脐带间充质干细胞对脐血CD34+细胞体外扩增的影响

背景：体外扩增是目前解决脐带血干细胞移植所面临的单份脐血造血干祖细胞数不足问题的主要手段。已有许多关于不同来源的间充质干细胞联合不同细胞因子支持脐血造血干祖细胞体外扩增的报道,而单独应用间充质干细胞对人脐血CD34+细胞体外扩增的报道仍很少。 目的：观察应用脐带源间充质干细胞作基质层的体外培养体系对脐血CD34+细胞体外扩增的支持作用。 设计、时间及地点：对比观察实验,于2006-03/2007-05在中国医学科学院中国协和医科大学血液学研究所、实验血液学国家重点实验室完成。 材料：经产妇知情同意,采集健康足月分娩胎儿脐带9份。 方法：应用贴壁培养的方法从正常足月出生儿脐带中分离培养间充质干细胞,通过细胞形态学、免疫表型、分化实验进行鉴定。利用免疫磁珠分离法从正常足月出生儿脐带血中分离CD34+细胞。应用3种不同培养体系进行脐血CD34+细胞的体外扩增：单独应用脐带源间充质干细胞作基质层、细胞因子培养体系和间充质干细胞联合细胞因子培养体系。 主要观察指标：应用细胞计数法、集落培养计数法和流式细胞学检测法对扩增后细胞数量、集落形成能力和免疫表型进行分析比较。RT-PCR检测间充质干细胞中细胞因子的表达情况。 结果：培养第14天后,间充质干细胞组的CD34+细胞比例明显高于细胞因子联合间充质干细胞组;CD34+细胞总数为培养前的(4.19±1.37)倍,明显高于细胞因子组。培养第7天和第14天,间充质干细胞组的CD34+CD38-细胞亚群比例均高于另两组。RT-PCR结果显示,脐带源间充质干细胞体外可表达干细胞因子和血小板生成素早期干细胞效应因子。 结论：单独应用脐带源间充质干细胞可有效地对脐血CD34+细胞进行体外扩增,更有利于保持较早期干、祖细胞的扩增。 关键词：间充质干细胞;脐带;脐血;CD34+细胞;体外扩增     

人脐血间充质干细胞抗缺氧诱导心肌细胞凋亡的作用☆

背景：抗细胞凋亡成为心力衰竭生物治疗的一个新方向。近年研究发现成体间充质干细胞在一定条件下可转化成心肌样细胞,亦可通过分泌多种细胞因子,促进心脏血管形成和减少细胞凋亡。 目的：观察人脐血间充质干细胞对缺氧诱人心肌细胞凋亡的保护作用。 方法：将人心肌细胞细胞复苏后,接种在6孔细胞培养板(对照组)和Transwell 3412培养板中;将人脐血间充质干细胞接种在Transwell插件的可渗透性滤膜上;将上述2组细胞置于体积分数95%N2+体积分数5%CO2缺氧环境下缺氧培2,4,12,24 h;检测各组心肌细胞的凋亡率。ELLES法检测细胞条件培养基内胰岛素样生长因子1的浓度。 结果与结论：第3代后人脐血间充质干细胞及原代心肌细胞均有胰岛素样生长因子分泌,但人脐血间充质干细胞条件培养基内胰岛素样生长因子1的浓度明显高于人心肌细胞。缺氧可以诱导心肌细胞凋亡,在短期和持续性缺氧的条件下,人脐血间充质干细胞对缺氧诱导的心肌细胞凋亡有保护作用,且人脐血间充质干细胞组心肌细胞凋亡率低于对照组( P < 0.05)。提示人脐血间充质干细胞对缺氧诱导的人心肌细胞凋亡有保护作用,这种保护作用可能与通过细胞间直接接触和旁分泌细胞因子有关。     

Umbilical cord blood stem cell therapy in premature brain injury: Opportunities and challenges

Preterm birth and associated brain injury are the primary cause of cerebral palsy and developmental disabilities and are among the most serious global health issues that modern society faces. Current therapy for infants suffering from premature brain injury is still mainly supportive, and there are no effective treatments. Thus there is a pressing need for comparative and translational studies on how to reduce brain injury and to increase regeneration and brain repair in preterm infants. There is strong supporting evidence for the use of umbilical cord blood (UCB)-derived stem cell therapy for treating preterm brain injury and neurological sequelae. UCB-derived stem cell therapy is effective in many animal models and has been shown to be feasible in clinical trials. Most of these therapies are still experimental, however. In this review, we focus on recent advances on the efficacy of UCB-derived stem cell therapy in preterm infants with brain injury, and discuss the potential mechanisms behind their therapeutic effects as well as application strategies for future preclinical and clinical trials.     

微囊微环境体外扩增人脐血造血干/祖细胞

背景：由于单份脐血所含的造血细胞数量有限,目前只能用于儿童或低体质量成人血液病和急性辐射损伤等疾病患者,有效扩增脐血造血干/祖细胞已成为研究热点。 目的：观察微囊微环境对脐血造血干/祖细胞扩增的影响,以及此过程中可否使造血干/祖细胞在扩增的同时仍维持其未分化状态。 设计、时间及地点：单一样本观察,于2006-06/2007-09在中国科学院大连化学物理研究所完成。 材料：正常足月产新生儿脐带血由大连市妇产医院提供,提供者知情同意。 方法：Ficoll法梯度分离人脐血单个核细胞,采用静电液滴法在生理条件下进行微囊化包封和体外培养。以同条件平面培养的脐血单个核细胞作为对照。 主要观察指标：观察微囊化脐血细胞的生长特点及脐血细胞总数的变化;流式细胞仪检测培养过程中CD34+细胞扩增情况;应用甲基纤维素半固体培养法观察扩增细胞的集落形成能力。 结果：脐血细胞在微胶囊内持续增殖,并以聚集成团的三维方式生长,两种培养方式对脐血细胞总数均无明显影响(P > 0.05)。对比CD34+细胞扩增和细胞集落的生成发现,微囊化培养脐血细胞的CD34+细胞数量和集落密度均在培养第6天达到高峰,明显高于平面培养3 d时所达到的峰值;之后逐渐下降,至12 d后平面培养细胞的CD34+细胞和集落形成能力几乎检测不到,而此时微囊化培养的CD34+细胞数量和集落密度仍与平面培养的扩增高峰相近。 结论：微囊化培养能有效扩增人脐血造血干/祖细胞,并显著减缓造血干/祖细胞的分化进程,维持其多分化潜能,提示微囊为造血干/祖细胞维持未分化状态的扩增提供了特殊的微环境。     

骨髓基质细胞联合细胞因子对脐血单个核细胞表面抗原表达的影响**

背景：课题组已建立胎儿骨髓基质细胞联合细胞因子的造血细胞体外培养体系,该培养体系能否有效扩增各个发育阶段的造血细胞有待验证。 目的：观察骨髓基质细胞联合细胞因子培养体系对脐血单个核细胞表面抗原CD133、CD34表达的影响。 方法：将从脐血标本中分离出来的单个核细胞接种于无血清培养体系,实验分为3组：①F组：干细胞因子+Flt3配体+促血小板生成素+单个核细胞。②S组：基质细胞+单个核细胞。③SF组：基质细胞+干细胞因子+Flt3配体+促血小板生成素+单个核细胞。在第0,6,10,14天检测有核细胞总数、CD133+、CD34+、CD133+CD34+细胞数以及集落形成单位数。 结果与结论：SF组有核细胞总数在各个检测时间点均比其他两组高;除了第14天外,第6、10天两个时间点SF组中CD133+、CD34+、CD133+CD34+细胞及集落形成单位数均高于其他组;含骨髓基质细胞的S组和SF组中CD133+细胞/有核细胞、CD34+细胞/有核细胞、CD133+CD34+细胞/有核细胞的比例保持在较高的水平。结果说明骨髓基质细胞联合细胞因子能有效的扩增脐血单个核细胞及其中的CD133+、CD34+、CD133+CD34+细胞,基质细胞对维持造血干细胞的原始性具有重要的作用。     

人脐血间充质干细胞向成心肌细胞诱导扩增后的生物安全性评价

背景：人脐血间充质干细胞在体外分离培养并向心肌细胞诱导分化的条件下，能否仍然维持原有的正常生理性状态，即是否达到种子细胞的生物安全性标准，目前尚少见研究。 目的：分析人脐血间充质干细胞在体外分离培养及向心肌细胞诱导分化的条件下，其染色体核型及端粒酶活性是否发生异常的改变及细胞是否产生致肿瘤性。 方法：采用染色体G显带处理方法体外分离、培养的第3代以后的人脐血间充质干细胞和向心肌细胞诱导培养至第7代后的样本进行核型分析，分析细胞的端粒酶活性，细胞周期相关基因cyclinA、cdk2、C-fos、h-TERT、c-myc和p53的表达，并通过裸鼠皮下致肿瘤试验对细胞的致肿瘤性进行分析。 结果与结论：人脐血间充质干细胞和向心肌细胞诱导体外培养至第7代，未发现染色体核型异常改变，相应的端粒酶活性也未出现异常增高。c-myc、p53、h-TERT、C-fos无表达。致肿瘤试验，实验裸鼠在观察期内均未见结节形成或可疑病灶产生，符合国家医疗产品生物学评价标准的要求。     

Transplantation of mesenchymal stem cells from human umbilical cord versus human umbilical cord blood for peripheral nerve regeneration

BACKGROUND: Mesenchymal stem cells (MSCs) appear to be a good alternative to Schwann cells in the treatment of peripheral nerve injury. Fetal stem cells, like umbilical cord blood (UCB) and umbilical cord (UC) stem cells, have several advantages over adult stem cells.OBJECTIVE: To assess the effects of UC-derived MSCs (UCMSCs) and UCB-derived MSCs (UCBMSCs) in repair of sciatic nerve defects. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the laboratory of Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, from July to December 2009. MATERIALS: UCMSCs were provided by the Research Institute of Biotechnology, Dongguk University. UCBMSCs were provided by the Laboratory of Stem Cells and Tumor Biology, College of Veterinary Medicine, Seoul National University. Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco-BRL, USA. METHODS: Seven-week-old Sprague-Dawley rats were randomly and evenly divided into three groups: DMEM, UCBMSCs, and UCMSCs. A 10-mm defect in the left sciatic nerve was constructed in all rats. DMEM (15 μL) containing 1 × 106 UCBMSCs or UCMSCs was injected into the gap between nerve stumps, with the surrounding epineurium as a natural conduit. For the DMEM group, simple DMEM was injected. MAIN OUTCOME MEASURES: At 7 weeks after sciatic nerve dissection, dorsal root ganglia neurons were labeled by fluorogold retrograde labeling. At 8 weeks, electrophysiology and histomorphometry were performed. At 2, 4, 6, and 8 weeks after surgery, sciatic nerve function was evaluated using gait analysis.RESULTS: The UCBMSCs group and the UCMSCs group exhibited similar sciatic nerve function and electrophysiological indices, which were better than the DMEM group, as measured by gait analysis (P < 0.05). Fluorogold retrograde labeling of sciatic nerve revealed that the UCBMSCs group demonstrated a higher number of labeled neurons; however, the differences were not significant. Histomorphometric indices were similar in the UCBMSCs and UCMSCs groups, and total axon counts, particularly axon density (P < 0.05), were significantly greater in the UCBMSCs and UCMSCs groups than in the DMEM group. CONCLUSION: Transplanting either UCBMSCs or UCMSCs into axotomized sciatic nerves could accelerate and promote sciatic nerve regeneration over 8 weeks. Both treatments had similar effects on nerve regeneration.     

In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133

The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133(+)), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we showed that both subfractions CD133(+)/SSEA4(+) and CD133(+)/CD15(+) isolated from the embryonic forebrain are enriched in neurosphere-initiating cells. In addition CD133, SSEA4, and CD15 expression is sustained in the expanded neurosphere cells and also mark subfractions of neurosphere-initiating cells. Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain.     

MCF7细胞中肿瘤干细胞的增殖特征

背景：肿瘤干细胞学说认为，肿瘤干细胞是肿瘤不断增殖的根源，并与肿瘤的浸润转移、耐药现象密切相关。 目的：在单细胞水平研究体外传代培养的MCF7细胞中肿瘤干细胞含量变化规律，认识肿瘤干细胞在体外培养环境下的增殖特点。 方法：利用单细胞分离种植-成瘤性克隆方法对传代培养后不同时间点MCF7细胞中肿瘤干细胞的比例连续检测，流式细胞仪同步检测相应细胞样本中CD44+CD24-/low亚群的含量变化。 结果与结论：传代后培养的MCF7细胞在不同时间点其肿瘤干细胞比例及CD44+/CD24-/low亚群比例均呈现有规律的变化。但CD44+/CD24-/low亚群变化更为显著(36.84%到81.95%)，而肿瘤干细胞含量仅在小范围波动(38.54%~47.39%)。结果提示传代培养的MCF7细胞中，肿瘤干细胞具有通过调节自身增殖来保持其比例相对稳定的特点；CD44+/CD24-/low亚群并不代表或者富集MCF7细胞株中的肿瘤干细胞亚群。     

Unconjugated bilirubin activates and damages microglia

Microglia are the resident immune cells of the brain and are the principal source of cytokines produced during central nervous system inflammation. We have previously shown that increased levels of unconjugated bilirubin (UCB), which can be detrimental to the central nervous system during neonatal life, induce the secretion of inflammatory cytokines and glutamate by astrocytes. Nevertheless, the effect of UCB on microglia has never been investigated. Hence, the main goal of the present study was to evaluate whether UCB leads to microglial activation and to the release of the cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6. Additionally, we investigated the effects of UCB on glutamate efflux and cell death. The results showed that UCB induces morphological changes characteristic of activated microglia and the release of high levels of TNF-alpha, IL-1beta, and IL-6 in a concentration-dependent manner. In addition, UCB triggered extracellular accumulation of glutamate and an increased cell death by apoptosis and necrosis. These results demonstrate, for the first time, that UCB is toxic to microglial cells and point to microglia as an important target of UCB in the central nervous system. Moreover, they suggest that UCB-induced cytokine production, by mediating cell injury, can further contribute to exacerbate neurototoxicity. Interestingly, microglia cells are much more responsive to UCB than astrocytes. Collectively, these data indicate that microglia may play an important role in the pathogenesis of encephalopathy during severe hyperbilirubinemia.     

人羊膜上皮细胞的干细胞特性***☆

背景：研究发现人羊膜上皮细胞具有类似胚胎干细胞或多能干细胞的多向分化潜能,说明其可能是未来组织工程重建的一种新型种子细胞。 目的：研究体外培养的人羊膜上皮细胞的干细胞特性。 方法：取足月剖宫产的人羊膜组织,经酶消化法和差异黏附法获得纯度高的人羊膜上皮细胞,接种于含体积分数为10%胎牛血清的DMEM/F12培养基中进行原代和传代培养,用免疫荧光法和流式细胞仪检测法检测人羊膜上皮细胞表面胚胎干细胞的表面标记蛋白OCT-4和干细胞表面分子标记CD29、CD34、CD44、CD45、CD105的表达。 结果与结论：人羊膜上皮细胞在体外培养条件下呈上皮细胞特有的铺路石样外观,其胞浆中有OCT-4免疫荧光表达,其干细胞标记分子CD29、CD34的表达是阳性,但干细胞标记分子CD44、CD45、CD105的表达为阴性。结果表明人羊膜上皮细胞具有干细胞的某些特性,其可能是未来组织工程重建的一种新型种子细胞。     

Elevated levels of bilirubin and long-term exposure impair human brain microvascular endothelial cell integrity

The pathogenesis of encephalopathy by unconjugated bilirubin (UCB) seems to involve the passage of high levels of the pigment across the blood-brain barrier (BBB) and the consequent damage of neuronal cells. However, it remains to be clarified if and how the disruption of BBB occurs by UCB. We used confluent monolayers of human brain microvascular endothelial cells (HBMEC) to explore the sequence of events produced by UCB. A cell line and primary cultures of HBMEC were exposed to 50 or 100 μM UCB, in the presence of 100 μM human serum albumin, to mimic moderate and severe jaundice, for 1-72 h. UCB caused loss of cell viability in a concentration-dependent manner. UCB inhibited the secretion of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor at early time points, but enhanced their secretion at later time points. Upregulation of mRNA expression, particularly by 100 μM UCB, preceded cytokine secretion. Other early events include the disruption of glutathione homeostasis and the increase in endothelial nitric oxide synthase expression followed by nitrite production. Prolonged exposure to UCB upregulated the expression of β-catenin and caveolin-1. In conclusion, elevated concentrations of UCB affect the integrity of HBMEC monolayers mediated by oxidative stress and cytokine release. UCB also induced increased expression of caveolin-1, which has been associated with BBB breakdown, and β-catenin, probably as an attempt to circumvent that impairment. These findings provide a basis for target-directed therapy against brain endothelial injury caused by UCB.     

Bilirubin-induced inflammatory response, glutamate release, and cell death in rat cortical astrocytes are enhanced in younger cells

Unconjugated bilirubin (UCB) encephalopathy is a predominantly early life condition resulting from the impairment of several cellular functions in the brain of severely jaundiced infants. However, only few data exist on the age-dependent effects of UCB and their association with increased vulnerability of premature newborns, particularly in a sepsis condition. We investigated cell death, glutamate efflux, and inflammatory cytokine dynamics after exposure of astrocytes at different stages of differentiation to clinically relevant concentrations of UCB and/or lipopolysaccharide (LPS). Younger astrocytes were more prone to UCB-induced cell death, glutamate efflux, and inflammatory response than older ones. Furthermore, in immature cells, LPS exacerbated UCB effects, such as cell death by necrosis. These findings provide a basis for the increased susceptibility of premature newborns to UCB deleterious effects, namely when associated with sepsis, and underline how crucial the course of cell maturation can be to UCB encephalopathy during moderate to severe neonatal jaundice.     

Unconjugated bilirubin differentially affects the redox status of neuronal and astroglial cells

We investigated whether nerve cell damage by unconjugated bilirubin (UCB) is mediated by oxidative stress and ascertained the neuronal and astroglial susceptibility to injury. Several oxidative stress biomarkers and cell death were determined following incubation of neurons and astrocytes isolated from rat cortical cerebrum with UCB (0.01-1.0 microM). We show that UCB induces a dose-dependent increase in neuronal death in parallel with the oxidation of cell components and a decrease in the intracellular glutathione content. Comparison of the results obtained in both cell types demonstrates that neurons are more vulnerable than astrocytes to oxidative injury by UCB, for which accounts the lower glutathione stores in neuronal cells. Moreover, neuronal oxidative injury is prevented by supplementation with N-acetylcysteine, a glutathione precursor, whereas astroglial sensitivity to UCB is enhanced by inhibition of glutathione synthesis, using buthionine sulfoximine. Collectively, we demonstrate that oxidative stress is involved in UCB neurotoxicity and depict a new therapeutic approach for UCB-induced oxidative damage.     

Apoptosis and impairment of neurite network by short exposure of immature rat cortical neurons to unconjugated bilirubin increase with cell differentiation and are additionally enhanced by an inflammatory stimulus

Nerve cell injury induced by unconjugated bilirubin (UCB) has been implicated in brain damage during severe neonatal hyperbilirubinemia, although the molecular mechanisms underlying UCB neurotoxicity are still not clarified. It has been suggested recently that there is an association between hyperbilirubinemia and long-term neurologic dysfunctions. We incubated immature neurons with UCB to evaluate the short- and long-term effects of UCB on apoptotic death and on neuritic outgrowth and ramification. We also evaluated whether mature neurons, exposed previously to UCB in an early stage of differentiation, are more sensitive to apoptosis or to neuritic breakdown when treated with inflammatory agents, such as lipopolysaccharide and tumor necrosis factor-alpha. Results show that exposure of immature neurons to UCB increased apoptosis and provoked a reduction of both neurite extension and number of nodes. These injurious effects observed in immature cells treated with UCB were increasingly perpetuated along cell differentiation, as compared to neurons incubated in the absence of UCB. In addition, neurons that were exposed to UCB when immature showed an increased susceptibility to death by apoptosis, as well as an additional decrease in neurite outgrowth when incubated with an inflammatory agent afterward. This work shows, for the first time, that UCB induces neurite changes consistent with neurodevelopment abnormalities. Furthermore, pre-exposure to UCB followed by an inflammatory stimulus leads to an enhanced susceptibility to long-term apoptosis, as well as a greater neuritic breakdown. These data support the association between neonatal hyperbilirubinemia and the later development of mental illness, such as schizophrenia.     

人脑胶质瘤干细胞初步研究

目的从人脑胶质瘤体外细胞系和胶质瘤组织中分离、鉴定肿瘤干细胞,为进一步研究其生物学特性奠定基础。方法将人胶质瘤SHG44细胞和手术标本制成的单细胞,分别用含血清培养基(DMEM 10% FBS)和无血清培养基(DMEM/F12,添加bFGF、LIF和EGF)培养。用CD133免疫磁珠筛选,流式细胞仪和免疫荧光共聚焦显微镜检测干细胞、祖细胞和分化细胞特异性标志物。结果SHG44细胞培养1周,用CD133磁珠分离得到的CD133~ 细胞的比例:血清组为0.021%,无血清组为1.2%。流式细胞仪检测:(1)Hoechst 33342~-细胞比例:血清组为1.5%,无血清组为16.4%;(2)nestin~ 细胞比例:血清组为7.2%,无血清组为51.05%;(3)免疫磁珠分离的CD133~ 细胞再用流式细胞仪测得的CD133~ 细胞为83.02%,CD133~-细胞群中有3.32%的CD133~ 细胞。标本源肿瘤细胞在无血清条件下培养两天后CD133磁珠分离CD133~ 细胞比例为4%。CD133~ 细胞在分化不同阶段共表达或分别表达祖细胞标志物nestin、神经元和胶质细胞特异性标志蛋白MAP2和GFAP。结论在胶质瘤细胞系和胶质瘤组织中均存在CD133~ 的脑肿瘤干细胞,具有自我更新和多向分化潜能、在无血清培养下细胞球体中CD133~ 细胞仍占少数,而nestin~ 细胞占多数,可作为进一步研究脑肿瘤干细胞生物学特性的实验材料在相关领域中的应用。     

干细胞相关因子在大肠癌组织及SW620细胞株的表达

背景：肿瘤细胞有多种干细胞标记的表达,深入研究其表达规律有助于揭示肿瘤发病机制、完善肿瘤干细胞理论。 目的：检测大肠癌实体组织中干细胞相关分子标记CD133、CD166、Oct4、Sox2、C-myc、Klf4、Bmi-1与癌旁组织的表达差异,以及CD133免疫磁珠分选阳性与阴性SW620细胞中上述基因的表达差异。 方法：选临床病理诊断清楚的29例大肠癌组织标本提取RNA,RT-PCR检测CD133、CD166、Oct4、Sox2、C-myc、Klf4、Bmi-1在大肠癌组织与癌旁对照组织中的表达。CD133免疫磁珠分选SW620,提取CD133细胞与CD133阴性细胞RNA,RT-PCR检测分选后上述基因的表达差异。 结果与结论：29例标本均诊断为低、中分化腺癌或黏液腺癌,干细胞相关分子标记癌组织与癌旁组织表达灰度相对值癌组织表达均高于癌旁组织(P < 0.05)。RT-PCR检测分选后CD133+细胞CD133,CD166,Oct4,Sox2,C-myc,Klf4,Bmi-1表达,结果均高于CD133-细胞。提示,CD133、CD166、Oct4、Sox2、C-myc、Klf4、Bmi-1相关干细胞标记可作为大肠癌肿瘤干细胞标记并有望用于诊断检测。