血管内皮生长因子对诱导多能干细胞向心肌细胞分化的影响

目的观察血管内皮生长因子(VEGF)对小鼠诱导多能干细胞(iPSCs)向心肌细胞分化的诱导作用。方法采用悬滴培养法,分别以10、20、50 ng/ml的VEGF对iPSCs细胞进行诱导分化,自然分化作为阴性对照组,加入1%二甲亚砜诱导剂为阳性对照组,倒置显微镜下观察细胞生长情况,记录跳动的拟胚体出现的时间和数目,计算心肌细胞分化率,细胞免疫荧光检测心肌细胞cTnT的表达,RT-PCR检测心肌发育基因α-MHC和β-MHC mRNA的表达。结果与自然分化组相比,三个浓度组的VEGF均可提高iPSCs的心肌细胞分化率(P<0.05),浓度为20 ng/ml时,iPSCs的心肌细胞分化率最高;与二甲亚砜组相比无统计学差异(P>0.05)。分化的心肌细胞可自发搏动,同时表达心肌特异蛋白cTnT和调控心肌发育的基因α-MHC和β-MHC;VEGF可上调α-MHC和β-MHC的表达(P<0.05)。结论 VEGF可以促进诱导iPSCs向心肌细胞分化。     

溶血磷脂酸对胚胎干细胞分化心肌细胞L型钙通道电流的影响

目的诱导小鼠胚胎干(ES)细胞向心肌细胞分化并观察溶血磷脂酸(LPA)对分化心肌细胞L型钙通道电流(ICa-L)的影响。方法采用悬滴培养法诱导小鼠ES细胞向心肌细胞分化,逆转录-聚合酶链式反应及免疫荧光检测心肌细胞特异性标志物,全细胞膜片钳记录LPA0.1,1.0和10μmol/L对小鼠ES细胞分化心肌细胞ICa-L的影响。结果小鼠ES细胞成功向心肌细胞分化。0.1,1.0和10.0μmol/LLPA使分化心肌细胞ICa-L峰电流密度分别由用药前-6.8±0.7pA/pF增加到-8.9±1.2,-12.6±2.9和-16.6±3.5pA/pF(P<0.01或P<0.05)。结论LPA呈浓度依赖性促进小鼠ES细胞分化的心肌细胞ICa-L。     

鼠拟胚体来源细胞向肝细胞及心肌细胞分化的初步观察

目的观察微重力生物反应器内拟胚体（EBs）的形成及其EBs来源细胞的肝细胞与心肌细胞分化。方法将未分化鼠胚胎干细胞（Es细胞）以1×10^6／mL移人微重力反应器内旋转培养，6d后取出反应器内形成的EBs接种于培养板培养，使用含DMSO、地塞米松等IMDM培养液继续培养10d。动态观察EBs形成和EBs来源细胞分化细胞形态特征，采用Western blot、免疫荧光染色方法检测EBs来源细胞肝细胞及心肌细胞标志物的表达。结果生物反应器旋转培养6d形成均一的拟胚体，接种后移行细胞中出现肝细胞特征样细胞和自发性搏动细胞，Western blot检测到EBs来源细胞肝细胞标志物Alb表达，免疫荧光染色分别检测出肝细胞标志物Alb、AFP和心肌细胞标志物GATA4的表达。结论微重力生物反应器可加快EBs的形成。EBs来源细胞可有效地向肝细胞及心肌细胞分化，两种细胞之间可能存在相互作用。     

甲状腺素促进miPS细胞向心肌细胞分化的非基因机制的作用

目的 探讨三碘甲腺原氨酸（thyroxine,T3）在诱导小鼠诱导多能干细胞(miPSC)向心肌细胞分化过程中非基因的机制作用。方法 培养Oct4-GFP+ miPS细胞,利用悬滴培养形成拟胚体(embryoid body,EB)的方法诱导miPSC向心肌细胞分化,在诱导过程中分别添加甲状腺素T3和甲状腺素类似物三碘甲腺乙酸（triiodothyroacetic acid,Triac）。实验共分对照组、T3处理组、T3+Triac处理组。分别在诱导分化第4、6和12天,在倒置显微镜下观察细胞的分化情况,采用免疫荧光染色法检测分化第12天心肌细胞特异性结构蛋白α-actinin及肌钙蛋白（cardiac troponin,cTn）T的表达,Western blot法检测cTnT和p-JNK/JNK的表达情况。结果 诱导分化第4、6和12天,T3处理组跳动EB数量明显多于对照组和T3+Triac组（P<0.05）;免疫荧光染色显示T3处理组的α-actinin和cTnT表达量较对照组和T3+Triac组明显增强（P<0.05）;Western blot结果表明T3显著提高cTnT的表达量（P<0.05）;细胞内信号分子JNK蛋白的磷酸化水平明显大于对照组和T3+Triac组（P<0.05）。结论 甲状腺素通过的非基因机制促进miPS细胞向心肌细胞分化,这种作用机制可能跟JNK信号通路有关。     

绿色荧光蛋白标记的小鼠胚胎干细胞系的建立及向心肌细胞分化

目的建立能够稳定表达绿色荧光蛋白（green fluorescent prote in,GFP）的小鼠胚胎干细胞系,并诱导其向心肌细胞分化。方法质粒pEGFP-N1脂质体复合体转染小鼠胚胎干细胞,经G418筛选后选取GFP强阳性克隆进行扩增建系。对稳定表达GFP的胚胎干细胞系进行畸胎瘤形成检测,观察其多向分化潜能。诱导GFP阳性胚胎干细胞向心肌细胞分化。免疫荧光及RT-PCR检测心肌细胞特异性标志物。结果转染后胚胎干细胞经20次传代后仍然表达GFP,裸鼠皮下接种胚胎干细胞后3050 d均可形成畸胎瘤。GFP阳性胚胎干细胞成功向心肌细胞分化。结论成功建立表达GFP的小鼠胚胎干细胞系,并可诱导其向心肌细胞分化。     

Integrin β1在维生素C诱导小鼠诱导性多能干细胞向心肌样细胞分化中的作用

目的:探讨整联蛋白β1（Integrin β1）在维生素C（Vc）诱导小鼠诱导性多能干细胞（induced pluripotent stem cells，iPSCs）向心肌样细胞(cardiomyocytes，CMs)分化中的作用。方法: 在立式显微镜下，分别摘取胚胎(12.5 d、14.5 d及16.5 d)BALB/c胎鼠、新生（出生1 d，P1）和成年BALB/c小鼠的心脏，提取总RNA，半定量及实时定量PCR分析心脏发育不同时期Integrin β1的表达。利用悬滴培养形成拟胚体(embryoid　body，EB)的方法体外诱导iPSCs向心肌样细胞分化，诱导过程中分别添加Vc和integrin β1抑制剂（HMβ1-1）处理，共分为3组:对照组、Vc处理组和Vc+HMβ1-1处理组。诱导分化的第3、5、7天，半定量及实时定量PCR检测Oct4、integrin β1和心肌特异性因子（α-MHC、MLC2a）。用免疫荧光染色法检测心肌特异性结构蛋白心肌肌钙蛋白 I（cTnI）的表达。结果: Integrin β1在心脏胚胎发育时期（E12.5、E14.5、E16.5、P1）的表达递增（P<0.01），成年期表达有所回落（P<0.01）。半定量及实时定量PCR的结果提示，Vc组α-MHC、MLC2a的表达显著高于对照组（P<0.01），加integrin β1抑制剂HM β1-1后表达量降低(P<0.01)。跳动EBs计数的结果提示，诱导分化第10、14、18、22、26天，Vc组跳动EBs的百分率显著高于对照组（P<0.01）；而Vc+ HMβ1-1组跳动EBs的百分率相对于Vc组显著降低（P<0.01）。免疫荧光染色显示，Vc组有较强的cTnI表达，添加integrin β1抑制后，Vc+HMβ1-1组cTnI的表达明显减弱。结论: Vc可促进iPSCs向心肌细胞分化，其机制可能是通过integrin β1介导。     

心肌营养素-1诱导小鼠诱导性多能干细胞心肌细胞定向分化的实验研究

目的:观察心肌营养素-1(CT-1)对小鼠诱导性多能干细胞(miPSCs)心肌细胞定向分化的诱导作用。方法:实验分为对照组与CT-1处理组。miPSCs经悬滴法诱导形成拟胚体,第3天培养基中加入1μmol/ml CT-1(CT-1组),对照组采用普通培养基。于第4、7、10和14天,收取细胞样本,采用实时PCR检测心脏特异标志物基因的表达。用免疫荧光染色及流式细胞术检测干细胞标志物Oct4和心肌特异性分化标志物肌钙蛋白I(cTnI)的表达。用透射电镜观察心肌样细胞的超微结构。结果:CT-1组第10天,样本中心肌特异性肌钙蛋白T(cTnT)基因表达的水平为同期对照组的1.75倍,有显著性差异(P<0.05)。免疫荧光染色法检测显示,两组均有cTnI表达。流式细胞术鉴定的结果显示,对照组与CT-1组cTnI的阳性率分别为28.5%和56.4%,有显著性差异(P<0.05)。电镜观察显示,CT-1组肌原纤维及胞内线粒体明显增多,肌纤维排列规则,可见细胞间桥粒连接和缝隙连接。结论:CT-1可显著提高miPSC向心肌细胞定向分化的效率。     

人双突变型低氧诱导因子α促进骨髓间充质干细胞向心肌细胞分化的实验研究

目的 探讨人双突变型低氧诱导因子1α(HIF-1α)基因(HIF-1α-402-564)对与心肌细胞共培养的骨髓间充质干细胞(MSC)向心肌细胞分化的影响.方法 实验分为4组:HIF-1α组(MSC+心肌细胞+Ad-HIF-1α)、LacZ组(MSC+心肌细胞+Ad-Lacz)、Sham组[MSC+心肌细胞+胎牛血清(PBS)]、MSC+HIF-1α组(MSC+Ad-HIF-1α),前三组中MSC与心肌细胞按1:2比例共同培养,各组细胞培养24 h后分别感染不同病毒(MOI=100)或加入PBS液.病毒感染后7 d,免疫细胞化学分析共培养的MSC表达心肌细胞特异性标志物心肌肌钙蛋白T(cTnT)的情况,逆转录聚合酶链式反应(RT-PCR)分析各组细胞HIF-1α、转化生长因子β1(TGF-β1)、Smad4、NKx2.5、GATA结合蛋白4(GATA-4)的mRNA表达量.结果 HIF-1α组MSc心肌细胞分化率为(32.68±6.52)%,显著高于LacZ组[(8.28±0.09)%]和Sham组[(10.25±2.20)%],P均<0.05,MSC±HIF-1α组仅为(0.32±0.05)%.LacZ组和Sham组间差异无统计学意义.MSC+HIF-1α组与Sham组比较,差异有统计学意义(P<0.05).HIF-1α组TGFβ1及Smad4 mRNA表达水平明显高于其余各组,P均<0.05.HIF-1α组NKx2.5和GATA-4 mRNA表达水平明显高于Sham组,P均<0.05.结论 HIF-1α能够促进与心肌细胞共培养的MSC向心肌细胞分化,TGF-β1/Smad4信号通路参与了该过程.     

3D 打印聚己内酯多级管道支架促进梗死后心肌修复

目的:探讨聚己内酯(PCL)多级管道支架对SD大鼠梗死后心肌修复的作用及潜在机制。方法:通过3D打印技术,制备PCL多级管道支架,选取体质量为200～250 g的SD大鼠24只,随机分为3组,分别为假手术组(Sham组)、急性心肌梗死组(MI组)以及治疗组(MI+scaffold组)。采用Masson染色,评价术后28 d各组SD大鼠纤维化程度及心梗面积大小;通过免疫荧光染色,评价术后28 d各组心肌梗死交界区和支架内部血管再生情况。结果:3D打印成功制备PCL多级管道支架;Masson染色表明,术后28 d MI组心肌梗死范围和瘢痕面积明显大于MI+scaftold组[(64.63±7.72)%对(42.01±8.68)%,P0.01;(13.85±1.98)mm~2对(9.82±1.47)mm~2,P0.01];免疫荧光染色表明,术后28 d MI+scaffold组心肌梗死交界区域心肌再血管化程度明显高于MI组[(15±3.13)个/HPF对(5.29±0.91)个/HPF,P0.01], MI+scaffold组补片内部血管化程度高于心肌梗死交界区[(17.7±2.71)个/HPF对(15±3.13)个/HPF,P0.05]。结论:3D打印PCL多级管道支架通过缩小SD大鼠心肌梗死范围及瘢痕区域,促进心肌再血管化来修复心肌。     

过表达GATA-4小鼠骨髓间充质干细胞向心肌细胞分化的初步探讨

目的:探讨过表达GATA-4小鼠骨髓间充质干细胞(BMSC)向心肌细胞分化的能力。方法:通过慢病毒载体GV308携带GATA-4转染小鼠BMSC构建过表达GATA-4小鼠BMSC并加入基因开启剂强力霉素(DOX)。各组BMSCs首先用Dil标记,而后将过表达GATA-4-BMSC+DOX与心肌细胞共同培养,空载体-BMSC+DOX与心肌细胞共同培养,BMSC+DOX与心肌细胞共同培养,BMSC单独培养作为对照组,各组培养48h。采用荧光染色定性检测心肌肌钙蛋白T(cTnT)、α肌动蛋白(α-actin)、连接蛋白43(Connexin 43)及结蛋白(Desmin)表达。而后采用Q-PCR定量检测上述心肌特异性抗原表达量。结果:GATA-4-BMSC与心肌细胞共培养组表达的cTnT、α-actin、Connexin-43与Desmin水平较其他组高,证实GATA-4可以有效促进BMSCs向心肌细胞转化。结论:过表达GATA-4的BMSCs可以更为有效地向心肌细胞分化。     

Bone Tissue Engineering with Adipose-Derived Stem Cells in Bioactive Composites of Laser-Sintered Porous Polycaprolactone Scaffolds and Platelet-Rich Plasma

Three-dimensional porous polycaprolactone (PCL) scaffolds with consistent inter-pore channels, 83% porosity and 300–400 μm pore size were fabricated via selective laser sintering. The PCL scaffold was combined with platelet-rich plasma (PRP) to form a bioactive composite and studied for potential application in bone tissue engineering using porcine adipose-derived stem cells (PASCs). The PCL/PRP/PASCs construct showed enhanced cell seeding efficiency and synergistically increased the differentiation capability of PASCs in osteogenic medium toward the osteoblast lineage, judging from elevated alkaline phosphatase activity and up-regulated osteogenic genes expression. For in vivo study, a 3 cm × 3 cm mandible defect was created in pigs and reconstructed by implanting acellular PCL scaffolds or PCL/PRP/PASCs constructs. Both groups showed new bone formation, however, the new bone volume was 5.1 times higher for PCL/PRP/PASCs 6 months post-operation. The bone density was less and loose in the acellular PCL group and the Young’s modulus was only 29% of normal bone. In contrast, continued and compact bone formation was found in PCL/PRP/PASCs and the Young’s modulus was 81% that of normal bone. Masson’s trichrome stain, immunohistochemical analysis of osteocalcin and collagen type I also confirmed new bone formation.     

Topological,Mechanical and Biological Properties of Ti6Al4V Scaffolds for Bone Tissue Regeneration Fabricated with Reused Powders via Electron Beam Melting

Cellularized scaffold is emerging as the preferred solution for tissue regeneration and restoration of damaged functionalities. However, the high cost of preclinical studies creates a gap between investigation and the device market for the biomedical industry. In this work, bone-tailored scaffolds based on the Ti6Al4V alloy manufactured by electron beam melting (EBM) technology with reused powder were investigated, aiming to overcome issues connected to the high cost of preclinical studies. Two different elementary unit cell scaffold geometries, namely diamond (DO) and rhombic dodecahedron (RD), were adopted, while surface functionalization was performed by coating scaffolds with single layers of polycaprolactone (PCL) or with mixture of polycaprolactone and 20 wt.% hydroxyapatite (PCL/HA). The mechanical and biological performances of the produced scaffolds were investigated, and the results were compared to software simulation and experimental evidence available in literature. Good mechanical properties and a favorable environment for cell growth were obtained for all combinations of scaffold geometry and surface functionalization. In conclusion, powder recycling provides a viable practice for the biomedical industry to strongly reduce preclinical costs without altering biomechanical performance.     

Cell-to-cell contact induces mesenchymal stem cell to differentiate into cardiomyocyte and smooth muscle cell

BACKGROUND: Recent evidences have suggested that stem cell can differentiate into cardiomyocyte and smooth muscle cell (SMC) in vivo or in vitro. But the mechanism on how stem cell differentiates is still unknown. We investigated whether intercellular interaction or soluble chemical factors would induce mesenchymal stem cells (MSCs) to acquire the phenotypical characteristics of cardiomyocytes or SMC. METHODS: MSCs were isolated from rat bone marrow with density gradient centrifugation and amplified in vitro. Flow cytometry was used to monitor the expression of surface antigen profile. After labeled by GFP (green fluorescent protein) transfection, rat MSCs were used to culture with adult rat cardiomyocytes and rat aortic SMCs in direct co-culture, indirect co-culture and conditioned culture, respectively. One week later, immunofluorescence staining against alpha-actin, desmin, and cardiac troponin T (cTnT) for cardiomyocyte, smooth muscle calponin and SM-alpha-actin for SMC were performed. RESULTS: Immunofluorescence staining was positive against alpha-actin, desmin, and cTnT on MSCs in co-culture group with adult cardiomyocytes, positive against smooth muscle calponin and SM-alpha-actin on MSCs in co-culture group with SMCs. In contrast, no alpha-actin, desmin, and cTnT expression was observed in the indirect co-culture group and conditioned culture group; no smooth muscle calponin and SM-alpha-actin in the indirect co-culture group and conditioned culture group. CONCLUSIONS: Direct cell-to-cell contact between MSC and adult cardiomyocyte or SMC, but not the soluble signaling molecules is obligatory in the differentiation of MSC into cardiomyocytes or SMC.     

3D PCL/Gelatin/Genipin Nanofiber Sponge as Scaffold for Regenerative Medicine

Recent advancements in tissue engineering and material science have radically improved in vitro culturing platforms to more accurately replicate human tissue. However, the transition to clinical relevance has been slow in part due to the lack of biologically compatible/relevant materials. In the present study, we marry the commonly used two-dimensional (2D) technique of electrospinning and a self-assembly process to construct easily reproducible, highly porous, three-dimensional (3D) nanofiber scaffolds for various tissue engineering applications. Specimens from biologically relevant polymers polycaprolactone (PCL) and gelatin were chemically cross-linked using the naturally occurring cross-linker genipin. Potential cytotoxic effects of the scaffolds were analyzed by culturing human dermal fibroblasts (HDF) up to 23 days. The 3D PCL/gelatin/genipin scaffolds produced here resemble the complex nanofibrous architecture found in naturally occurring extracellular matrix (ECM) and exhibit physiologically relevant mechanical properties as well as excellent cell cytocompatibility. Samples cross-linked with 0.5% genipin demonstrated the highest metabolic activity and proliferation rates for HDF. Scanning electron microscopy (SEM) images indicated excellent cell adhesion and the characteristic morphological features of fibroblasts in all tested samples. The three-dimensional (3D) PCL/gelatin/genipin scaffolds produced here show great potential for various 3D tissue-engineering applications such as ex vivo cell culturing platforms, wound healing, or tissue replacement.     

Synthesis and Characterization of Electrospun Composite Scaffolds Based on Chitosan-Carboxylated Graphene Oxide with Potential Biomedical Applications

This research study reports the development of chitosan/carboxylated graphene oxide (CS/GO-COOH) composite scaffolds with nanofibrous architecture using the electrospinning method. The concept of designed composite fibrous material is based on bringing together the biological properties of CS, mechanical, electrical, and biological characteristics of GO-COOH with the versatility and efficiency of ultra-modern electrospinning techniques. Three different concentrations of GO-COOH were added into a chitosan (CS)-poly(ethylene oxide) (PEO) solution (the ratio between CS/PEO was 3/7 (w/w)) and were used in the synthesis process of composite scaffolds. The effect of GO-COOH concentration on the spinnability, morphological and mechanical features, wettability, and biological properties of engineered fibrous scaffolds was thoroughly investigated. FTIR results revealed the non-covalent and covalent interactions that could take place between the system’s components. The SEM micrographs highlighted the nanofibrous architecture of scaffolds, and the presence of GO-COOH sheets along the composite CS/GO-COOH nanofibers. The size distribution graphs showed a decreasing trend in the mean diameter of composite nanofibers with the increase in GO-COOH content, from 141.40 nm for CS/PG 0.1% to 119.88 nm for CS/PG 0.5%. The dispersion of GO-COOH led to composite scaffolds with increased elasticity; the Young’s modulus of CS/PG 0.5% (84 ± 4.71 MPa) was 7.5-fold lower as compared to CS/PEO (662 ± 15.18 MPa, p < 0.0001). Contact angle measurements showed that both GO-COOH content and crosslinking step influenced the surface wettability of scaffolds, leading to materials with ~1.25-fold higher hydrophobicity. The in vitro cytocompatibility assessment showed that the designed nanofibrous scaffolds showed a reasonable cellular proliferation level after 72 h of contact with the fibroblast cells.     

生长分化因子-11促进小鼠诱导性多能干细胞向心肌细胞定向分化的研究

目的 明确生长分化因子（GDF）-11对小鼠诱导多能干细胞（miPSCs）向心肌细胞定向分化的促进作用,为心肌再生的细胞生物学治疗提供种子细胞和实验依据。方法 常规培养小鼠miPSCs,分为对照组和GDF-11组。GDF-11组在普通分化培养基中加用10 ng/ml的GDF-11。悬滴法诱导培养形成拟胚体（EBs）,每日观察搏动拟胚体数目。采用实时荧光定量PCR检测多能干细胞标志物Oct-4、心脏中胚层标志物Flk-1、心脏祖细胞标志物Nkx2.5、和心肌特异性标志物cTnT的表达变化。用免疫荧光染色观察心肌结构蛋白cTnI的表达水平。结果 GDF-11组的Oct-4表达水平在诱导分化的第3、7天分别为同期对照组的（0.55±0.31）倍（P<0.01）和（0.41±0.57）倍（P<0.05）;诱导分化的第10天,GDF-11组的Flk-1和Nkx2.5表达相分别为对照组的（2.09±0.8）倍（P<0.05）和（2.47±0.22）倍（P<0.01）;cTnT的表达在分化后第10天和第14天分别为对照组的（1.81±0.19）倍（P<0.01）和（1.61±0.20）倍（P<0.01）;两组均出现了心肌样搏动细胞团,GDF-11组搏动EBs百分比要显著高于对照组（P<0.01）。免疫荧光染色显示,GDF-11组的cTnI阳性率要显著高于对照组（P<0.01）。结论 GDF-1能够显著促进miPSCs的心肌定向分化。