural features of sarcomere formation and inward rectifier potassium current (I(K1)). It is concluded that BMSCs possess the potential to differentiate into cardiomyocytes in the cardiac environment. BMSCs provide an excellent model for development of stem cell therapeutics, and their potential in the cardiac repair under various pathological conditions.     

Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells

OBJECTIVE: We have previously shown the simultaneous generation of CD73(+) mesenchymal stromal cells (MSCs) along with CD34(+) hematopoietic cells from human embryonic stem cells (ESCs) when they are cocultured with OP9 murine stromal cells. We investigated whether MSCs can be derived from human ESCs without coculturing with OP9 cells, and if such cells exhibit immunological properties similar to MSCs derived from adult human bone marrow (BM). MATERIALS AND METHODS: Our starting populations were undifferentiated human ESCs cultured on Matrigel-coated plates without feeder cells. The differentiated fibroblast-looking cells were tested for expression of MSC markers and their potential for multilineage differentiation. We investigated surface expression of human leukocyte antigen (HLA) molecules on these MSCs before and after treatment with interferon-gamma (IFN-gamma). We also tested the proliferative response of T-lymphocytes toward MSCs and the effects of MSCs in mixed lymphocyte reaction (MLR) assays. RESULTS: We derived populations of MSCs from human ESCs with morphology, cell surface marker characteristics, and differentiation potential similar to adult BM-derived MSCs. Similar to BM-derived MSCs, human ESC-derived MSCs express cell surface HLA class I (HLA-ABC) but not HLA class II (HLA-DR) molecules. However, stimulation with IFN-gamma induced the expression of HLD-DR molecules. Human ESC-derived MSCs did not induce proliferation of T-lymphocytes when cocultured with peripheral blood mononuclear cells. Furthermore, ESC-derived MSCs suppressed proliferation of responder T-lymphocytes in MLR assays. CONCLUSIONS: MSCs can be derived from human ESCs without feeder cells. These human ESC-derived MSCs have cell surface markers, differentiation potentials, and immunological properties in vitro that are similar to adult BM-derived MSCs.     

骨髓基质干细胞种植体外修复内皮及对血管平滑肌细胞增生的影响

目的探讨骨髓基质干细胞(MSCs)种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔血管内皮、平滑肌和人MSCs,通过细胞共培养模拟血管内皮修复过程,用流式细胞仪分析MSCs分子表型特征,免疫荧光细胞化学法观察与内皮共培养的MSCsFlk1和vWF蛋白表达,根据下室内皮生长状态及是否接种MSCs将其分为对照组、单纯MSCs组、融合内皮组、对数内皮组和MSCs种植组。氚胸腺嘧啶脱氧核苷(3HTdR)掺入检测平滑肌细胞DNA合成,Westernblot检测平滑肌细胞中增殖细胞核抗原蛋白表达。结果分离的MSCs表达基质细胞标志CD105和CD166,不表达造血干祖细胞和内皮细胞标志CD34、Flk1、vWF;与内皮共培养5天时,vWF染色仍为阴性,但约25.71%MSCs开始表达Flk1;MSCs种植组平滑肌细胞3HTdR掺入虽高于融合内皮组,但与对数内皮组比较显著降低;MSCs种植组平滑肌细胞PCNA蛋白吸光度相对值虽高于融合内皮组,但与对数内皮组比较明显减少。结论MSCs种植能抑制平滑肌细胞增生,种植在成熟内皮中的MSCs具有微环境依赖向内皮分化的能力。     

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p < 0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.     

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。