脐带间充质干细胞与许旺细胞共移植修复大鼠坐骨神经缺损

Previous research has demonstrated that cotransplantation of umbilical cord mesenchymal stem cells (UCMSCs) and Schwann cells (SCs) can repair spinal nerve injury, but few studies have investigated their use in peripheral nerve regeneration. In the present study, we cotransplanted UCMSCs and SCs to repair 5-mm left sciatic nerve defects in rats, and compared the effects of UCMSCs + SCs transplantation with UCMSCs or SCs transplantation alone. After UCMSCs + SCs transplantation, nerve conduction velocity of the left sciatic nerve and gait were both improved. Retrograde tracing analysis demonstrated that the mean count of fluorogold-labeled neurons, as well as the mean axon count and axon density, were significantly greater in the left sciatic nerve after UCMSCs + SCs transplantation, compared with UCMSCs or SCs transplantation alone. Improvements in conduction velocity and increased sheath thickness in the left sciatic nerve were similar after UCMSCs transplantation and UCMSCs + SCs transplantation. These findings suggest that UCMSCs transplantation can promote the repair of sciatic nerve defects to some extent, but that combined UCMSCs + SCs transplantation has a significantly greater regenerative effect.     

Transplantation of human umbilical cord blood mesenchymal stem cells to treat a rat model of traumatic brain injury

In the present study, human umbilical cord blood mesenchymal stem cells were injected into a rat model of traumatic brain injury via the tail vein. Results showed that 5-bromodeoxyuridine-labeled cells aggregated around the injury site, surviving up to 4 weeks post-transplantation. In addition, transplantation-related death did not occur, and neurological functions significantly improved. Histological detection revealed attenuated pathological injury in rat brain tissues following human umbilical cord blood mesenchymal stem cell transplantation. In addition, the number of apoptotic cells decreased. Immunohistochemistry and in situ hybridization showed increased expression of brain-derived neurotrophic factor, nerve growth factor, basic fibroblast growth factor, and vascular endothelial growth factor, along with increased microvessel density in surrounding areas of brain injury. Results demonstrated migration of transplanted human umbilical cord blood mesenchymal stem cells into the lesioned boundary zone of rats, as well as increased angiogenesis and expression of related neurotrophic factors in the lesioned boundary zone.     

Human umbilical cord blood-derived stem cells and brain-derived neurotrophic factor protect injured optic nerve：viscoelasticity characterization

The optic nerve is a viscoelastic solid-like biomaterial.Its normal stress relaxation and creep properties enable the nerve to resist constant strain and protect it from injury.We hypothesized that stress relaxation and creep properties of the optic nerve change after injury.Moreover,human brain-derived neurotrophic factor or umbilical cord blood-derived stem cells may restore these changes to normal.To validate this hypothesis,a rabbit model of optic nerve injury was established using a clamp approach.At 7 days after injury,the vitreous body received a one-time injection of 50 μg human brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood-derived stem cells.At 30 days after injury,stress relaxation and creep properties of the optic nerve that received treatment had recovered greatly,with pathological changes in the injured optic nerve also noticeably improved.These results suggest that human brain-derived neurotrophic factor or umbilical cord blood-derived stem cell intervention promotes viscoelasticity recovery of injured optic nerves,and thereby contributes to nerve recovery.     

体外诱导脐血间充质干细胞向胰岛β样细胞的分化

背景：目前,国内外对骨髓间充质干细胞、胰腺干细胞向胰岛β样细胞分化的研究报道较多,但关于脐血间充质干细胞向胰岛β样细胞分化的研究报道较少。 目的：验证脐血间充质干细胞能否在体外向胰岛β样细胞分化,并探索其诱导条件。 方法：在无菌条件下采集正常产妇脐血,用羟乙基淀粉沉淀法分离脐血中的有核细胞,进而采用贴壁筛选法获得脐血间充质干细胞。纯化后的脐血间充质干细胞用表皮生长因子、β-巯基乙醇、高糖、激活素A和肝细胞生长因子进行诱导。观察诱导后的细胞形态变化,采用胰岛素免疫荧光染色对诱导后的细胞进行鉴定,定量检测胰岛素分泌水平及其对葡萄糖刺激的反应性。 结果与结论：经过诱导后,细胞形态发生明显变化,形态变圆而且聚集成团;诱导后细胞的胰岛素免疫荧光染色为阳性;而且细胞能分泌少量胰岛素,并对糖刺激具有反应性。在体外,脐血间充质干细胞具有向胰岛β样细胞分化的潜能。     

中药单体黄芩苷体外诱导人脐血间充质干细胞向神经元样细胞的分化

背景: 抗氧化剂法及细胞因子法是体外诱导脐血间充质干细胞定向分化为神经元的2种方法,怎样选择既可广泛保护神经,又具有较强抗氧化作用的诱导剂？通过广泛筛选,应用中药干预脐血间充质干细胞的体外纯化、扩增并向神经元细胞分化。 目的：观察中药单体黄芩苷对人脐血间充质干细胞体外纯化、扩增及向神经元样细胞诱导分化的作用。 设计,时间,地点：随机对照,细胞学体外观察,于2005-02/06在南昌大学第一附属医院神经内科实验室完成。 材料：年龄为23~35岁的健康孕妇足月顺产儿的脐带血10份;黄芩苷,纯度> 95%,由中南大学湘雅医学院药剂科提供;抗氧化添加剂β-巯基乙醇为SABC公司产品。 方法：采集胎儿脐带血,肝素抗凝,分离出脐血单个核细胞,调整细胞浓度为1×109 L-1,以含体积分数为20%胎牛血清、谷氨酰胺、B27、粒巨噬细胞集落刺激因子、干细胞因子的DMEM液体培养基进行纯化和扩增。按照抗氧化添加剂的不同分为4组：黄芩苷组加入黄芩苷;空白对照组不添加抗氧化剂;β-巯基乙醇组添加β-巯基乙醇;共培养组添加黄芩苷、β-巯基乙醇。连续培养4周。 主要观察指标：①脐血冷冻保存后7,14,21,28 d CD34及CD29阳性细胞的表达。②细胞形态学观察。③流式细胞仪检测间充质干细胞表面标志。④免疫细胞化学染色观察间充质干细胞扩增培养4周后神经细胞特异性烯醇化酶、神经微管相关蛋白2和胶质纤维酸性蛋白阳性细胞的表达。 结果：①与冻存前相比,冻存后7,14,21,28 d脐血单个核细胞锥虫蓝拒染率均明显降低(P < 0.05)。②细胞形态学观察：脐血单个核细胞培养第2~3天开始贴壁生长,2周左右达高峰,3周后细胞生长达80%~90%融合,此时脐血原始间充质干细胞呈较均一的长梭形。4周后,黄芩苷组原来呈梭形的胞体一部分发生收缩,细胞边缘出现细的突起; 一部分胞体逐渐近似圆形、锥形、三角形,伪足有多个细长突起。β-巯基乙醇组、共培养组有少量细胞存在脱落、死亡现象,且随着培养时间延长呈逐渐增加的趋势。③培养4周后,空白对照组以CD45阳性细胞为主;其余3组均以CD29和CD83阳性细胞为主,其中共培养组最多,黄芩苷组次之,β-巯基乙醇组最少,组间两两比较差异具有显著性意义(P < 0.01);各组贴壁生长细胞中均未见CD34阳性细胞。④扩增培养4周后与黄芩苷组比较,空白对照组、β-巯基乙醇组神经细胞特异性烯醇化酶及神经微管相关蛋白2阳性细胞表达率均显著降低(P < 0.01)。各组胶质纤维酸性蛋白阳性细胞表达率均低于1%。 结论：100 μmol/L黄芩苷可促进脐血间充质干细胞体外扩增,且随抗氧化作用时间的延长效果显著增强,同时在一定程度上还能够诱导其向神经元样细胞方向分化。     

睫状神经营养因子和丹参注射液体外诱导肌源性干细胞向神经元样细胞的分化

背景：在干细胞异体移植时神经细胞的低存活率成为异体细胞移植失败的主要诱因。核因子κB是细胞信号传导的主要转录因子之一，参与细胞的增殖和分化过程。 目的：观察睫状神经营养因子和丹参注射液联合诱导肌源性干细胞向神经元样细胞的分化情况，以及分化过程中核因子κB的表达。 设计、时间及地点：细胞学基因水平实验，于2008-03/05在辽宁医学院附属第二医院口腔科学实验室完成。 材料：新生7 d龄SD乳鼠10只，由辽宁医学院实验动物中心提供。睫状神经营养因子为Sigma公司产品，丹参注射液购自正大青春宝药业有限公司。 方法：体外分离培养鼠肌源性干细胞，差速贴壁法和酶消化法纯化后接种于6孔板内，诱导组用含睫状神经营养因子的DMEM预诱导24 h，更换培养液，洗涤3次，再加入含丹参注射液的无血清DMEM诱导5 h；对照组使用无血清DMEM培养基处理。 主要观察指标：RT-PCR及Western blotting法检测神经丝蛋白、核因子κB抑制蛋白的表达。 结果：诱导前肌源性干细胞未见神经丝蛋白表达，诱导后的神经元样细胞神经丝蛋白呈阳性表达。凝胶电泳及Western blot检测结果显示，与诱导前比较，诱导后对照组核因子κB抑制蛋白的表达无明显变化，诱导组核因子κB抑制蛋白的表达明显下降。 结论：睫状神经营养因子和丹参注射液可联合诱导肌源性干细胞分化为神经元样细胞，分化过程中核因子κB的活化被抑制。     

人骨髓间充质干细胞向神经元样细胞诱导分化

骨髓间充质干细胞具备取材方便、对组织损伤小等优点,其低免疫原性及易于诱导机体的免疫耐受性,使得在不需要HLA配型的前提下也可以进行异体移植,从而减少免疫抑制剂的副作用。目前常采用密度梯度离心法体外分离培养人骨髓间充质干细胞,可根据生物学特性、细胞表面标记、多分化潜能、低免疫原性和免疫调节功能对其进行鉴定。骨髓间充质干细胞虽来源于中胚层,但在相应的诱导下可以向内胚层或外胚层的方向分化,一般选取传至第5代的骨髓间充质干细胞,除在培养液中加入传统的诱导剂如神经生长因子、维甲酸、脑源性生长因子、碱性成纤维生长因子、表皮生长因子外,向培养液中加入二十二碳六烯酸和花生四烯酸可诱导加速骨髓间充质干细胞向神经元样细胞分化,并促进神经元轴突的生长。但是骨髓间充质干细胞移植的安全性,尤其是致瘤性的问题仍有待进一步研究。     

预诱导与全反式维甲酸并脑源性神经营养因子体外诱导骨髓基质干细胞向神经元样细胞的分化

背景：骨髓基质干细胞可定向诱导分化为神经元样细胞,但目前培养的方法尚不统一。 目的：采用预诱导与全反式维甲酸和脑源性神经营养因子为培养体系,拟在体外诱导大鼠骨髓基质干细胞向神经细胞分化。 设计、时间及地点：细胞学体外观察,于2007-12/2008-06在新疆医科大学第一附属医院医学研究中心干细胞室完成。 材料：清洁级雄性SD大鼠2只,由新疆医科大学动物试验中心提供。 方法：采用贴壁法体外分离培养大鼠骨髓基质干细胞,经反复传代细胞逐渐纯化。取生长状态良好的第3代骨髓基质干细胞,按8×107 L-1密度接种后,改换含10 μg/L碱性成纤维细胞生长因子、20 μg/L表皮生长因子的神经干细胞培养体系进行预诱导,48 h后去除预诱导液,加入含10 μg/L脑源性神经生长因子、1 μmol/L全反式维甲酸的神经干细胞培养体系定向诱导骨髓基质干细胞分化为神经细胞。以未诱导的骨髓基质干细胞作为对照。 主要观察指标：流式细胞仪检测第3代骨髓基质干细胞表面标志的表达,诱导后免疫荧光染色和流式细胞仪检测巢蛋白阳性的表达,免疫荧光染色鉴定神经元特异性烯醇化酶和胶质纤维酸性蛋白的表达。 结果：第3代骨髓基质干细胞CD29,CD44表达率分别为97.1%,99%,CD45表达率为0.5%,CD34呈阴性表达。预诱导48 h后巢蛋白阳性率为24%,而对照组仅为4.6%。诱导48 h后,巢蛋白染色呈阳性,并可见大量神经元特异性烯醇化酶和胶质纤维酸性蛋白阳性细胞出现;对照组均呈阴性表达。 结论：神经干细胞培养体系联合全反式维甲酸与脑源性神经生长因子,可在体外高效、稳定地诱导骨髓基质干细胞转化为神经元样细胞,并进一步向神经元和神经胶质细胞方向分化。     

体外诱导人脐带间充质干细胞向胰岛β样细胞的分化

背景：体外实验中人们发现,常规诱导骨髓间充质干细胞分化的胰岛β样细胞由于种种原因限制了其进一步的应用。关于人脐带间充质干细胞是否可以成功诱导的胰岛β样细胞尚未见系统报道。 目的：进一步验证人脐带间充质干细胞向胰岛β样细胞分化的可行性。 方法：采用胶原酶消化法分离人脐带细胞进行贴壁培养;传2代后,用高浓度葡萄糖(25 mmol/L)培养液DMEM(含体积分数为10％胎牛血清)以及碱性成纤维细胞和尼克酰胺诱导脐带间充质干细胞向胰岛β样细胞分化。倒置显微镜下观察间充质干细胞诱导后的形态变化,用胰岛β细胞特异染色方法双硫腙染色鉴定诱导后细胞游离锌离子浓度;免疫细胞化学鉴定诱导后细胞内是否储存有胰岛素。 结果与结论：第2代脐带间充质干细胞经过高糖诱导后,间充质干细胞形成细胞团;并且形成了双硫腙染色阳性的细胞团;免疫细胞化学表明诱导后细胞内的细胞胰岛素染色阳性。结果表明人脐带分离出的间充质干细胞在体外可以定向诱导分化为胰岛β样细胞,这种胰岛β样细胞具有表达、储存胰岛素的功能。     

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.     

5-氮杂胞苷诱导人脐带间充质干细胞向心肌样细胞的分化☆

背景：5-氮杂胞苷能诱导人脐带间充质干细胞分化为心肌细胞。 目的：以5-氮杂胞苷诱导人脐带间充质干细胞分化为心肌细胞。 方法：采用贴壁培养法分离、纯化人脐带间充质干细胞,以5-氮杂胞苷诱导第3代人脐带间充质干细胞分化为心肌样细胞。 结果与结论：诱导前人脐带间充质干细胞呈典型的梭形;诱导后一二周细胞体积变大,三四周后相邻细胞间胞膜有接触,逐渐相连呈肌管状,细胞胞质内可见细丝样结构。诱导4周后免疫组织化学鉴定人脐带间充质干细胞cTnI表达阳性,未诱导细胞cTnI表达阴性;诱导组Nkx2.5、GATA 4 mRNA表达水平较未诱导组显著增加(P < 0.05)。提示5-氮杂胞苷可能通过调控GATA4、Nkx2.5基因的表达促进人脐带间充质干细胞分化为心肌样细胞,并促进其成熟。     

人脐血间充质干细胞的体外成骨

背景：用免疫细胞阴性筛选法可以排除造血干细胞对于间充质干细胞生长的影响,较快的获得具有表达间充质干细胞的细胞群。 目的：探讨人脐静脉血间充质干细胞向成骨样细胞分化的能力。 方法：用免疫磁珠阳性筛选法分离脐血间充质干细胞后进行培养。取P2代细胞行成骨诱导分化。 结果与结论：人脐血间充质干细胞贴壁生长,长梭形,3周长满瓶底,传代后细胞增殖迅速。细胞表型为高表达CD44+,CD29+和CD166+。在成骨诱导后碱性磷酸酶染色阳性,Von-Kossa染色提示钙化结节形成。提示脐血间充质干细胞能够在体外分化为成骨样细胞。     

CM-Dil体外标记人脐带间充质干细胞传代示踪的可行性

背景：掌握人脐带间充质干细胞的移植示踪方法是研究其生物学特性的关键。 目的：观察用CM-Dil标记人脐带间充质干细胞及在体外传代示踪的可行性。 方法：采用酶消化法体外分离培养人脐带间充质干细胞,通过流式细胞仪检测细胞免疫表型和细胞周期、体外成脂成骨诱导鉴定该细胞。将第5代细胞用CM-Dil标记,并将细胞传代,荧光显微镜观察体外标记情况。 结果与结论：第3代人脐带间充质干细胞强表达CD44,CD29,低表达CD106,不表达CD34、CD40;有80%以上的细胞处在G0/G1期,成脂成骨诱导后,油红O染色和碱性磷酸酶染色分别阳性。CM-Dil标记人脐带间充质干细胞细胞标记率达90%以上,体外传代后荧光强度逐渐减退,传8代后,荧光基本消失。说明人脐带间充质干细胞增殖、分化能力强,CM-Dil标记细胞示踪方法简单易行。     

Human umbilical cord blood stem cells and brain-derived neurotrophic factor for optic nerve injury:a biomechanical evaluation

Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.     

Acetylcholine secretion by motor neuron-like cells from umbilical cord mesenchymal stem cells

Umbilical cord mesenchymal stem cells were isolated by a double enzyme digestion method.The third passage of umbilical cord mesenchymal stem cells was induced with heparin and/or basic fibroblast growth factor.Results confirmed that cell morphology did not change after induction with basic fibroblast growth factor alone.However,neuronal morphology was visible,and microtubule-associated protein-2 expression and acetylcholine levels increased following induction with heparin alone or heparin combined with basic fibroblast growth factor.Hb9 and choline acetyltransferase expression was high following inductive with heparin combined with basic fibroblast growth factor.Results indicate that the inductive effect of basic fibroblast growth factor alone was not obvious.Heparin combined with basic fibroblast growth factor noticeably promoted the differentiation of umbilical cord mesenchymal stem cells into motor neuron-like cells.Simultaneously,umbilical cord mesenchymal stem cells could secrete acetylcholine.     

黄芩甙诱导人脐血间充质干细胞分化为神经元样细胞

目的探讨中药黄芩甙体外诱导人脐血间充质干细胞（MSCS）分化为神经元样细胞的可行性及其可能的机制。方法无菌条件下采集正常足月胎儿的脐带血,经肝素抗凝,用相对密度1．077的淋巴细胞分离液分离脐血单个核细胞,加入含黄芩甙50μmol/L的液体培养体系中进行扩增培养。取扩增培养2周的人脐血MSCS进行诱导实验。实验共分3组：诱导组（诱导液和维持液均含黄芩甙300～400μmol/L）；对照1组（诱导液和维持液均不含黄芩甙及其它抗氧化剂）；对照2组（诱导液和维持液均含3mmol/Lβ-巯基乙醇、20g/L二甲基亚砜和20mmol/L丁化羟基苯甲醚,不含黄芩甙）。诱导30min后开始在倒置显微镜下动态观察人脐血MSCS生长情况及诱导前后形态学变化。各组分别在诱导6h、24h、7d留取标本制作细胞爬片,用免疫细胞化学染色法评价神经细胞特异性烯醇化酶（NSE）和微管相关蛋白2（MAP-2）阳性细胞的表达率。用Hoechest 33258染色法评价各组细胞存活率。结果黄芩甙诱导7d后,人脐血MSCS形成较典型的神经元样细胞形态,免疫细胞化学染色显示黄芩甙诱导组NSE、MAP-2阳性细胞表达率及细胞存活率分别为77．2％±9．8％、76．6％±6．2％、86．5％±5．2％,显著高于对照1、2组（P＜0．01）。分别为4．6％±0．7％、0．7％±0．5％、45．7％±8．3％和68．9％±4．5％、51．5％±5．2％、71．6％±6．4％。结论黄芩甙能诱导人脐血MSCS分化为神经元样细胞,其诱导作用温和、稳定而持久,其诱导机制可能与黄芩甙的抗氧化、调控细胞NF-κB的活性从而刺激多种细胞因子的生成有关。     

人脐带间充质干细胞生物学特性及其研究进展

背景：人脐带间充质干细胞是一类具有自我更新和多向分化潜能的成体干细胞,具有来源丰富,对供者无影响,易于采集和运输,无异体排斥反应,避免伦理争议等诸多优点。 目的：综述人脐带间充质干细胞的生物学特性及其应用进展。 方法：以“人脐带间充质干细胞,生物学特征,基因分析,诱导分化, human umbilical cord mesenchymal stem cells(hUMSCs),biocharacteristics, gene analysis,induce differentiation”为关键词应用计算机检索CNKI数据库、万方数据库、PubMed数据库文章。 结果与结论：人脐带间充质干细胞呈典型的成纤维状,其表达的表面标志抗原具有非单一性,高表达间质细胞标志、整合素受体,不表达造血系标志、协同刺激分子CD80、CD86 和CD40、人白细胞抗原HLA-DR,HLA-G,HLA-DP,HLA-DQ、内皮标志CD31或CD33、CD14、CD56等。人脐带间充质干细胞与造血干细胞和胚胎干细胞类似,在体外可以分化为骨细胞、软骨细胞、肝细胞、心肌细胞等;在体内可以分化为多巴胺能神经元、骨骼肌细胞、内皮细胞、胰岛细胞等。但人脐带间充质干细胞的研究仍存在亟需解决的问题,如分离方法、培养条件的规范化,如何控制其生长和分化等。