脐血血清对急性白血病化疗后骨髓抑制期CFU-GM生长的影响

探讨脐血血清对急性白血病化疗后骨髓抑制期造血细胞增殖的促进作用。采用体外CFU-GM培养法观察正常成人血清和脐血血清对正常造血细胞和急性白血病化疗后骨髓抑制期造血细胞增殖的影响。结果显示：脐血血清能明显促进正常CFU－GM和急性白血病化疗后骨髓抑制期CFU－GM生长。因此：脐血在体外能够刺激正常骨髓和急性白血病化疗后骨髓抑制期骨髓造血祖细胞增殖,提示急性白血病强化疗和干细胞移植时输注脐血可促进骨髓造血功能恢复。     

干细胞因子联合粒细胞集落刺激因子对单侧输尿管梗阻大鼠骨髓干细胞及内皮祖细胞的动员作用

目的：探讨干细胞因子(SCF)联合粒细胞集落刺激因子（G-CSF）对单侧输尿管梗阻(UUO)大鼠骨髓干细胞及内皮祖细胞的动员作用。方法：56只Wistar大鼠随机分为7组：正常对照组、SCF组、G-CSF组、SCF联合G-CSF组（SCF-G组）、假手术组、UUO组、SCF联合G-CSF用于UUO组（UUO+SCF-G组）。实验第5天采集血标本后：①流式细胞仪检测静脉血单个核细胞中CD34+、CD34+/CD133+细胞;②检测血清谷丙转氨酶、谷草转氨酶、尿素氮、肌酐水平。结果：①对照组大鼠静脉血单个核细胞中CD34+细胞为（0.13±0.01）%,假手术组CD34+细胞为（0.24±0.06）%与对照组比较差异无显著性（P>0.05）;其余各组与对照组比较CD34+细胞百分率均明显增高(P<0.05),以UUO+SCF-G组（3.04±0.42）%及SCF-G组（2.10±0.28）%增高最为明显;②对照组大鼠静脉血单个核细胞中CD34+/CD133+细胞为（0.02±0.01）%,假手术组CD34+/CD133+细胞（0.05±0.02）%与对照组比较差异无显著性（P>0.05）;其余各组与对照组比较CD34+/CD133+细胞百分率均明显增高(P<0.05),以UUO+SCF-G组（0.73±0.17）%增高最为明显;③7组血清尿素氮,肌酐、谷丙转氨酶水平无明显增高,UUO组谷草转氨酶水平较其他6组增高,差异有显著性。结论：SCF和G-CSF对干细胞和内皮祖细胞的动员效果并非完全呈平行关系,联合使用可提高内皮祖细胞和干细胞的动员率,短期内未见肝、肾毒副作用。［中国当代儿科杂志,2007,9（2）：144-148］     

环磷酰胺对小鼠骨髓造血干/祖细胞作用及机制研究

目的探讨环磷酰胺(CY)对小鼠骨髓造血干/祖细胞损伤的作用特点与机制。方法分别以大(380mg/kg)、中(200mg/kg)、小(100mg/kg)三种剂量CY腹腔注射BALB/C小鼠,注射后一周内动态检测外周血白细胞(WBC)、骨髓及外周血CD34+细胞含量、骨髓有核细胞数(NC)与细胞凋亡及病理学改变。结果①在CY处理后3～4d内,骨髓病理损伤逐渐加重,外周血WBC和骨髓NC逐渐下降直至最低值,受抑程度与CY剂量呈显著正相关。②外周血CD34+细胞与骨髓CD34+细胞水平变化基本一致,均在CY注射后1～3d进行性降低,随后中、小剂量组先一过性迅速上升至高出正常水平,然后再下降,而大剂量组则一直处于缓慢回升。③CY处理后1～3d内骨髓细胞凋亡明显增加。结论CY短期内对小鼠造血干/祖细胞损伤存在剂量—时间效应关系,中、小剂量起“动员”作用,大剂量起“摧毁”作用,诱导细胞凋亡是造血损伤的机制之一。     

造血因子对被化疗药物抑制的骨髓造血功能的影响

将２５例正常骨髓标本，经阿糖胞苷（Ａｒａ－ｃ）和柔红霉素（ＤＮＲ）抑制后，再以不同方式给予造血生长因子（ＨＧＦ），即重组人粒一单细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）和白细胞介素３（ｒｈＩＬ一３）。观察其对骨髓造血功能恢复的影响。结果表明，用ＨＧＦ组骨髓集落形成数量高于不用ＨＧＦ组；在使用化疗药物前后均用ＨＧＦ，骨髓造血功能的恢复能力比仅在化疗药物后用好。提示ＨＧＦ对被细胞毒药物抑制的正常骨髓造血功能有明显的促进作用。     

全反式维甲酸对脐血粒-单系祖细胞HOXB6基因表达的影响

目的探讨脐血造血干祖细胞(HSPC)向粒-单系(CFU-GM)增殖过程中HOXB6mRNA表达及全反式维甲酸(ATRA)对HOXB6mRNA表达的影响。方法1.采用造血祖细胞体外培养技术,以ATRA持续干扰人类造血干祖细胞,观察人脐血HSPC经人粒细胞-单核细胞集落刺激因子(GM-CSF)诱导后,在培养过程第3天,7天和12天的CFU-GM集落生成情况。2.采用实时荧光定量PCR技术(FQ-RT-PCR)检测造血祖细胞增殖分化过程中HOXB6基因的表达水平。3.结果用DNA相对拷贝数和RNA表达相对量(2-△△Ct)表示HOXB6基因相对表达量。结果1.人类造血干祖细胞向粒单系增殖分化过程中,各组细胞HOXB6基因均表达;2.随时间延长,CFU-GM-HOXB6-mRNA在增殖分化的第7天表达最强烈,第12天表达明显减弱;3.与正常对照组比较,ATRA可上调HOXB6基因的表达。结论1.在脐血CFU-GM祖细胞的不同增殖阶段,HOXB6基因呈现持续稳定的表达,提示HOXB6是人类造血干祖细胞向粒单系正常增殖分化过程中的调控基因之一;2.ATRA能显著上调HOXB6基因的表达。     

碱性成纤维生长因子对HIBD新生大鼠脑组织nestin表达的影响

目的：碱性成纤维生长因子(bFGF)对缺氧缺血脑损伤(HIBD)的重要修复作用已引起国内外学者普遍关注,但bFGF对于增强脑损伤后神经再生和修复能力的机制尚不完全清楚。巢蛋白(nestin)是一种中间丝蛋白,是胚胎神经干细胞的特征性标记,神经系统发生病变或损伤引起再生时可诱导其再表达。该研究旨在探讨外源性bFGF对HIBD新生大鼠脑组织nestin表达的影响及bFGF对新生大鼠HIBD后的神经修复机制,为临床应用bFGF奠定理论基础。方法：84只新生7日龄SD大鼠,分为3组,每组28只①假手术组;②缺氧缺血组;③bFGF干预组。结扎大鼠左侧颈总动脉和行8%低氧暴露制备新生大鼠HIBD模型。假手术组仅游离左颈总动脉但不结扎和行缺氧处理。bFGF干预组大鼠缺氧缺血后立即给予bFGF腹腔注射,4000U/kg,每天1次。每组再随机分为缺氧缺血后3,12,24h,3,7,14d组处死,每组4只。采用SABC免疫组化方法检测nestin蛋白的表达。结果：假手术组nestin蛋白在海马、室管膜弱表达,在皮质不表达。nestin蛋白在大鼠缺氧缺血脑损伤后的表达增强,nestin蛋白7d达高峰。经bFGF干预后nestin蛋白表达较缺氧缺血组增高,缺氧缺血后1d时有统计学差异,持续至14d(P<0.05)。结论：外源性bFGF可以明显增加新生大鼠缺氧缺血脑损伤后nestin蛋白表达,在缺氧缺血脑损伤后的神经元再生与修复中发挥一定保护作用。     

碱性成纤维细胞生长因子对新生鼠缺氧缺血性脑损伤胶质纤维酸性蛋白表达的影响

目的探讨外源性碱性成纤维细胞生长因子（bFGF）对新生大鼠缺氧缺血性脑损伤（HIBD）后海马胶质纤维酸性蛋白（GFAP）表达的影响。方法通过结扎并剪断7日龄新生Wistar大鼠右侧颈总动脉，吸入8％氧气和92％氮气2h制备新生鼠HIBD模型，设假手术组、生理盐水对照组、bFGF治疗组。通过免疫组织化学方法和计算机图像分析技术检测3组大鼠不同时点（术后d4、7、10、17、24）海马CAI区GFAP表达强度变化。结果假手术组海马内GFAP阳性细胞数量和染色强度术后d7达高峰：对照组、治疗组GFAP表达较假手术组增多，治疗组增多更明显，术后d10达高峰，GFAP阳性细胞主要分布于海马CAI、CA3区．术后d4、10、17组比较差异有显著性（P均〈0．05）。结论1．新生鼠HIBD后脑缺血易损伤区GFAP表达增加，可能与脑损伤后神经细胞再生有关；2．外源性给予bFGF可增强新生鼠脑缺氧缺血后中枢神经系统GFAP表达，在神经细胞损伤的修复中发挥一定保护作用。     

脐血单个核细胞移植对缺氧缺血性脑损伤新生大鼠促红细胞生成素蛋白及少突胶质祖细胞的影响

目的 探讨脐血单个核细胞(UCBMC)移植对缺氧缺血性脑损伤(HIBD)新生大鼠脑促红细胞生成素(EPO)蛋白及少突胶质祖细胞的影响。方法 7 日龄健康 Sprague-Dawley 新生大鼠40只随机分为正常对照组、HI组、UCBMC对照组和HI+UCBMC组,每组10只。采用经典Rice法制成HIBD模型,造模24 h后,正常对照组和HI组经侧脑室注入2 μL PBS,UCBMC对照组和HI+UCBMC组经侧脑室注入3×106 UCBMC。移植后7 d,采用EPO/DAPI与NG2/DAPI免疫荧光双标法观察损伤侧室管膜下区(SVZ)EPO蛋白及少突胶质祖细胞的变化,并进行相关性分析。结果 移植后7 d,HI+UCBMC组NG2+DAPI+及EPO+DAPI+细胞数均显著高于UCBMC对照组(均P<0.05)、HI组及正常对照组(均P<0.01),UCBMC对照组NG2+DAPI+及EPO+DAPI+细胞数均显著高于正常对照组和HI组(均P<0.01),正常对照组NG2+DAPI+细胞数显著高于HI组(P<0.01)。HI+UCBMC组NG2+DAPI+细胞数与EPO+DAPI+细胞数呈正相关(r=0.898)及直线回归(β=1.4604,P<0.01)。结论 UCBMC可促进HIBD新生大鼠少突胶质祖细胞的表达,其机制与EPO蛋白的表达增加相关,从而修复脑白质损伤。     

血管紧张素Ⅱ对新生大鼠心肌成纤维细胞增殖及其信号转导机制的影响

目的 研究血管紧张素Ⅱ(Ang Ⅱ)对新生大鼠心肌成纤维细胞(MFs)增殖及蛋白激酶Cε(PKCε)和蛋白激酶Cα(PKCα)的表达及转位的影响,了解AngⅡ的促增殖和信号转导机制.方法 以差速贴壁法原代分离提纯培养SD乳鼠MFs为实验模型,免疫组织化学法鉴定后传代培养,第2-4代MFs分为实验组(加AngⅡ 10-6 mol/L)和对照组(不加AngⅡ)培养;四氮唑盐(MTT)比色法检测MFs增殖;通过间接免疫荧光法观察实验组和对照组PKC亚型ε和α在细胞内的分布和定位,Image-Pro-Plus 4.0版专业图像处理软件对荧光强度进行半定量统计.结果 1.实验组MFs数量较对照组显著增加(P<0.001);2.实验组和对照组荧光显微镜下初步观察:对照组PKC亚型ε存在于细胞膜、胞质和核-细胞骨架3个部分,但均较弱;实验组PKC亚型ε在细胞膜、胞质和核-细胞骨架3个部分强度明显增强,尤以核-细胞骨架为甚;对照组PKC亚型α主要存在于核-细胞骨架;实验组PKC亚型α在细胞膜、胞质和核-细胞骨架3个部分强度均明显增加,尤以核-细胞骨架为甚;且PKC亚型ε和α荧光强度半定量比较,实验组均显著高于对照组(Pa<0.001).结论 AngⅡ有促进MFs增殖的作用,PKC亚型ε、ε可能在MFs信号转导过程中起重要作用.     

黄芪对心肌成纤维细胞增殖及分泌转化生长因子-β1的影响#br#

目的观察黄芪注射液对血管紧张素Ⅱ(AngⅡ)诱导的心肌成纤维细胞分泌转化生长因子(TGF-β1)的影响。方法体外培养乳鼠心脏成纤维细胞并制成单细胞悬液,分为对照组,黄芪低、中、高剂量组,AngⅡ组,AngⅡ联合黄芪低、中、高剂量组,分别加入50、100、200 mg/ml黄芪注射液,以及10-7 mol/L AngⅡ,共同培育2 d。用四甲基偶氮哇盐法测定各组细胞增殖情况,ELISA法测定培养上清TGF-β1分泌情况。结果各组间细胞增殖率差异有统计学意义(F=71.84,P=0.000);AngⅡ组的细胞增殖率明显升高,与其余各组比较均有统计学意义(P均<0.05);AngⅡ联合黄芪后,随着黄芪剂量的逐渐增加,细胞增殖率逐渐下降,差异均有统计学意义(P<0.05)。各组间TGF-β1分泌量差异也有统计学意义(F=786.81,P=0.000);黄芪低、中、高剂量组的TGF-β1分泌量均低于对照组,差异有统计学意义(P均<0.05);AngⅡ组的TGF-β1分泌量为各组中最高,与其余各组比较均有统计学意义(P均<0.05);AngⅡ联合黄芪后,随着黄芪剂量的逐渐增加,TGF-β1分泌量逐渐下降,差异均有统计学意义(P<0.05)。结论 AngⅡ可刺激心肌成纤维细胞增殖,促进心肌成纤维细胞分泌TGF-β1,而黄芪注射液可明显抑制AngⅡ诱导心肌成纤维细胞的增殖作用,并且减少心肌成纤维细胞分泌TGF-β1。     

Analysis and characterization of hematopoietic progenitor cells from fetal bone marrow, adult bone marrow, peripheral blood, and cord blood.

Hematopoietic stem cell transplantation has been increasingly used to replace a defective hematopoietic system and to treat various genetic defects as well as malignant diseases. However, the limitations of conventional bone marrow transplantation have stimulated an intense interest in exploring the use of alternative sources of hematopoietic stem cells, including peripheral blood mononuclear cells (PBMC) and cord blood (CB). A major investigative effort of our laboratory has been focused on evaluating fetal bone marrow (FBM) for transplantation. The current study compares and characterizes the functional and phenotypic characteristics of FBM, CB, adult bone marrow (ABM), and PBMC by clonogenicity assays, immunogenicity, and the quantification of progenitor cells. There was a striking difference in the proportion of CD34+ cells in FBM, ABM, PBMC, and CB (24.6%, 2.1%, 0.5%, and 2.0%, respectively). The clonogenic potential, as measured by colony forming unit in culture (CFU-C) assay, was significantly higher in FBM when compared with ABM, PBMC, and CB (202.5, 73.5, 40.8, and 65.5 colonies/10(5) cells, respectively). There was a significant decrease in proliferative responsiveness in mixed lymphocyte reaction (MLR) assay of FBM and CB compared with ABM and PBMC. These observations indicate that each source of hematopoietic stem cells has different intrinsic properties closely correlated with ontogenetic age that is a vital determinant for phenotypic characteristics, lineage commitments, immunogenicity, and proliferative potentials.     

In vitro hepatic differentiation of human umbilical cord blood and bone marrow cells

The purpose of the present study was to investigate whether human umbilical cord blood (UCB) as well as bone marrow (BM) can generate hepatocyte lineage cells in a simple culture condition. Mononuclear cells (MNCs) separated from UCB and BM were cultured in the presence of fibroblast growth factor (FGF)-1, FGF-2, stem cell factor (SCF), and hepatocyte growth factor (HGF). The cultured cells were analyzed for morphology and for the expression of mRNAs and/or proteins of hepatocyte lineage markers. Both the UCB and BM MNCs grown in the given culture condition yielded large, round cells that were adherent to the culture dishes. RT-PCR analysis showed that mRNAs of albumin (ALB), cytokeratin (CK)-18, and alpha-fetoprotein were expressed from day 7 in both the UCB- and BM-derived cells. Immunofluorescent staining showed that the large, round cells expressed not only ALB and CK-19 but also proliferating cell nuclear antigen, implying the proliferative potential of hepatocyte lineage cells. Therefore, UCB as well as BM can give rise to hepatocyte lineage cells in the simple culture condition with HGF, SCF, FGF-1, and FGF-2. These cells may be one of the potential candidates of cell sources for the cytotherapy of hepatic disease, although it remains to be determined if the hepatocyte lineage cells are derived from plastic hematopoietic stem cells or from liver stem cells that reside in UCB or BM.     

含人胚胎骨髓基质细胞层体系对脐血造血干/祖细胞的扩增效果

目的 研究含人胚胎骨髓基质细胞层体系对脐血造血干／祖细胞的扩增效果。方法 分别建立人胚胎骨髓基质细胞层和成人骨髓基质细胞层,观察他们单独及其与造血生长因子联合,在不同时间对脐血ＣＤ＾＋３４细胞,ＣＤ＾＋３４ＣＤ＾－３８细胞,粒单系祖细胞,多向祖细胞,早期红系祖细胞的扩增作用。结果 （１）人胚胎骨髓基质细胞单独作用及其与ＨＧＦｓ「包括脐血浆,干细胞因子（ＳＣＦ）,ＩＬ－３,ＩＬ－６,白细胞介素１β（     

Effects of cytokines on hematopoietic progenitor cells in cord blood,in bone marrow,and in peripheral blood mobilized by chemotherapy and G-CSF

We compared the effects of various combinations of cytokines (stem cell factor [SCF], interleukin [IL] ?3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO]) among the growth of human hematopoietic progenitor cells from cord blood (CB), bone marrow (BM), and peripheral blood mononuclear cells (MNC) mobilized by chemotherapy and G-CSF (PB) in a semi-solid medium. Macroscopic colonies, that were visible to the naked eye, were formed from PB-MNC within 1 week even without cytokines. They consisted of blasts containing macrophage-like cells with immature nuclei on Wright stain, and were strongly accelerated by IL-3. Macroscopic colonies were also formed from CB-MNC. However, they appeared after 1–3 weeks and synergistic effects of SCF with other cytokines, especially EPO, were prominent. Macroscopic colonies were not formed from BM-MNC. Granulocyte-colony stimulating factor was effective in increasing colony forming units of granulocyte macrophage from BM-MNC and they appeared between 1 and 2 weeks. These results suggested that the quality of hematopoietic progenitor cells was different among blood sources. This might lead to different bone marrow recovery patterns after transplantation of each blood source. The appropriate cytokines should be added to evaluate their exact potential.     

人类巨细胞病毒感染对脐血造血祖细胞增殖的影响(英文)

目的：探讨人类巨细胞病毒(HCMV)感染对脐血造血祖细胞(CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk)体外增殖的抑制作用及其机制。方法：20例脐血标本收集于正常足月顺产新生儿。实验共分5组:(1)3个HCMV感染组,每个感染组分别加入0.1 mL的103、104及105空斑形成单位(PFU)HCMV-AD169病毒液于培养体系中;(2)灭活对照组,加入同体积灭活HCMV病毒液;(3)空白对照组,不加HCMV病毒液,代之以同体积的IMDM。采用造血祖细胞体外半固体培养技术,培养、观察、计数HCMV-AD169株对脐血CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk集落数、抑制率和集落维持时间;并用聚合酶链反应(PCR)技术检测集落细胞内HCMV-DNA。结果：(1)在造血祖细胞培养体系中加入不同滴度的HCMV-AD169后,104和105PFU滴度感染对CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk集落形成均有显著的抑制作用,103PFU滴度感染对CFU-Mix及CFU-Mk集落形成有显著的抑制作用,与空白对照组和灭活对照组比较,差异有显著性(P<0.05)。病毒滴度越高,抑制程度越明显(P<0.05)。(2)104和105PFU滴度感染组CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk集落维持时间较对照组明显缩短(P<0.01),103PFU滴度感染组CFU-Mix和CFU-Mk集落维持时间较对照组明显缩短(P<0.01)。(3)PCR显示3个感染组的CFU-GM、CFU-E、CFU-Mix及CFU-Mk集落细胞内均有HCMV-AD169DNA存在。结论：HCMV-AD169能直接感染CFU-GM、CFU-E、BFU-E、CFU-Mix及CFU-Mk造血祖细胞,并抑制造血祖细胞的增殖,这可能与HCMV感染患儿出现粒细胞减少、血小板减少和贫血等造血功能紊乱有关。     

群体反应性抗体干扰脐血CD34+细胞增殖、分化的实验研究

目的 探讨血清特异性群体反应件抗体(PRA)对脐血CD34+细胞增殖、分化能力的影响.方法 取含PRA(经实验证实)的β地中海贫血患儿血清,与脐血CD34+细胞、补体孵育,观察PRA对CD34+细胞增殖、分化的影响,分A组(不加血清组)、B组(PRA血清组)、C组(PRA血清加补体组)、D组(补体组)、E组(PRA阴性血清组)共5组.孵育后以3H-TaR掺入法测定细胞DNA合成及流式细胞仪检测Annexin V和CD95表达;并进行集落培养,于第10天计数集落.结果 A组为(20.71±2.81)U/L,低于B组(64.28±5.12)U/L、C组(84.29±4.99)U/L,B组低于C组;D、E组均为(22.86±2.91)U/L和(22.86±2.91)U/L,均低于B、C组;各组氚每分钟β射线释放量(cpm):A组为(22629±3288),高于B组(4598±2178)和C组(1626±1192),A组和D、E组之间的差异无统计学意义(P＞0.05);A组的总集落数、粒-巨噬细胞集落形成单位(CFU-GM)、混合系集落形成单位(CFU-GEMM)及爆式红系集落形成单位(BFU-E)数均高于B、C组,B组的总集落数、CFU-GM及CFU-GEMM数均高于C组;D组和E组的各种集落数与A组的差异无统计学意义(P＞0.05);各组CD34+细胞Annexin V及CD95表达百分率差异无统计学意义(P＞0.05).结论 特异件PRA血清对脐血CD34+细胞的增殖和分化有抑制作用,补体可增强上述作用;特异性PRA血清对脐血CD34+细胞的凋亡无明显影响.     

Unrelated cord blood and bone marrow transplantation in pediatric leukemia

To investigate the role of cord blood as an alternative stem cell source for hematopoietic stem cell transplantation for pediatric acute leukemia, we retrospectively analyzed the outcomes of 35 unrelated cord blood transplantations (UCBT) and 56 unrelated bone marrow transplantations (UBMT) with myeloablative conditioning. The 5 year overall survival (OS) probability was 49.8% (95% confidence interval [95%CI]: 35.6–62.4%) for UBMT and 53.8% (95%CI: 34.0–70.1%) for UCBT (P = 0.92). The 5 year event‐free survival (EFS) probability was 47.3% (33.6–59.8%) for UBMT and 33.0% (15.9–51.2%) for UCBT (P = 0.38). OS and EFS were not significantly different between the groups. On multivariate analysis there was no significant difference between the groups. In conclusion, UCBT can have a role as important as that of UBMT in pediatric acute leukemia.