大鼠肌肉注射移植人脐带间充质干细胞的安全性

背景：目前国内关于间充质干细胞肌肉注射安全性方面的研究报道较少。 目的：观察人脐带间充质干细胞肌肉注射后,移植大鼠各项生理指标及注射局部肌肉组织的病理学变化。 方法：SPF级雄性Wistar大鼠51只,取3只作为空白对照,剩余48只随机均分为4组：低、中、高浓度细胞移植组分别于大鼠左下肢腓肠肌外侧肌肉注射2.5×109 L-1,5×109 L-1,1.5×1010 L-1人脐带间充质干细胞悬液,共注射2个位点,每个位点注射0.1 mL,2个位点间隔约0.5 cm;溶媒对照组同法注射50 g/L葡萄糖溶液。分别于注射后1 d及1,2,4周进行大鼠尿常规、血液学、血液生化学和病理组织学检查。 结果与结论：人脐带间充质干细胞肌肉注射后对大鼠尿常规及肝脏、肾脏均无明显影响;仅引起总胆红素一过性升高,以及血小板、乳酸脱氢酶和肌酸激酶同工酶轻度炎症反应性升高;高浓度细胞移植可引起肌肉注射局部明显炎症反应。提示人脐带间充质干细胞免疫原性极低,在掌握了细胞移植的适宜浓度及剂量的前提下,以肌肉注射的方式进行异种移植不会引起受者严重的免疫排斥反应。     

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。     

Protective effects of human umbilical cord mesenchymal stem cell vein transplantation against spinal cord ischemia/reperfusion injury in rats

BACKGROUND: The majority of studies addressing spinal cord ischemia/reperfusion injury (SCIRI) have focused on drugs, proteins, cytokines, and various surgical techniques. A recent study reports that human umbilical cord mesenchymal stem cell (hUCMSC) transplantation achieves good therapeutic effects, but the mechanisms underlying nerve protection remain poorly understood.OBJECTIVE: To observe survival of transplanted hUCMSCs in SCIRI rat models and the influence on motor function in the hind limbs, to determine interleukin-8 expression and cellular apoptosis in spinal cord tissues, and to verify the hypothesis that hUCMSC transplantation exhibits protective effects on SCIRI.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Laboratory of the Department of Orthopedics in the First Affiliated Hospital of Soochow University,China between January 2007 and December 2008.MATERIALS: hUCMSCs were harvested from umbilical cord blood of healthy pregnant women after parturition in the Obstetrical Department of the First Affiliated Hospital of Soochow University, China. Rabbit anti-human BrdU monoclonal antibody was provided by DAKO, USA. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kit and enzyme-linked immunosorbent assay (ELISA) Kit were purchased by Wuhan Boster, China. METHODS: A total of 72 healthy, Wistar, adult rats were randomly assigned to three groups: sham-surgery, model, and transplantation, with 24 rats in each group. SCIRI was induced in the model and transplantation groups via the abdominal aorta block method. The inf rarenal abdominal aorta was not blocked in the sham-surgery group. Prior to abdominal aorta occlusion, 0.2-0.3 mL bromodeoxyuridine (BrdU)-labeled hUCMSCs suspension (cell concentration 5 × 10~3/μL) was injected through the great saphenous vein of the hind limb, and an equal volume of physiological saline was administered to the model and sham-surgery groups.MAIN OUTCOME MEASURES: Pathological observation of rat spinal cord tissues was performed by hematoxylin-eosin staining at 6, 24, and 48 hours post-surgery. Immunohistochemistry was applied to determine hUCMSCs survival in the spinal cord. The amount of cellular apoptosis and interleukin-8 expression in spinal cord tissues was assayed utilizing the TUNEL and ELISA methods, respectively. Motor function in the hind limbs was evaluated according to Jacob's score. RESULTS: Numerous BrdU-positive cells were observed in spinal cord tissues from the transplantation group. The number of apoptotic cells and interleukin-8 levels significantly decreased in the transplantation group (P < 0.05), pathological injury was significantly ameliorated, and motor function scores significantly increased (P < 0.05) compared with the model group. CONCLUSION: Via vein transplantation, hUCMSCs were shown to reach and survive in the injury area. Results suggested that the transplanted hUCMSCs contributed to significantly improved pathological changes in the injured spinal cord, as well as motor function, following SCIRI. The protective mechanism correlated with inhibition of cellular apoptosis and reduced production of inflammatory mediators.     

Effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein

We have discovered that bradykinin is effective in causing the synthesis of prostacyclin in endothelial cells cultured from calf and pig aorta and human umbilical cord vein. Maximal stimulation is attained at 10 ng bradykinin per ml with a 10 fold increase of PGI₂ as assayed by radioimmunoassay for 6-k-PGF_1α. Ionophore A23187, trypsin, and the precursor arachidonic acid are also stimulatory. We have confirmed the study of Weksler $\text{et al}$ ($\text{J. Clin. Invest., 62}$, 923, 1978) and Czervionke $\text{et al}$ ($\text{Thrombosis Research, 14}$, 781, 1979) that thrombin is effective in stimulating PGI₂ synthesis in endothelial cells from human umbilical cord vein. However, we found that cells from calf or pig aorta are not stimulated as well by thrombin. Thus, there appears to be a difference in the response to thrombin between these cells. When calf cells were grown in the presence of [³H]arachidonic acid, the radioactivity is incorporated mainly into the phospholipids. Bradykinin, ionophore A23187, and trypsin stimulated the release of radioactive materials into medium from [³H]arachidonic acid labeled calf cells. Arachidonic acid is the major radioactive substance released and PGI₂ is the major known arachidonic acid metabolite formed.     

人脐血干细胞移植治疗大鼠脑缺血的实验研究

目的研究人脐血干细胞(HUCBCs)移植治疗脑缺血大鼠的疗效及HUCBCs在缺血大鼠脑内的状况。方法采集足月新生儿脐带血60～100ml,分离出其中的单个核细胞,体外培养并予5溴脱氧嘧啶尿苷(Brdu)标记48h。采用线栓法制作大鼠脑缺血再灌注模型,1d后将3×106HUCBCs经缺血侧侧脑室注射入大鼠脑内。在移植后不同时间对大鼠进行神经损害严重程度评分(NSS),用免疫组化技术观察移植后的HUCBCs的存活、迁徙、分化状况。结果HUCBCs在体外具有增殖能力;移植组大鼠自移植后3周起其NSS显著低于对照组(均P<0.05);移植后2周、4周、6周脑组织切片中均可见Brdu染色阳性细胞,缺血侧明显多于对侧(均P<0.05),移植后4周、6周明显多于移植后2周(均P<0.05);移植组各时间点脑组织切片中均可见神经巢蛋白阳性细胞;植入的HUCBCs在大鼠脑内能向损伤区域迁徙并能分化为星形胶质细胞、少突胶质细胞和神经元。结论HUCBCs能在缺血大鼠脑内存活、迁徙和分化,并能改善其神经功能。HUCBCs移植可作为脑梗死的有效治疗手段。     

人脐带间充质干细胞复合聚左旋乳酸多孔支架材料异位成骨的实验研究

摘要 背景：聚左旋乳酸材料有良好的支撑作用,具有三维模板作用,为细胞黏附、增殖和分化提供场所。 目的：以人脐带间充质干细胞作为种子细胞、多孔聚左旋乳酸作为支架材料构建组织工程化骨异位成骨的可行性及效果。 方法：制备聚左旋乳酸多孔支架材料。用酶消化法分离培养人脐带间充质干细胞,传代培养、鉴定及诱导成骨,ALP染色检测骨向分化。将人脐带间充质干细胞与聚左旋乳酸材料复合培养,MTT及扫描电镜检测细胞增殖和细胞材料复合情况,应用矿化诱导7 d的细胞材料复合物植入兔大腿肌袋模型观察组织工程骨的异位成骨能力。4周后应用组织学观察新骨的形成情况。 结果与结论：与聚左旋乳酸多孔支架材料复合的人脐带间充质干细胞生长良好,细胞增殖未受影响,扫描电镜示细胞在支架材料上吸附、生长良好。体外对人脐带间充质干细胞骨向诱导后ALP染色阳性。矿化诱导的人脐带间充质干细胞复合聚左旋乳酸材料植入动物4周时,形成明显的块状组织,质地坚硬。组织学检查见新形成的组织有成骨细胞及其周围有血管长入。提示聚左旋乳酸多孔材料对种子细胞人脐带间充质干细胞的增殖无影响;人脐带间充质干细胞细胞与聚左旋乳酸多孔材料复合体可在异位形成骨组织。 关键词：人脐带间充质干细胞;聚乳酸;生物相容性;异位成骨;兔 doi:10.3969/j.issn.1673-8225.2011.12.008     

两步法分离人脐血单个核细胞的最佳条件

背景：如何获得较为纯化、高活性的干细胞,目前未见深入研究报告,也未见一个标准化操作流程方案。 目的：探讨两步法分离人脐血单个核细胞最佳分离条件。 方法：观察羟乙基淀粉在20,30,40,50,60,70 min不同时间沉淀脐血中红细胞的效果;使用人淋巴细胞分离液,分别在800,700,600,500,400 g/min,离心30,25,20 min的条件下分离人脐血单个核细胞。 结果与结论：6%羟乙基淀粉沉淀脐血60 min效果最好;使用密度为(1.077 0±0.000 1) g/mL人淋巴细胞分离液,在4 ℃条件下以700 g/min离心力,离心30 min,洗涤3次,这样获得的人脐血单个核细胞效果最好,所得细胞沉淀中混杂细胞如红细胞及其他细胞碎片较少,人脐血单个核细胞的细胞得率及活力比较高。提示应用羟乙基淀粉沉淀和人淋巴细胞分离液分离两步法,在最佳时间条件下可提高脐血干细胞的回收率。     

Neural differentiation and potential use of stem cells from the human umbilical cord for central nervous system transplantation therapy

The human umbilical cord is a rich source of autologous stem and progenitor cells. Interestingly, subpopulations of these, particularly mesenchymal-like cells from both cord blood and the cord stroma, exhibited a potential to be differentiated into neuron-like cells in culture. Umbilical cord blood stem cells have demonstrated efficacy in reducing lesion sizes and enhancing behavioral recovery in animal models of ischemic and traumatic central nervous system (CNS) injury. Recent findings also suggest that neurons derived from cord stroma mesenchymal cells could alleviate movement disorders in hemiparkinsonian animal models. We review here the neurogenic potential of umbilical cord stem cells and discuss possibilities of their exploitation as an alternative to human embryonic stem cells or neural stem cells for transplantation therapy of traumatic CNS injury and neurodegenerative diseases.     

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。     

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。     

Stem cell transplantation for treatment of cerebral ischemia in rats Effects of human umbilical cord blood stem cells and human neural stem cells

BACKGROUND:Exogenous neural stem cell transplantation promotes neural regeneration. However, various types of stem cells transplantation outcomes remain controversial. OBJECTIVE:To explore distribution, proliferation and differentiation of human neural stem cells (hNSCs) and human umbilical cord blood stem cells (hUCBSCs) following transplantation in ischemic brain tissue of rats, and to compare therapeutic outcomes between hNSCs and hUCBSCs. DESIGN, TIME AND SETTING:Randomized controlled animal studies were performed at the Experimental Animal Center of Nanjing Medical University and Central Laboratory of Second Affiliated Hospital of Nanjing Medical University of China from September 2008 to April 2009. MATERIALS:hNSCs were harvested from brain tissue of 10-13 week old fetuses following spontaneous abortion, and hUCBSCs were collected from umbilical cord blood of full-term newborns at the Second Affiliated Hospital of Nanjing Medical University of China. hNSCs and hUCBSCs were labeled by 5-bromodeoxyuridine (BrdU) prior to transplantation. METHODS:Rat models of cerebral ischemia were established by the suture method. A total of 60 healthy male Sprague Dawley rats aged 7-9 weeks were randomly assigned to hNSC transplantation, hUCBSC transplantation and control groups. The rat models in the hNSC transplantation, hUCBSC transplantation and control groups were infused with hNSC suspension, hUCBSC suspension and saline via the caudal vein, respectively. MAIN OUTCOME MEASURES:The distribution, proliferation and differentiation of hNSCs and hUCBSCs in ischemic brain tissue were observed using immunohistochemical methods. Neurological function in rats was assessed using the neurological severity score. RESULTS:The number of BrdU-positive cells was significantly greater in the hNSC transplantation group compared with hUCBSC transplantation group at 14 days following transplantation (P < 0.05). The number of BrdU-positive cells reached a peak at 28 days following transplantation. Nestin-positive, glial fibrillary acidic protein-positive, cyclic nucleotide 3' phosphohydrolase-positive and neuron specific enolase-positive cells were visible following transplantation. No significant difference was determined in the constituent ratio of various cells between hNSC and hUCBSC transplantation groups (P > 0.05). The neurological severity score was significantly decreased in rats at 21 days following transplantation (P < 0.05). No significant difference was detected in neurological severity score between hNSC and hUCBSC transplantation groups at various time points (P > 0.05). CONCLUSION:The transplanted hNSCs and hUCBSCs can migrate into ischemic brain tissue, proliferate and differentiate into neuron-like, astrocyte-like and oligodendrocyte-like cells, and improve neurological function in rats with cerebral ischemia.     

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分离培养及向脂肪与成骨细胞的分化**△○

背景：研究报道显示人脐带间充质干细胞体外分离方法及效率、培养条件各不相同,并且尚未有统一的鉴定标准。因此建立高效、经济的培养体系十分必要。 目的：本研究旨在建立从人脐带组织分离培养间充质干细胞的方法,并进行向脂肪细胞和成骨细胞的诱导分化。 方法：首先采用组织块贴壁法或酶消化法分离纯化得到脐带间充质干细胞(Umbilical cord mesenchymal stem cells,UCMSCs),分析其细胞形态、增殖方式和某些免疫表型,并在体外诱导其向脂肪细胞和成骨细胞分化。 结果和结论:结果发现,hUCMSCs在适当的诱导条件下能向脂肪和成骨细胞分化,体外能连续传代40次以上,且形态和表型保持稳定。这证实了人脐带组织存在间充质干细胞,且具有向脂肪和成骨细胞分化的潜能,该细胞可用于后续的应用研究。     

纤维蛋白(原)及其降解产物对共培养人脐静脉内皮细胞的细胞间黏附分子-1表达的影响

目的探讨纤维蛋白原(Fg)、纤维蛋白(Fb)及其降解产物(FDPs)对人脐静脉内皮细胞(HU-VECs)/兔平滑肌细胞(SMCs)共培养模型中细胞间黏附分子-1(ICAM-1)表达的影响。方法建立原代HU-VECS与兔SMC共培养体系,根据干预物及其浓度的不同分为Fg、Fb、FDPs组及相应的0 mg/ml、0.5 mg/ml、3.0 mg/ml和6.0 mg/ml亚组。应用核因子B抑制蛋白(I-B)拮抗剂BAY 11-7082、蛋白激酶C(PKC)拮抗剂Staurosporine分别对0.5 mg/ml、3.0 mg/ml和6.0 mg/ml亚组进行干预。采用反转录(RT)-PCR法检测HU-VECS内ICAM-1 mRNA水平;酶联免疫吸附法(ELISA)检测培养上清液的ICAM-1蛋白含量。结果 Fg 3.0mg/ml和6.0 mg/ml亚组ICAM-1 mRNA水平和蛋白含量显著高于0 mg/ml亚组及相应浓度Fg+BAY 11-7082亚组(均P<0.05);Fg 3.0 mg/ml亚组ICAM-1 mRNA水平和蛋白含量显著高于3.0 mg/ml+Staurosporine亚组(均P<0.05)。Fb 6.0 mg/ml亚组ICAM-1 mRNA水平显著高于0 mg/ml、6.0 mg/ml+BAY11-7082和6.0 mg/ml+Staurosporine亚组(均P<0.05),但Fb 6.0 mg/ml亚组ICAM-1蛋白含量显著高于0 mg/ml亚组(P<0.05)。FDPs 6.0 mg/ml亚组ICAM-1 mRNA水平和蛋白含量显著高于0 mg/ml、相应浓度BAY11-7082和Staurosporine亚组(均P<0.05)。结论中、高浓度Fg、Fb、FDPs能上调血管内皮细胞ICAM-1的表达。     

Therapeutic effect of human umbilical cord mesenchymal stem cells on neonatal rat hypoxic–ischemic encephalopathy

The therapeutic potential of umbilical cord blood mesenchymal stem cells has been studied in several diseases. However, the possibility that human umbilical cord Wharton's jelly‐derived mesenchymal stem cells (hUCMSCs) can be used to treat neonatal hypoxic–ischemic encephalopathy (HIE) has not yet been investigated. This study focuses on the potential therapeutic effect of hUCMSC transplantation in a rat model of HIE. Dermal fibroblasts served as cell controls. HIE was induced in neonatal rats aged 7 days. hUCMSCs labeled with Dil were then transplanted into the models 24 hr or 72 hr post‐HIE through the peritoneal cavity or the jugular vein. Behavioral testing revealed that hUCMSC transplantation but not the dermal fibroblast improved significantly the locomotor function vs. vehicle controls. Animals receiving cell grafts 24 hr after surgery showed a more significant improvement than at 72 hr. More hUCMSCs homed to the ischemic frontal cortex following intravenous administration than after intraperitoneal injection. Differentiation of engrafted cells into neurons was observed in and around the infarct region. Gliosis in ischemic regions was significantly reduced after hUCMSC transplantation. Administration of ganglioside (GM1) enhanced the behavioral recovery on the base of hUCMSC treatment. These results demonstrate that intravenous transplantation of hUCMSCs at an early stage after HIE can improve the behavior of hypoxic–ischemic rats and decrease gliosis. Ganglioside treatment further enhanced the recovery of neurological function following hUCMSC transplantation. © 2013 Wiley Periodicals, Inc.     

Functional recovery and microenvironmental alterations in a rat model of spinal cord injury following human umbilical cord blood-derived mesenchymal stem cells transplantation

BACKGROUND: Transplantation of human umbilical cord blood-derived mesenchymal stem cells (MSCs) has been shown to benefit spinal cord injury (SCI) repair. However, mechanisms of microenvironmental regulation during differentiation of transplanted MSCs remain poorly understood. OBJECTIVE: To observe changes in nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and interleukin-8 (IL-8) expression following transplantation of human umbilical cord-derived MSCs, and to explore the association between microenvironment and neural functional recovery following MSCs transplantation.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Soochow University from April 2005 to March 2007. MATERIALS: Human cord blood samples were provided by the Department of Gynecology and Obstetrics, First Affiliated Hospital of Soochow University. Written informed consent was obtained. METHODS: A total of 62 Wister rats were randomly assigned to control (n = 18), model (n = 22, SCI + PBS), and transplantation (n = 22, SCI + MSCs) groups. The rat SCI model was established using the weight compression method. MSCs were isolated from human umbilical cord blood and cultured in vitro for several passages. 5-bromodeoxyuridine (BrdU)-labeled MSCs (24 hours before injection) were intravascularly transplanted. MAIN OUTCOME MEASURES: The rats were evaluated using the Basso, Beattie and Bresnahan (BBB) locomotor score and inclined plane tests. Transplanted cells were analyzed following immunohistochemistry. Enzyme-linked immunosorbant assay was performed to determine NGF, BDNF, and IL-8 levels prior to and after cell transplantation.RESULTS: A large number of BrdU-positive MSCs were observed in the SCI region of the transplantation group, and MSCs were evenly distributed in injured spinal cord tissue 1 week after transplantation. BBB score and inclined plane test results revealed significant functional improvement in the transplantation group compared to the model group (P< 0.05), which was maintained for 2-3 weeks. Compared to the model group, NGF and BDNF levels were significantly increased in the injured region following MSCs transplantation at 3 weeks (P < 0.05), but IL-8 levels remained unchanged (P > 0.05).CONCLUSION: MSCs transplantation increased NGF and BDNF expression in injured spinal cord tissue. MSCs could promote neurological function recovery in SCI rats by upregulating NGF expression and improving regional microenvironments.     

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。     

Functional recovery and local microenviromental change after in vivo transplantion of human umbilical cord blood mesenchymal stem cells into spinal cord injury rats

目前的体内实验研究发现,成体干细胞移植是促进中枢神经系统损伤如脊髓损伤(spinal cord injury, SCI)等难治性疾病的有益尝试,成体干细胞如间充质干细胞具有可塑性,移植的细胞可以在损伤部位存活、整合入宿主组织中,分化出神经元、星形胶质细胞和少突胶质细胞,从而促使中枢神经系统的功能得到部分恢复。近年来也有一部分学者指出,成体干细胞具有可塑性证据不足,成体干细胞并不能跨胚层分化,而只是简单地与宿主细胞的融合。那么,如果并没有或极少有干细胞的转分化,还有哪些因素有助于干细胞移植后神经损伤功能的恢复呢？本文究其相关机制作了初步探讨     

Engraftment of freshly isolated or cultured human umbilical cord blood cells and the effect of cyclosporin A on the outcome

Human umbilical cord blood (HUCB) is a potentially valuable resource for cell therapy. The present study investigated the short-term survival of intrastriatal grafts of either freshly isolated or cultured HUCB cells and the effect of the immunosuppressive agent cyclosporin A (CSA) in host rat brains. The group injected with either freshly isolated or cultured HUCB cells was subdivided into CSA or saline controls. Freshly isolated and cultured HUCB cells displayed surface markers CD33, CD44, CD45, CD51/61 and CD90/Thy-1. The hematopoietic progenitor marker CD34 was expressed only in freshly isolated cells. The majority of injected HUCB cells were localized within a 500-mum radius from the injection site in the striatum; however, a subpopulation migrated along the corpus callosum. There was no significant statistical difference in the cell count between freshly isolated and cultured HUCB cells with or without CSA. Some grafted HUCB cells expressed either a neural or microglial marker. There was weak up-regulation of major histocompatibility complex (MHC) class I antigen in rats either with or without CSA. However, there were considerably fewer positive cells labeled with an MHC class II antigen in CSA groups. These results suggest that neither freshly isolated nor cultured HUCB cells induce acute rejection after intrastriatal transplantation up to 14 days. CSA suppressed up-regulation of MHC class II antigen in the host brain.     

Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron‐like cells

Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron‐like cells from hUCB‐MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB‐MSCs with k252a, the Trk receptor inhibitor of brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron‐like cells was significantly decreased compared with ACM‐treated hUCB‐MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron‐like cell differentiation compared with control. The transplantation of ACM‐induced hUCB‐MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron‐like cells. In conclusion, we propose that hUCB‐MSCs are a good source of DA neuron‐like cells and that ACM is a potential inducer to obtain DA neuron‐like cells from hUCB‐MSCs in vitro for an ethical and legal cell therapy for PD. © 2013 Wiley Periodicals, Inc.