早期作用造血因子对人脐血CD34+细胞的体个扩增作用

为了观察早期作用造血细胞因子SCF、FL、IL-3、IL-6、TPO单独及联合应用,对脐血CD34+细胞的体外扩增作用.我们用吸附单克隆抗体-磁珠分离系统富集人脐血CD34+细胞,在体外液体培养体系中加入不同的细胞因子扩增4周,每周取样计数有核细胞总数及集落形成细胞(CFC)数.结果表明:用磁性细胞分离仪富集脐血CD34+细胞纯度为80%～87%;一些细胞因子有明显的协同效应,其联合应用的扩增作用显著高于单因子作用;SCF+FL存在下,IL-3是有效扩增有核细胞总数及CFC的关键因子;细胞因子SCF+FL+IL-3和SCF+FL+IL-3+IL-6组合对有核细胞总数及CFC均有良好的扩增效应,培养2周时对CFC的扩增倍数分别为38.3±4.4 和29.6±2.7倍,可满足成人移植及基因治疗等的需要.     

人骨骼肌源性血管外膜细胞对人脐血CD34+细胞的体外造血支持作用#br#

文题释义： 血管外膜细胞：是血管周围星状细胞,分布于全身的毛细血管和微血管的管壁,是血管周围微环境的重要核心组成成分。它们表达CD146、NG2、PDGFRβ、LepR、Nestin等标记物,而不表达内皮细胞标记物CD144、vWF、CD31及造血细胞标记物CD34、CD45、CD14。研究显示血管外膜细胞是间充质干细胞的前体细胞,其表达间充质干细胞表面标记物,具有多向分化潜能,并可支持造血。 造血干细胞微环境：是造血组织中造血干细胞赖以生存并进行自我更新、多向分化的场所。它由骨内膜微环境和血管微环境组成,前者维持造血干细胞的静止及自我更新,后者促进造血干细胞动员、增殖、分化。 背景：研究显示血管外膜细胞是间充质干细胞的前体细胞,其通过细胞接触或旁分泌效应调节造血干细胞的行为并支持造血,人骨骼肌源性血管外膜细胞对造血的支持作用有待于研究。 目的：从人骨骼肌中分离培养血管外膜细胞并进行生物学特性鉴定,研究其对脐血CD34⁺细胞的体外支持作用。 方法：①利用多参数流式细胞术从人骨骼肌中分选表型为CD146⁺CD56^-CD34^-CD144^-CD45^-的血管外膜细胞,并对其进行生物学鉴定;②建立以CD146⁺人骨骼肌源性血管外膜细胞为滋养层的脐血CD34⁺细胞体外培养体系(实验组),以人骨髓间充质干细胞为滋养层的脐血CD34⁺细胞体外培养体系为阳性对照组,共培养1,2,4周检测培养体系的细胞数量、集落形成能力及免疫表型并进行统计分析。 结果与结论：①通过多参数流式细胞术分选出CD146⁺人骨骼肌源性血管外膜细胞,回测纯度为(91.5±1.85)% (n=5);其表达间充质干细胞表面抗原CD73、CD90、CD105、CD44,不表达造血细胞及内皮细胞表面抗原CD45、CD34、CD31;经诱导培养可向骨细胞、软骨细胞、脂肪细胞及肌细胞分化;②实验组与阳性对照组相比,在细胞数、集落形成能力及免疫表型方面(CD45⁺、CD34⁺CD33^-、CD14⁺、CD10⁺/CD19)差异均无显著性意义(P > 0.05,n=6);无滋养层的空白对照组培养1周时细胞数量明显减少,2周时几乎无细胞存活;③结果表明,CD146⁺人骨骼肌源性血管外膜细胞与人骨髓间充质干细胞一样对脐血CD34⁺细胞具有体外支持作用。 ORCID: 0000-0002-6768-5273(郑波) 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     

FL、rIL-6、rIL-6R对脐血CD34+细胞的体外分化增殖作用

目的探讨FL、rIL-6、rIL-6R对CD34+细胞的体外分化增殖作用.方法用磁性细胞分离仪富集人脐血CD34+细胞.加入FL、rIL-6、rIL-6R及其不同组合因子,不同时间取样计数CD34+细胞集落数及细胞数.结果FL、rIL-6、rIL-6R扩增细胞形成集落的主要成分为CFU-GM,但在rIL-6+rIL-6R及FL+rIL-6+rIL-6R作用下,也可形成一定数量的BFU-E及CFU-M,而CFU-MK则未见形成.FL+rIL-6+rIL-6R可刺激红细胞生成.可使CD34+细胞总数在第7天时扩增6.4倍.结论FL+rIL-6+rIL-6R可扩增脐血CD34+细胞数量并可促进其分化.     

人脐血单个核细胞中CD133+细胞移植治疗心力衰竭的评价

BACKGROUND:Cell purification can eliminate the biological variability of cells, providing new insight into cell regeneration therapy. OBJECTIVE:To study the Influence of CD133⁺ cells on human umbilical cord blood mononuclear cell transplantation for treatment of heart failure. METHODS:Human cord blood mononuclear cells were isolated using lymphocyte separation medium method, and CD133⁺ and CD133^- cells were sorted using immunomagnetic beads at a cell density of 1×10⁸/L. Forty Sprague-Dawley rats were randomized into five groups: sham group, model group, CD133⁺ cell group, CD133^- cell group and mononuclear cell group. Animal models of heart failure were made using intraperitoneal injection of isoproterenol in all the groups except for the sham group. Rats in the CD133⁺ cell group and CD133^- cell group were given 1 mL CD133⁺ cells plus 1 mL PBS and 1 mL CD133^- cells plus 1 mL PBS via the tail vein, respectively. Rats in the mononuclear cell group were given 1 mL CD133⁺ cells plus 1 mL CD133^- cells via the tail vein, and those in the sham and model groups given 2 mL PBS via the tail vein. After 4 weeks, cardiac pathology, degree of myocardial fibrosis and colonization of CD133⁺ cells in myocardial tissues were observed. RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that myocardial tissues arranged disorderly in the model group, but regularly in the sham group; myocardial disorders were mildest in the CD133⁺ cell group, successively followed by the mononuclear cell group, and severest in the CD133^- cell group and model group. Masson staining showed that in the model group, collagen fibers were proliferated, arranged irregularly and even broken, while in the sham group, the collagen fibers were less in number and arranged in order. Additionally, there was less reduction in collagen fibers and milder myocardial disorders in the CD133⁺ cell group compared with the other groups. Area of collagen fibers was increased significantly in all the groups except for the sham group (P < 0.05), but this increment was the minimal in the CD133⁺ cell group. Findings from immunohistochemistry and immunofluorescence staining showed that there were no CD133⁺ cells in the myocardial tissues of rats. Therefore, our data indicate that compared with the mononuclear cell transplantation, CD133⁺ cell transplantation exerts superiorities in relieving myocardial damage and reducing myocardial fibrosis. However, CD133⁺ cells are not colonized in the myocardial tissue after transplantation.     

甘露醇促进人脐血CD34+细胞通过血脑屏障静脉迁移至高血压脑梗死大鼠脑组织

背景：单纯脐血细胞经静脉移植治疗脑梗死疗效有限,移植细胞经血脑屏障迁入脑内数量不足为重要原因之一。 目的：观察血脑屏障开放剂甘露醇对人脐血CD34⁺细胞经静脉移植治疗高血压大鼠脑梗死疗效的影响。 方法：分离人脐血CD34⁺细胞,脂质体方法转染pEGFPF质粒,制备pEGFP-CD34⁺细胞;45只雄性SD大鼠经线栓法建立大脑中动脉栓塞模型,造模后24 h随机分为3组：实验组注射1×10⁶ GFP-CD34⁺细胞,继之注射20%甘露醇2 g/kg;阳性对照组注射1×10⁶ GFP-CD34⁺细胞;空白对照组注射等量生理盐水。 结果与结论：①荧光显微镜下实验组每张切片脑组织GFP标记阳性的绿色荧光细胞计数平均值显著多于阳性对照组。②移植后第7天治疗组与其他各组神经功能损害评分差异无显著性意义;移植后28 d,各组神经功能均有不同程度恢复,实验组神经功能恢复明显优于阳性对照组和空白对照组(P < 0.05)。③实验组与其他两组相比,大鼠脑梗死体积均显著减少。④实验组脑组织匀浆胶质细胞源性神经营养因子水平较阳性对照组、空白对照组明显增高。提示血脑屏障开放剂甘露醇促进静脉移植CD34⁺细胞通过血脑屏障迁入至脑组织,并增强静脉移植CD34⁺细胞治疗脑梗死的疗效。     

几种细胞因子对人脐血CD34+干细胞在体外诱导扩增为树突状细胞的研究

多种疾病用树突状细胞 (dendriticcell,DC)作免疫治疗具有一定的疗效。由于DC在人体组织中含量甚微 ,限制了DC的研究及临床应用。因此如何获得足够的成熟DC便成为DC广泛应用的关键技术问题。我们比较了不同细胞因子组合对脐血CD34 造血干细胞在体外诱导扩增为DC的效率及功能的影响 ,提供一种体外获得大量成熟DC的有效途径。脐血采自本院正常分娩志愿者 ,Fi coll淋巴细胞分离液离心获得单个核细胞 ,按CD34 细胞MACS免疫磁性分离试剂盒操作说明分离纯化CD34 细胞。纯化的CD34 细胞配制成含 5× 10 4 ml细胞的悬液 ,按以下 5组…     

CD34基因与血管内皮细胞

D34基因有 8个外显子 ,其产物CD34抗原为 1 0 5～ 1 2 0kD的跨膜细胞表面糖蛋白 ,可选择性地在造血干 /祖细胞中表达 ,而且也在血管内皮细胞以及其它组织中表达。它具有粘附、加速血管前内皮细胞聚集形成血管、调控造血细胞的增生和分化的功能。可用来测量肿瘤内及缺血组织微血管的密度和面积检测早期的新生微血管形成及分离CD34 造血干 /祖细胞等。     

P19细胞移植改善心肌梗死大鼠心功能

目的观察P19细胞植入大鼠心肌后成活、分化及对大鼠心功能的影响。方法SD大鼠30只,随机分为移植组(n=20)和对照组(n=10)。用液氮冷冻方法建立心肌梗死模型,梗死后立即将培养的P19细胞植入心肌梗死区及周边区,8周后观察植入细胞的成活、分化,并通过血流动力学各项指标评价细胞移植对心功能的改善情况。结果移植组免疫组化染色GATA-4、α-肌节肌动蛋白(-αsarcomeric actin)及肌细胞生成蛋白(myogenin)在成活移植细胞呈阳性,左室收缩压(LVSP)、左室等容期压力最大变化速率(±dp/dtmax)明显高于对照组(P<0.01),左室舒张末期压(LVEDP)明显低于对照组(P<0.01)。移植组有1例检测心功能时发现有偶发室性早搏。结论P19细胞移植入心肌梗死区后能够成活并向心肌分化,可明显改善心肌梗死后大鼠的心功能;P19细胞可以作为研究心肌梗死细胞移植疗法的模型细胞。     

瑞舒伐他汀联合脐血间充质干细胞移植改善急性心肌梗死后心功能

BACKGROUND:In recent years, it has been a hot topic that stem cell transplantation is used to improve cardiac insufficiency after acute myocardial infarction by inducing regeneration of cardiomyocytes in the infarction regions. OBJECTIVE:To observe the effect of rosuvastatin combined with umbilical cord blood mesenchymal stem cells transplantation on rat cardiac function after acute myocardial infarction. METHODS:Forty-five Sprague-Dawley rats were enrolled to prepare myocardial infarction models by ligaturing the left anterior descending coronary artery. Then they were equivalently divided into model group, transplantation group and combination group. At 7 days after modeling, rats in the combination group were given injection of 300 μL umbilical cord blood mesenchymal stem cells (15.0×10⁸) via the tail vein and by gavage once a day for 28 days with 1 mg/kg rosuvastatin; rats in the transplantation group and model group were injected with 300 μL umbilical cord blood mesenchymal stem cell suspension through the tail veins or the same amount of LG-DMEM medium, respectively, followed by intragastrical administration of the same amount normal saline. At 5 weeks after modeling, indexes of cardiac function, level of plasma Lp-PLA2 and heat shock protein 70 in the infarction regions were detected by color Doppler ultrasound, enzyme-linked immunosorbent assay and western blot assay, respectively. In addition, pathological changes of myocardial tissues were observed using hematoxylin-eosin staining. RESULTS AND CONCLUSION:Left ventricular ejection fraction and left ventricular end-systolic pressure were significantly higher in the combination group than in the transplantation group as well as higher in the transplantation group than the model group (P < 0.05); compared with the transplantation group, left ventricular end-diastolic pressure was significantly decreased in the combination group, but significantly increased in the model group (P < 0.05); the number of cardiomyocytes in the infarction regions was significantly higher in the combination group than the other groups. Additionally, expression of heat shock protein 70 in the infarction regions was significantly increased in the combination group (P < 0.05). To conclude, rosuvastatin combined with umbilical cord blood mesenchymal stem cell transplantation can significantly improve rat cardiac function after myocardial infarction.     

转染IL-21的CD34~+脐血造血干细胞抗裸鼠卵巢癌作用研究

目的 探讨IL-21转染的脐血造血干细胞(CD34~+UBSC·IL-21)对荷卵巢癌裸鼠的治疗作用.方法 从脐血分离CD34~+造血干细胞,体外培养扩增后用于重组体pIRES2-IL-21-EGFP转染.以肿瘤大小、荷瘤鼠生存期判断CD34~+UBSC-IL-21对荷瘤裸鼠的治疗效应.以RT-PCR、免疫荧光、ELISA、Western blot、脾细胞增殖试验及免疫组化法分别鉴定CD34~+UBSC和肿瘤组织中IL-21的表达及活性.裸鼠脾细胞中NK细胞含量及脾细胞的杀伤效应、血清中IFN-γ和TNF-α水平分别用FCM与ELISA检测.结果 pIRES2-IL-21-EGFP成功转染CD34~+UBSC.CD34~+UBSC-IL-21能抑制肿瘤生长,延长荷瘤裸鼠生存期,治疗鼠肿瘤局部能表达IL-21、血清IFN-γ和TNF-α水平升高,NK细胞含量及NK细胞杀伤活性明显增强,与其他组相比,差异有统计学意义(P<0.01).结论 转染IL-21的CD34~+UBSC有良好的抗裸鼠卵巢癌作用,该结果为临床使用UBSC为载体的基因治疗卵巢癌研究奠定了基础.     

Platelet-derived growth factor enhances expansion of umbilical cord blood CD34+ cells in contact with hematopoietic stroma

Stem cell expansion remains an elusive but highly desirable goal. Here we show that platelet-derived growth factor (PDGF), along with cultured endothelial or stromal cells, significantly enhances expansion of human CD34+ cells in vitro. In media supplemented with thrombopoietin, stem cell factor, flt-3 ligand, and granulocyte-colony stimulating factor, CD34+ cells, as well as CFU-GM, BFU-CFU-E, CFU-GEMM, and CFU-MK, increased by 34.3-, 138-, 59.7-, 38.4-, and 86.0-fold, respectively. Co-culturing of CD34+ cells with cultured stromal cells or human umbilical cord vein endothelial cells (HUVECs) greatly enhanced expansion efficiency. The presence of PDGF (50 ng/ml) further augmented expansion, such that increases of 77.0-, 262-, 90.0-, 93.0-, and 200-fold, respectively, were achieved. Six weeks after infusion of expanded cells into NOD/SCID mice, human CD45+ cells were detected in recipients' bone marrow, spleen, and peripheral blood. Our results provide a rationale for development of a stem cell expansion protocol for clinical applications.     

脐血浆神经营养活性蛋白成分对CD34+细胞增殖分化的影响

背景：迄今为止,人们对于脐血浆中是否存在活性成分及其对于神经发育及神经损伤修复的作用认识尚不足,有待深入研究。 目的：检测脐血浆生物活性成分,观察其在神经发育及神经损伤修复中的作用。 方法：采用抗体芯片技术对脐血浆和健康青年女性静脉血浆活性成分进行对比分析,采用免疫磁珠从脐血中分选出CD34⁺细胞,经体外培养计数观察CD34⁺细胞增殖能力,通过免疫组化法观察细胞分化情况。 结果与结论：脐血浆中有31种蛋白分子的含量显著高于静脉血浆,其中具有促进神经再生和神经修复潜能的活性蛋白10种,包括FGF4、Frizzled-3、IL-3、RAGE、CRIM-1、Neuritin、Neuropilin-2、Neurturin、SFRP-3、Tomoregulin-1;在CD34+细胞培养液中加入脐血浆能显著促进细胞增殖,促进细胞向神经细胞分化。     

Co-culture of umbilical cord blood CD34+ cells with human mesenchymal stem cells

Insufficient numbers of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) sometimes limit allogenic transplantation of umbilical cord blood (UCB). Ex vivo expansion may overcome this limitation. Mesenchymal stem cells (MSCs), as non-hematopoietic, well-characterized skeletal and connective-tissue progenitor cells within the bone marrow stroma, have been investigated as support cells for the culture of HSCs/HPCs. MSCs are attractive for the rich environmental signals that they provide and for immunological compatibility in transplantation. Thus far, HSC/MSC co-cultures have mainly been performed in 2-dimensional (2D) configuration. We postulate that a 3-dimensional (3D) culture environment that resembles the natural in vivo hematopoietic compartment might be more conducive for regulating HSC expansion. In this study, we compared the co-culture of HSCs and MSCs in 2D and 3D configurations. The results demonstrated the benefit of MSC inclusion in HSC expansion ex vivo. Direct contact between MSCs and HSCs in 3D cultures led to statistically significantly higher expansion of cord blood CD34+ cells than in 2D cultures (891- versus 545-fold increase in total cells, 96- versus 48-fold increase of CD34+ cells, and 230- versus 150-fold increase in colony-forming cell assay [CFC]). Engraftment assays in non-obese diabetic/severe combined immunodeficiency mice also indicated a high success rate of hematopoiesis reconstruction with these expanded cells.     

Chemoattraction of femoral CD34+ progenitor cells by tumor-derived vascular endothelial cell growth factor

Patients and animals with GM-CSF-producing tumors have an increased number of mobilized CD34⁺ progenitor cells within their peripheral blood and tumor tissue. These CD34⁺ cells are inhibitory to the activity of intratumoral T-cells. The present study used the murine Lewis lung carcinoma (LLC) model to assess mechanisms that could lead to the accumulation of CD34⁺ cells within the tumor tissue. In vitro analyses showed that LLC tumor explants released chemoattractants for normal femoral CD34⁺ cells. The LLC tumor cells contributed to the production of this activity since CD34⁺ cell chemoattractants were also released by cultured LLC cells. Antibody neutralization studies showed that most, although not all, of the chemotactic activity that was produced by LLC cells could be attributed to VEGF. In vivo studies with fluorescent-tagged CD34⁺ cells showed their accumulation within the tumor tissue, but not within the lungs, spleen or bone marrow, suggesting a selective accumulation within the tumor. Whether or not VEGF could chemoattract CD34⁺ cells in vivo was measured with a VEGF-containing Matrigel plug assay. Infusion of fluorescent-tagged CD34⁺ cells into mice after the plugs became vascularized revealed the accumulation of fluorescent-tagged cells within the plugs. However, these CD34⁺ cells failed to accumulate within the VEGF-containing Matrigel plugs when they were infused together with neutralizing anti-VEGF antibody. Through a combination of in vitro and in vivo analyses, the LLC cells were shown to be capable of chemoattracting CD34⁺ cells, with most of the tumor-derived chemotactic activity being due to tumor release of VEGF. This revised version was published online in July 2006 with corrections to the Cover Date.     

脐带间充质干细胞移植治疗心肌梗死大鼠心功能变化的Meta分析

文题释义： 脐带间充质干细胞：是新生儿脐带组织中的一种具有自我复制能力的多功能干细胞,因其低免疫原性和较强的增殖分化能力,目前被广泛应用于心肌梗死方面的研究。在一定条件下它可以分化成多种组织细胞,同时也可以分泌一些细胞因子,为新生细胞的生长提供良好的微环境,进而促进心肌新生,改善血供和心功能,降低死亡率。 心功能：利用超声心动图检测心功能是目前评价心肌梗死疗效的有效手段,具有无创、安全、简便、可重复检查等优势。大量实验研究表明,脐带间充质干细胞移植治疗心肌梗死可以提高心室射血分数,减少心室重构,改善心功能,降低心力衰竭的病死率。 背景：研究显示,相对于其他来源的干细胞,脐带间充质干细胞这类多功能干细胞的免疫原性较低,将其应用到心肌梗死大鼠基础研究中具有显著疗效。 目的：系统评价脐带间充质干细胞对心肌梗死大鼠心功能的影响。 方法：计算机检索PubMed、Cochrane、Embase、CBM、中国知网、万方、维普、中国期刊数据库,检索时限从建库起至2019年6月,收集关于脐带间充质干细胞治疗大鼠心肌梗死的系列研究。由2位研究者按照纳入标准独立完成文献筛选、资料提取及方法学质量评价,采用Stata14.0进行Meta分析。 结果与结论：最终9篇文献纳入研究,共计216只大鼠。Meta分析结果显示：①脐带间充质干细胞可显著升高心肌梗死大鼠的左室射血分数[95%CI (3.16,3.76),P < 0.001];②脐带间充质干细胞对心肌梗死大鼠左室短轴缩短率有显著增加作用[95%CI (0.18,0.54),P < 0.001];③脐带间充质干细胞对心肌梗死大鼠的左室舒张末期内径[95%CI (-1.90,-0.99),P=0.042]和收缩末期内径[95%CI (-6.56,-4.65),P < 0.001]有明显缩短作用;④脐带间充质干细胞可显著改善心肌梗死大鼠的左室舒张末期容积[95%CI (-2.01,-1.11),P < 0.001]和收缩末期容积[95%CI (-3.44,-2.17),P < 0.001];⑤结果表明,脐带间充质干细胞移植治疗对心肌梗死大鼠的心功能有明显改善作用,且安全性较高。受纳入文献质量的限制,以上结论需更高质量、更大样本的随机对照实验加以验证。 ORCID: 0000-0002-9025-9352(陈思宇) 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     

Effects of intravenous human umbilical cord blood CD34+ stem cell therapy versus levodopa in experimentally induced Parkinsonism in mice

Introduction

Parkinsonism is a neurodegenerative disease with impaired motor function. The current research was directed to investigate the effect of CD34+ stem cells versus levodopa in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism.

Material and methods

Mice were divided into 4 groups; saline-injected, MPTP: received four MPTP injections (20 mg/kg, i.p.) at 2 h intervals, MPTP groups treated with levodopa/carbidopa (100/10 mg/kg/twice/day for 28 days) or single intravenous injection of 10⁶ CD34+ stem cells/mouse at day 7 and allowed to survive until the end of week 5.

Results

Levodopa and stem cells improved MPTP-induced motor deficits; they abolished the difference in stride length, decreased percentage of foot slip errors and increased ambulation, activity factor and mobility duration in parkinsonian mice (p < 0.05). Further, they significantly (p < 0.05) increased striatal dopamine (85.3 ±4.3 and 110.6 ±5.3) and ATP levels (10.6 ±1.1 and 15.5 ±1.14) compared to MPTP (60.1 ±3.9 pmol/g and 3.6 ±0.09 mmol/g, respectively) (p < 0.05). Moreover, mitochondrial DNA from mice treated with levodopa or stem cells was in intact form; average concentration was (52.8 ±3.01 and 107.8 ±8.6) and no appreciable fragmentation of nuclear DNA was found compared to MPTP group. Regarding tyrosine hydroxylase (TH) immunostaining, stem cell group showed a marked increase of percentage of TH-immunopositive neurons (63.55 ±5.2) compared to both MPTP (37.6 ±3.1) and levodopa groups (41.6 ±3.5).

Conclusions

CD34+ cells ameliorated motor, biochemical and histological deficits in MPTP-parkinsonian mice, these effects were superior to those produced by levodopa that would be promising for the treatment of PD.