MACS系统对小鼠造血干细胞定向分化为未成熟树突状细胞的实验研究

目的:探讨经MACS系统在体外获得大量高纯度未成熟树突状细胞(imDC)的方法,并对其细胞表面标志、形态和功能进行鉴定。方法:对健康C57小鼠的骨髓通过MACS系统分离、纯化CD117 造血干细胞(HSC);使用SCF IL-3行体外扩增,并采用GM-CSF IL-4 IL-10诱导其定向分化为imDC;进而在倒置显微镜、扫描电镜以及透射电镜下观察其形态、功能,采用流式细胞计数法检测、鉴定其表面标志物的表达。结果:SCF IL-3可以分别在3、5、7d时体外扩增HSC达10.34±1.43倍、22.65±2.71倍、54.39±3.08倍;小鼠HSC可被成功诱导分化为imDC,且具有吞噬功能,表面树突呈毛刺状、较为短小,imDC并表达CD11c 、I-A/I-Elow、CD40-、CD80-、CD86-。结论:本方法可稳定、有效地获得大量高纯度的imDC。     

兔骨髓间充质干细胞的生物学特征及原代培养

背景：兔来源的骨髓间充质干细胞是具有较强的体外增殖和多系统分化潜能的成体干细胞,在组织工程及生物治疗领域蕴藏着巨大的潜能。 目的：体外培养扩增、鉴定兔骨髓间充质干细胞,观察骨髓间充质干细胞的生物学特征。 方法：无菌条件下抽取兔骨髓,分别运用全骨髓贴壁法和Percoll密度梯度离心法体外分离骨髓间充质干细胞,利用差速贴壁原理对细胞进行纯化扩增。在显微镜下观察细胞形态特征及生长规律,流式细胞技术检测细胞表面抗原标记物的表达。 结果与结论：兔骨髓间充质干细胞贴壁时间短,增殖快,经过细胞传代后能够获得进一步纯化的细胞,杂质细胞减少。原代细胞形态即呈现三角形、长梭形、纺锤形的贴壁细胞特征。第 5代骨髓间充质干细胞呈典型的极性漩涡状生长,形态单一均匀,不具有表达造血前体细胞表面标志抗原CD34和白细胞表面标志抗原CD45的表面标记物功能,但是具有能够表达出整合素家族的成员 CD29 及黏附分子CD44的特点。说明经全骨髓贴壁法和Percoll密度梯度离心法体体外分离培养的细胞在形态学、细胞表面标志物表达和多向分化能力方面具有干细胞生物学特性,经流式细胞分析鉴定为兔骨髓间充质干细胞。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Notch信号介导共培养脐带间充质干细胞和造血干细胞的相互作用

目的:探讨脐带间充质干细胞(UC-MSC)体外与造血干细胞共培养后Notch信号分子的改变。方法:通过胶原酶消化方法分离UC-MSC,通过流式细胞仪检测以及成脂、成骨和成软骨诱导鉴定UC-MSC具备间充质干细胞的特性。进而,将UC-MSC与脐血CD34+造血干细胞(HSC)体外培养,实时PCR方法检测MSC及CD34+细胞表面Notch配体及受体表达以及表达是否存在变化;在共培养体系中加入Notch信号阻滞剂DAPT(γ-secretase抑制剂),比较Hes-1基因活化状态的改变。结果:体外实验显示:UC-MSC在形态学、细胞表面表型和诱导分化能力上均具备间充质干细胞的特性。UC-MSC及CD34+细胞表面存在Notch信号配体及受体的表达,共培养后Jagged 1、Notch1基因表达明显增加;共培养后CD34+细胞中的Hes-1基因表达明显增加而加入DAPT后Hes-1基因表达未检出明显改变。结论:UC-MSC支持造血中,Notch信号可能发挥重要作用。     

脐血单个核细胞与骨髓间充质干细胞滋养层的共培养

背景：骨髓间充质干细胞具有维持骨髓正常造血功能,在造血调控中发挥重要的作用。 目的：观察骨髓间充质干细胞的生物学性状和多向分化能力,并检测其在体外支持造血的能力。 方法：利用密度梯度培养法分离骨髓单个核细胞进行培养,流式细胞仪检测骨髓间充质干细胞表型;接种脐血单个核细胞于骨髓间充质干细胞滋养层培养板上共培养,观察粒-单系祖细胞集落变化。 结果与结论：骨髓间充质干细胞呈典型的成纤维样细胞形态,强表达CD44,CD29,不表达CD34和CD106。可促进脐血单个核细胞扩增并形成造血祖细胞集落。提示骨髓间充质干细具有造血支持作用。     

小鼠毛囊来源间充质干细胞体外诱导成软骨的细胞标记物

背景：小鼠毛囊来源的间充质干细胞有很强的多向诱导分化能力,但少有研究关注其在诱导分化为软骨细胞过程中的细胞表面标记物。 目的：观察小鼠毛囊来源的间充质干细胞分化为软骨细胞过程中的形态、细胞表面标记物和硫酸黏多糖含量的变化。 方法：体外分离培养ICR新生小鼠皮肤干细胞,用成软骨细胞诱导液诱导培养7,14 d观察各项指标。 结果与结论：经成软骨细胞诱导液处理后,小鼠毛囊来源的间充质干细胞中CD44(+)细胞数量无明显增加, 硫酸黏多糖含量无变化,CD54(+)细胞和CD166(+)细胞则显著增加,两者硫酸黏多糖含量亦明显升高。表明小鼠毛囊来源的间充质干细胞能诱导形成软骨样细胞;CD44不能作为小鼠毛囊来源的间充质干细胞向软骨细胞分化的细胞表面标记物;CD54和CD166以及硫酸黏多糖可作为对小鼠毛囊来源的间充质干细胞向软骨细胞分化的监测指标。     

从骨髓和脐血造血干细胞中诱导成骨样细胞

骨髓有2个重要的功能:造血和成骨。骨髓中存在造血干细胞(hem atopoietic stem cells,HSCs)(CD34 细胞)和间充质干细胞(m esenchym al stem cells,MSCs)。这2种干细胞共同存在于骨髓腔中,MSC对HSC不仅有空间位置的机械支持作用,还分泌多种造血因子支持其造血功能,有助于HSC未分     

致敏小鼠骨髓间充质干细胞体外培养及多向分化功能的测定

目的：评价异基因脾细胞输注致敏的小鼠骨髓源性间充质干细胞(MSCs)的体外培养生长能力及其多向分化功能。方法：应用贴壁培养法体外培养间充质干细胞,流式检测其表面标志以及检测其成骨、成脂和成肌多向分化状况;结果：致敏小鼠骨髓源性MSCs与非致敏小鼠骨髓源性MSCs比较,形态学无差异且均表达CD29+、CD105+、CD44+和Sca-1+ ;CD34-、CD11b-;同时在相应的诱导条件下具有向成骨、成脂、成肌多向分化的能力。结论：异基因脾细胞输注致敏的小鼠,其骨髓源性MSCs的形态学和功能与正常小鼠的MSCs比较评估未见异常。     

脐血间充质干细胞生物学特性及其成骨成脂分化潜能

背景:对于脐血来源间充质干细胞的研究是干细胞研究领域的重点和热点,但目前国内外研究对其报道尚较少,许多方面存在争议。目的:观察人脐血间充质干细胞的生物学特性,并探讨其向成骨、成脂肪细胞诱导分化的能力。方法:从不同胎龄脐血中分离培养间充质干细胞;通过倒置相差显微镜及透射电镜观察细胞的形态、生长增殖情况及其超微结构;并描绘细胞生长曲线;用流式细胞仪对细胞表面标志物进行检测。分别用成骨及成脂诱导液对脐血间充质干细胞进行诱导,通过茜素红染色检测脐血间充质干细胞向成骨细胞诱导分化的能力;通过油红O染色检测其向脂肪细胞诱导分化的能力。结果与结论:倒置相差显微镜下观察脐血间充质干细胞贴壁生长,呈成纤维细胞样外观,细胞呈螺旋状排列;透射电镜观察脐血间充质干细胞细胞核大,胞核比例大,细胞器少,为低分化细胞;原代及传代培养的脐血间充质干细胞生长曲线均呈S型,第3,5代细胞增殖能力最强。流式细胞仪检测结果显示,脐血间充质干细胞均稳定表达与间充质干细胞相关的表面抗原标志物CD29,CD44和CD90等,不表达造血标志CD34和CD45。脐血间充质干细胞成骨诱导后3周时茜素红染色可检测到大量钙化基质的形成;成脂诱导3周时油红O染色可检测到胞质中脂滴的形成。提示脐血间充质干细胞的形态特征、生长增殖特点及细胞表面标志物等生物学特性与骨髓间充质干细胞相似,具有强大的生长增殖与自我更新能力。脐血间充质干细胞在体外诱导条件下可以向成骨细胞及脂肪细胞等间质组织细胞定向分化。     

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。     

凝血因子Ⅸ基因敲除对小鼠骨髓间充质干细胞生物学特性无直接影响

背景：凝血因子Ⅸ基因敲除有可能通过影响造血系统的稳定性从而影响骨髓造血微环境中骨髓间充质干细胞的生物学特性。 目的：鉴定凝血因子Ⅸ基因敲除C57BL/6J-F9 tm1. Dws小鼠骨髓间充质干细胞的生物学特征。 方法：采用改进的全骨髓贴壁细胞分离法分离培养凝血因子Ⅸ基因敲除C57BL/6J-F9 tm1. Dws小鼠骨髓间充质干细胞,流式细胞术鉴定其表面标记的表达,以特殊诱导培养基诱导骨髓间充质干细胞干向成骨细胞,脂肪细胞,软骨细胞方向分化。 结果与结论：分离的骨髓间充质干细胞形态均一为梭形,增殖能力和自我更新能力强。流式细胞术检测细胞表面标识CD44、CD56、CD73、CD90、CD105、CD106、CD166及CD271表达呈阳性,CD34、CD11b表达呈阴性。分离的骨髓间充质干细胞具有向成骨细胞,脂肪细胞,软骨细胞方向分化的能力。说明凝血因子Ⅸ基因敲除C57BL/6J-F9 tm1. Dws小鼠的骨髓间充质干细胞仍具备良好的干细胞的生物学特性,初步确定,凝血因子Ⅸ基因敲除对骨髓间充质干细胞的生物学特性并无直接影响。     

Immunology of stem cells and cancer stem cells

The capacity of pluri-potent stem cells to repair the tissues in which stem cells reside holds great promise in development of novel cell replacement therapeutics for treating chronic and degenerative diseases. However, numerous reports show that stem cell therapy, even in an autologous setting, triggers lymphocyte infiltration and inflammation. Therefore, an important question to be answered is how the host immune system responds to engrafted autologous stem cells or allogeneous stem cells. In this brief review, we summarize the progress in several related areas in this field, including some of our data, in four sections： （1） immunogenicity of stem cells; （2） strategies to inhibit immune rejection to allograft stem cells; （3） immune responses to cancer stem cells; and （4） mesenchymal stem cells in immune regulation. Improvement of our understanding on these and other aspects of immune system-stem cell interplay would greatly facilitate the development of stem cell-based therapeutics for regenerative purposes. Cellular ＆ Molecular Immunology.     

人诱导性多能干细胞定向分化为神经干细胞的实验研究

目的 观察人诱导性多能干细胞（iPS细胞）定向分化为神经干细胞（NSCs）的潜能。方法 用维甲酸（RA）诱导人iPS细胞向NSCs分化。倒置显微镜下观察iPS细胞的形态变化,RT-PCR检测NANOG, OCT4, SOX2的表达;免疫组织化学方法检测NESTIN、SOX2、β-TUBULIN Ш和GFAP的表达。结果RA诱导后第4天,贴壁的拟胚体出现了早期神经祖细胞特有的神经管样结构并不断增多,细胞表达神经巢蛋白NESTIN,而对照组未观察到神经管样结构。神经管样结构内的细胞能分化为β-TUBULIN Ш阳性的神经元,但GFAP阳性的星形胶质细胞少见。结论 人iPS细胞具有定向分化为NSCs的潜能,并能模拟神经发育过程。     

Mesenchymal-like stem cells derived from human parthenogenetic embryonic stem cells

Human parthenogenetic embryonic stem cells (hpESCs) established from artificially activated oocytes have a wider immune-matching ability because of their homozygosity in the major histocompatibility complex alleles. Whether these cells possess the differentiation capacity similar to regular human embryonic stem cells (hESCs) derived from fertilized eggs is unclear. The aims of this study were to determine whether hpESCs could be differentiated into multipotent mesenchymal stem cell (MSC)-like cells in vitro and then compare these cells with those derived from hESCs. MSC-like cells were obtained from both hpESCs and hESCs, which exhibited similar cell surface marker expression profiles. Further analyses revealed that cells derived from hpESCs possessed stronger osteogenic but weaker adipogenic potentials compared with cells derived from hESCs. This is the first work that demonstrates the differentiation of hpESCs into multipotent MSC-like cells. These hpESCs could be a potential source for cell-based therapies.     

表皮干细胞与毛囊干细胞生物学特性的比较

背景：获取更合适的组织工程皮肤种子细胞可使皮肤功能得到更好的修复。 目的：分离、培养表皮干细胞和毛囊干细胞,并比较两种细胞的生物学特性。 方法：体外分离培养2月龄新西兰兔表皮干细胞和毛囊干细胞,取生长良好的第2,3,6代细胞观察其生物学特性。 结果与结论：毛囊干细胞较表皮干细胞贴壁快,具有更高的增殖活性。免疫组化及荧光定量PCR检测显示毛囊干细胞β1整合素、角蛋白19蛋白及mRNA表达均明显高于表皮干细胞。提示作为皮肤组织工程的种子细胞,毛囊干细胞较表皮干细胞更具优势。     

Muscle stem cells

Since its discovery four decades ago, the satellite cell of skeletal muscle has been implicated as the major source of myogenic cells involved in growth and repair of muscle fibres. This review not only looks at the role of the satellite cell in these processes but discusses how cells derived from other sources and tissues have recently been implicated in muscle formation and regeneration. Muscle itself also yields cells that contribute to other cell lineages although it is currently debated as to whether these cells originate within muscle or have migrated there from other tissues. The reality of using cells from muscle or other tissues to repair diseased muscle fibres is also addressed.     

Gastrointestinal stem cells

Turnover of the epithelial cell lineages within the gastrointestinal tract is a constant process, occurring every 2-7 days under normal homeostasis and increasing after damage. This process is regulated by multipotent stem cells, which give rise to all gastrointestinal epithelial cell lineages and can regenerate whole intestinal crypts and gastric glands. The stem cells of the gastrointestinal tract are as yet undefined, although it is generally agreed that they are located within a 'niche' in the intestinal crypts and gastric glands. Studies of allophenic tetraparental chimeric mice and targeted stem cell mutations suggest that a single stem cell undergoes asymmetrical division to produce an identical daughter cell, and thus replicate itself, and a committed progenitor cell which further differentiates into an adult epithelial cell type. The discovery of stem cell plasticity in many tissues, including the ability of transplanted bone marrow to transdifferentiate into intestinal subepithelial myofibroblasts, provides a potential use of bone marrow cells to deliver therapeutic genes to damaged tissues, for example, in treatment of mesenchymal diseases in the gastrointestinal tract, such as fibrosis and Crohn's disease. Studies are beginning to identify the molecular pathways that regulate stem cell proliferation and differentiation into adult gastrointestinal cell lineages, such as the Wnt and Notch/Delta signalling pathways, and the importance of mesenchymal-epithelial interactions in normal gastrointestinal epithelium and in development and disease.     

Hepatic stem cells

The liver in an adult healthy body maintains a balance between cell gain and cell loss. Though normally proliferatively quiescent, hepatocyte loss such as that caused by partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and 'wear and tear' renewal is largely achieved by hepatocyte self-replication. Furthermore, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes can undergo significant clonal expansion, suggesting that hepatocytes themselves are the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential so-called oval cells within the lobules that can differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells can contribute to the albeit low renewal rate of hepatocytes, make a more significant contribution to regeneration, and even completely restore normal function in a murine model of hereditary tyrosinaemia. How these three stem cell populations integrate together to achieve a homeostatic balance is not known. This review focuses on two major aspects of liver stem cell biology: firstly, the identity of the liver stem cells, and secondly, their potential value in the treatment of major liver disease.