骨髓间充质干细胞移植后在mdx鼠体内的分布研究

目的： 研究骨髓间质干细胞移植治疗Duchenne型肌营养不良鼠(mdx鼠)后体内各主要器官的分布状况。方法: 取第5代SD大鼠骨髓间质干细胞，应用［3H］－TdR标记后以2×107cells／只细胞尾静脉移植到经7Gy γ射线预处理后的7-9周龄mdx鼠体内，分别于移植后24 h、48 h、4周、8周和16周测定血液、肺、肝、骨髓、骨骼肌及心肌的放射性分布计数。结果: 经7Gy γ射线放疗预处理的mdx鼠，在尾静脉移植骨髓干细胞后，24 h肺的放射性分布计数最高，为36.70±3.04；48 h肝的放射性分布计数最高，为36.74±3.28；骨髓分布计数随移植时间延长增多，2周时达到高峰，计数为38.43±4.99，以后逐渐下降，但4月时仍明显高于其它组织器官，计数为13.45±1.37；骨骼肌、心肌的放射性分布计数随移植时间延长明显增高，16周时分别达到4.79±0.94和9.55±1.53。结论: 在MSCs移植后早期，MSCs主要分布于肺、肝等血流丰富的器官，随移植时间延长逐渐归巢到骨髓定居，2周时达到高峰，此后逐渐向损伤器官如骨骼肌、心肌等定向迁移，并且随时间延长分布增加，从而为MSCs定居于肌组织并且分化为肌细胞提供了证据。     

Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration

Cell therapy for degenerative muscle diseases such as the muscular dystrophies requires a source of cells with the capacity to participate in the formation of new muscle fibers. We investigated the myogenic potential of human fetal mesenchymal stem cells (hfMSCs) using a variety of stimuli. The use of 5-azacytidine or steroids did not produce skeletal muscle differentiation, whereas myoblast-conditioned medium resulted in only 1%-2% of hfMSCs undergoing muscle differentiation. However, in the presence of galectin-1, 66.1% +/- 5.7% of hfMSCs, but not adult bone marrow-derived mesenchymal stem cells, assumed a muscle phenotype, forming long, multinucleated fibers expressing both desmin and sarcomeric myosin via activation of muscle regulatory factors. Continuous exposure to galectin-1 resulted in more efficient muscle differentiation than pulsed exposure (62.3% vs. 39.1%; p < .001). When transplanted into regenerating murine muscle, galectin-1-exposed hfMSCs formed fourfold more human muscle fibers than nonstimulated hfMSCs (p = .008), with similar results obtained in a scid/mdx dystrophic mouse model. These data suggest that hfMSCs readily undergo muscle differentiation in response to galectin-1 through a stepwise progression similar to that which occurs during embryonic myogenesis. The high degree of myogenic conversion achieved by this method has relevance for the development of therapies for muscular dystrophies.     

小鼠胎肝间质干细胞的纯化与分化多能性研究

目的： 分离纯化小鼠胎肝间质干细胞（flMSCs）并探讨其分化潜能。方法： 通过改良贴壁法分离BABL/c小鼠胎肝间质干细胞；流式细胞术测定细胞周期和表型；体外诱导贴壁细胞向脂肪、软骨、骨组织以及神经细胞分化；化学染色、RT-PCR和免疫细胞化学染色鉴定分化。结果： 改进贴壁法分离的flMSCs呈均一梭形，(83.76±2.88)%处于G₀/G₁期,表达CD44、CD29,不表达造血细胞表面标志CD45、CD11b,诱导后能向脂肪、软骨、骨以及神经方向分化。结论： 贴壁法可以分离纯化小鼠flMSCs，小鼠flMSCs具有较强的分化潜能和“可塑性”，可用于干细胞治疗疾病的进一步研究。     

骨髓干细胞移植后mdx鼠腓肠肌病理变化

目的 研究骨髓干细胞移植后mdx鼠腓肠肌组织病理变化. 方法 7～9周龄mdx鼠20只平均分为4组,放射处理后移植1.2×107细胞/只同种异基因全骨髓干细胞,于移植后4、8、12及16周用HE染色观察腓肠肌组织细胞形态及核中心移位纤维(CNF).C57鼠和未治疗mdx鼠各5只作阳性和阴性对照. 结果 CS7鼠腓肠肌横切面可见肌细胞大小形态基本一致,无核中心移位现象.各细胞移植治疗组和阴性对照组mdx鼠均有大量的炎细胞浸润,核中心移位明显.未治疗mdx鼠CNF最高,约达70%;移植后4、12和16周,CNF分别为55%、50%和44%. 结论 骨髓干细胞移植后mdx鼠腓肠肌CNF随移植时间延长逐渐减少,提示骨髓干细胞移植后长久持续参与受损骨骼肌的修复与再生.     

Mechanical and energetic properties of dystrophic (mdx) mouse muscle

The mechanical and energetic properties of extensor digitorum longus (EDL) and soleus muscles of X chromosome-linked muscular dystrophic mutant (mdx) mice aged 4-6 weeks were studied and compared with those of the muscles of normal mice. Maximum tetanic tension, the speed of contraction of relaxation, and the heat production of mdx soleus muscles were not significantly different from those of the normal muscles. However, in mdx EDL muscles, the tension and heat production were significantly reduced, and relaxation was prolonged. To study the cause of these changes in mdx EDL muscles, tension and heat production were measured at various muscle lengths greater than optimum for tension. Both the amount of twitch heat and the heat rate for a tetanus were linearly related to the tension and had non-zero intercepts at zero tension, the activation heat. The twitch activation heat and the tension-related heat in tetani of mdx EDL muscles were not different from those in normal muscles. On the other hand, the tetanus activation heat of mdx EDL muscles was significantly smaller than that of normal muscles. Assuming that the degenerated fibers do not contribute to the active force produced, these results suggest that the amount of Ca2+ released in a contraction is not significantly different between normal and mdx muscles, but the Ca-ATPase activity of the salcoplasmic reticulum is reduced in mdx EDL, which could cause the slowing of relaxation.     

人骨髓胎儿间充质干细胞在小鼠体内的分化

目的：探讨人骨髓胎儿间充质干细胞（ hfMSC）的特性及其在X染色体- 连锁肌萎缩（ mdx）小鼠体内分 化的可能性。 方法：从胎儿骨髓中分离扩增hfMSC,检测hfMSC Oct-4 和Nanog-3 的表达。经DIR 标记后注射 hfMSC 到mdx小鼠腹股沟三角皮下,采用活体成像法观察小鼠离体后肢肌肉植入细胞的存活状态,观察是否出 现畸胎瘤,并采用免疫荧光染色法检测dystrophin 的表达。结果：从胎儿骨髓中分离到表达Oct-4 和Nanog-3 的 hfMSC。在注射hfMSC 的mdx小鼠离体后肢肌肉中检测到明显的荧光信号,并在肌肉组织中检测到人dystrophin 的 表达,而没有发现畸胎瘤的出现。结论： hfMSC 表达多潜能抗原标志Oct-4 和Nanog-3,hfMSC 在mdx小鼠体内可 以产生dystrophin,且不会形成畸胎瘤。     

Endothelial differentiation by multipotent fetal mouse lung mesenchymal cells

During fetal lung development, cells within the mesenchyme differentiate into vascular endothelia. This process of vasculogenesis gives rise to the cells that will eventually form the alveolar capillary bed. The cellular mechanisms regulating lung vasculogenesis are poorly understood, partly due to the lack of experimental systems that model this process. Here, we have developed and characterized a novel fetal mouse lung cell model of mesenchymal to endothelial differentiation. Using mesenchymal cells from the lungs of embryonal day 15 Immortomice, we show that endothelial growth media containing fibroblast growth factor-2 and vascular endothelial growth factor can stimulate formation of vascular endothelial cells in culture. These newly formed endothelial cells retain plasticity, as removing endothelial growth media causes loss of vascular markers and renewed formation of α-smooth muscle actin positive stress fibers. Cells with the highest Flk-1 expression differentiated into endothelia more efficiently. Individual mesenchymal cell clones had varied ability to acquire an endothelial phenotype. These fetal lung mesenchymal cells were multipotent, capable of differentiating into not only vascular endothelia, but also osteogenic and chondrongenic cell lineages. Our data establish a cell culture model for mesenchymal to endothelial differentiation that could prove useful for future mechanistic studies in the process of vasculogenesis both during normal development and in the pathogenesis of pulmonary vascular disease.     

Improved contractile function of the mdx dystrophic mouse diaphragm muscle after insulin-like growth factor-I administration

Limited knowledge exists regarding the efficacy of insulin-like growth factor I (IGF-I) administration as a therapeutic intervention for muscular dystrophies, although findings from other muscle pathology models suggest clinical potential. The diaphragm muscles of mdx mice (a model for Duchenne muscular dystrophy) were examined after 8 weeks of IGF-I administration (1 mg/kg s.c.) to test the hypothesis that IGF-I would improve the functional properties of dystrophic skeletal muscles. Force per cross-sectional area was approximately 49% greater in the muscles of treated mdx mice (149.6 +/- 9.6 kN/m(2)) compared with untreated mice (100.1 +/- 4.6 kN/m(2), P < 0.05), and maintenance of force over repeated maximal contraction was enhanced approximately 30% in muscles of treated mice (P < 0.05). Diaphragm muscles from treated mice comprised fibers with approximately 36% elevated activity of the oxidative enzyme succinate dehydrogenase, and approximately 23% reduction in the proportion of fast IId/x muscle fibers with concomitant increase in the proportion of type IIa fibers compared with untreated mice (P < 0.05). The data demonstrate that IGF-I administration can enhance the fatigue resistance of respiratory muscles in an animal model of dystrophin deficiency, in conjunction with enhancing energenic enzyme activity. As respiratory function is a mortality predictor in Duchenne muscular dystrophy patients, further evaluation of IGF-I intervention is recommended.     

人胎骨髓MSCs的分离鉴定及其向肝细胞样细胞的分化

目的: 分离鉴定人胎骨髓中的间质干细胞(MSCs),探索其体外培养的生物学特性，并在化学因子作用下诱导其向肝细胞分化。方法: 利用细胞差速贴壁生长特性分离纯化人胎骨髓MSCs；利用流式细胞仪检测其细胞周期和表面标志；添加常规诱导液诱导其向脂肪、成骨方向分化;采用DMSO、β-Me和5-aza联合预诱导24 h,换用H-DMEM和 rh-HGF正式诱导人胎骨髓MSCs向肝细胞分化，并从形态学和特异性细胞化学染色等方面加以鉴定。结果: 从人胎骨髓中成功分离、纯化得到MSCs，P4代MSCs有92.3%的细胞处于G₀/G₁期；P5代MSCs有96.1%的细胞处于G₀/G₁期。流式细胞仪检测P3代MSCs结果显示：人胎骨髓MSCs表达CD29、CD44、CD105和CD106 ,不表达造血细胞标志CD34、CD45,不表达与GVHD相关的HLA-DR、CD80、CD86、CD40、CD40L。在经典的诱导条件下，人胎骨髓MSCs可快速向脂肪及成骨细胞分化；在上述诱导条件下，人胎肝MSCs可分化为类肝细胞,表达特异性抗原甲胎蛋白（AFP）和白蛋白（ALB）。结论: 人胎骨髓中含有丰富的MSCs,人胎骨髓来源的MSCs具有较强的多向分化潜能，经DMSO、β-Me和5-aza联合预诱导及rh-HGF、nictinion等化学因子的作用，易向肝细胞样细胞分化，且免疫原性弱,是组织工程（生物型人工肝）的较为理想的种子细胞。     

Osteogenic differentiation of human marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) are adherent cells that differentiate into chondroblasts, osteoblasts and adipocytes. In this short review, we summarize the molecular mechanisms that are known to control osteoblast differentiation and osteogenic potential of MSCs in vitro. We discuss the advances made in gene-based therapy to promote osteogenic differentiation of MSCs and the perspectives for an optimal use of MSCs for bone tissue regeneration or repair. One important challenge at the present time is to identify factors and pathways that promote osteogenic commitment of MSCs in order to use MSCs with functional potential for optimal bone repair in humans. In this context, genomic and proteomic analyses may help to identify molecules that could be used to promote osteogenic differentiation of human MSCs. In the future this may lead to selective therapeutic strategies for tissue engineering application in bone regeneration and repair in humans.     

Expansion and chondrogenic differentiation of human mesenchymal stem cells

The potential of human mesenchymal stem cells (hMSC) to differentiate into various types of mesenchymal tissue, such as chondrocytes, makes them a potential cell source in cartilage tissue engineering. Because of the requirement of high cell amounts for the generation of cartilage implants or for the extensive experimental studies to investigate the culture parameters, the initial cells have to be expanded, which leads to high population doubling numbers. It is known that hMSC can differentiate into chondrocytes at least up to the 15th population doubling. To monitor the differentiation status, the protein MIA (melanoma inhibitory activity), which is only synthesized by malignant melanomas and chondrocytes, can be used. In this study the chondrogenic differentiation potential of hMSC beyond the 15th population doubling was investigated using MIA as a chondrocyte marker. A chondrogenic potential of hMSC at higher population doubling numbers may be of interest due to the requirement of less frequent isolations of cells. Therefore hMSC were cultured in a monolayer until the 37th population doubling. Cells of different passages were cultured as pellets for two weeks in transforming growth factor (TGF)-beta3 containing differentiation medium. The MIA contents in medium on the last three cultivation days were measured for each case using an MIA-ELISA-kit. A significant difference between MIA content in medium of the pellet and nonstimulated monolayer reference cultures was detectable until the 32nd population doubling. In addition, the hMSC were seeded at lower densities to investigate whether the cells may be expanded faster and with less amount of work due to higher population doubling numbers per passage. The reduced inoculation density led to an increased growth rate.     

小鼠胎肝间充质干细胞的分离及鉴定

目的 分离培养小鼠胎肝间充质干细胞（flMSCs）。方法 改良贴壁法分离BABL/c胎鼠肝脏间充质干细胞，测定生长曲线检测增殖能力；流式细胞术分析细胞周期和表型；体外诱导贴壁细胞向脂肪、软骨、骨组织分化并鉴定。结果 贴壁法分离的flMSCs呈均一梭形，细胞倍增时间约为24 h。83.76%±2.88% 的flMSCs处于G0/G1期，表达间质系列表面标志CD44, CD29, 不表达造血细胞表面标志CD45, CD11b; 诱导后能向脂肪、软骨、骨组织方向分化。结论 贴壁法可以分离flMSCs, 传代有助其纯化；flMSCs具有强增殖能力和分化潜能，是理想的干细胞治疗来源。     

Engraftment potential of human placenta-derived mesenchymal stem cells after in utero transplantation in rats

BACKGROUND: Human placental mesenchymal stem cells (hPMCs) are thought tobe multipotent, but their fate after in utero transplantationis not known. METHODS: hPMCs isolated from term placenta were assessed for their phenotypemarkers, mutilineage capacity, and immunomodulatory properties.Their engraftment potential was analyzed in a pregnant rat modelafter in utero transplantation at embryonic day 17. Immunohistochemistry,tracing of labeled cells, fluorescence in situ hybridizationand real-time PCR were used to assess post-transplant chimerism. RESULTS: In vitro, lineage-negative, CD34-negative hPMCs differentiatedinto osteocytes, adipocytes, hepatocytes and endothelial cellswith tube formation, and actively suppressed the rat lymphocyteproliferative response to allogeneic lymphocyte stimulation(P < 0.0001). After in utero transplantation into pregnantrats, a low level of engraftment was achieved in various fetaltissues. Engraftment occurred in more than 60% of the fetalrats. Cells persisted for at least 12 weeks after delivery andevidence was obtained to suggest differentiation into specificlineages, including hepatocytes and hematopoietic cells. However,a greater number of hPMCs migrated to the placenta than to thefetus, thus limiting the degree of cell engraftment in fetalorgans. CONCLUSIONS: We conclude that hPMCs are mutipotent cells that can be engraftedlong-term in immunocompetent rats after in utero transplantation.     

Neuronal differentiation potential of human adipose-derived mesenchymal stem cells

Adult mesenchymal stem cells derived from adipose tissue (A-MSC) have the capacity to differentiate in vitro into mesenchymal as well as endodermal and ectodermal cell lineages. We investigated the neuronal differentiation potential of human A-MSC with a protocol which included sphere formation and sequential culture in brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). After 30 days, about 57% A-MSC showed morphological, immunocytochemical and electrophysiological evidence of initial neuronal differentiation. In fact, A-MSC displayed elongated shape with protrusion of two or three cellular processes, selectively expressed nestin and neuronal molecules (including GABA receptor and tyroxine hydroxilase) in the absence of glial phenotypic markers. Differentiated cells showed negative membrane potential (-60 mV), delayed rectifier potassium currents and TTX-sensitive sodium currents. Such changes were stable for at least 7 days after removal of differentiation medium. In view of these results and the easy availability of adipose tissue, A-MSC may be a ready source of adult MSC with neuronal differentiation potential, an useful tool to treat neurodegenerative diseases.     

Characterization of mesenchymal stem cells isolated from mouse fetal bone marrow

Mesenchymal stem cells (MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells. MSCs have some features (surface molecules and cytokine production, etc.) common to so-called traditional bone marrow (BM) stromal cells, which have the capacity to support hemopoiesis. In the present study, we isolated murine MSCs (mMSCs) from the fetal BM using an anti-PA6 monoclonal antibody (mAb) that is specific for bone marrow stromal cells. The mMSCs, called FMS/PA6-P cells, are adherent, fibroblastic, and extensively expanded and have the ability to differentiate not only into osteoblasts and adipocytes but also into vascular endothelial cells. The FMS/PA6-P cells produce a broad spectrum of cytokines and growth factors closely related to hemopoiesis and show good hemopoiesis-supporting capacity both in vivo and in vitro, suggesting that they are a component of the hemopoietic stem cell niche in vivo. Interestingly, although the FMS/PA6-P cells express a high level of the PA6 molecule, which is reactive with anti-PA6 mAb, they gradually lose their ability to express this molecule during the course of differentiation into osteoblasts and adipocytes, indicating that the PA6 molecule might serve as a novel marker of mMSCs.     

胎肝间充质干细胞和皮肤成纤维细胞有限细胞系的建立及肝向分化研究

通过体外长期培养胎肝间充值干细胞和皮肤成纤维细胞,建立两种细胞长期培养体系并比较此两种细胞的表型特征及向肝细胞分化的潜能。方法 从人胎肝中分离贴壁生长的间充质干细胞,同时取同一个体的皮肤组织,分离培养成纤维细胞。采用免疫细胞化学法及流式细胞术检测上述两种细胞的CD34,CD90,CD105等细胞表型;染色体分析及软琼脂...     

骨髓间充质干细胞体外诱导向内皮和上皮细胞的分化

背景：骨髓来源的间充质干细胞在体外具有多系分化潜能,但其在体外向肺组织细胞的分化能力尚存在争议。 目的：体外诱导验证小鼠骨髓间充质干细胞向内皮细胞和上皮细胞分化的能力。 方法：分离小鼠骨髓来源的间充质干细胞,以内皮诱导液向内皮细胞分化。另外将小鼠骨髓间充质干细胞分别在以下诱导培养基中进行上皮诱导3周：单纯上皮诱导培养液,上皮诱导培养液加10 μg/L 转化生长因子β1,并以未经诱导的小鼠骨髓间充质干细胞作为阴性对照,肺泡上皮作为阳性对照。 结果与结论：小鼠骨髓间充质干细胞在上皮诱导培养液中诱导培养3周后,部分细胞由梭形变为典型的卵石样上皮细胞形态,诱导后约60%细胞表达广谱上皮细胞标志pan-CK,RT-PCR结果显示分化后的细胞表达上皮细胞特异标志CK18,未经诱导的间充质干细胞未表达。小鼠骨髓间充质干细胞在内皮诱导24 h后即出现了典型的血管网状结构,vWF免疫荧光染色显示约70%的细胞呈阳性,RT-PCR显示分化后的细胞表达内皮细胞特异性标志CD31、vWF和CD34。提示骨髓来源的间充质干细胞体外诱导培养具有跨胚层多系分化能力。