人脐血单个核细胞向神经细胞分化

目的：观察培养前后人脐血单个核细胞(MNCs)的形态学及免疫反应性变化,探讨其能否向神经细胞的分化及机制。方法：密度梯度离心脐血中单个核细胞,接种并用。EGF和bFGF刺激细胞生长,倒置显微镜下观察培养前后细胞形态变化,并行免疫细胞化学鉴定。结果：培养前脐血MNCs胞体小呈圆形,nestin阳性细胞、AP2阳性细胞散在分布(阳性率为1．5％和3．4％)无GFAP阳性细胞着色。培养14d后,细胞群中相邻细胞突起连成网状;AP2、GFAP染色阳性细胞成片状分布(阳性率33．5％和24．6％),未见nestin阳性细胞。结论：脐血细胞中可能有多能干细胞,经体外培养后能分化为具有一定形态的神经细胞。     

脐血和外周血单个核细胞培养上清影响HIV-1感染的体外实验研究

目的：体外研究脐血和外周血单个核细胞（CBMC和PBMC）培养上清对HIV-1感染的影响，为发现抗HIV-1的可溶性因子奠定基础。 方法： PHA刺激CBMC和PBMC后5 h和12 h收集上清，加入荧光标记的HIV-1ⅢB/H9和MT-2细胞培养体系中，2 h后在倒置荧光显微镜下观察其对细胞融合的影响；用Luminex 100TM分析仪检测收集上清中细胞因子含量。 结果： PHA刺激CBMC和PBMC后5 h和12 h的上清均可抑制HIV-1ⅢB/H9和MT-2细胞融合，同一时间收集的PBMC和CBMC上清对HIV-1ⅢB/H9和MT-2细胞融合的抑制作用无差异，但5 h上清的抑制作用强于12 h的上清；CBMC 5 h上清中促炎症细胞因子比PBMC 5 h上清为低，而CCR5配体MIP-1α和RANTES则比PBMC 5 h上清高，差异显著（P＜0.05）。 结论： 通过脐血和外周血单个核细胞可以制备有效抗HIV感染的可溶性因子，可能为艾滋病治疗药物提供新的来源。     

Phenotypic and functional heterogeneity of natural killer cells from umbilical cord blood mononuclear cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-gamma and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the na?ve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56(hi)CD16- NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-gamma expression by CD56(hi)CD16- and CD56(lo)CD16- subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.     

人脐带血与周围血中NKT细胞频率、亚群、表型及功能的比较

目的比较足月产新生儿脐带血和正常人周围血NKT细胞的频率、亚群、表型特征及功能。方法分离足月产新生儿脐带血CBMCs和正常成年人周围血PBMCs,流式细胞术检测TCRvβ11、CD4、CD8、CD45RA、CD62L、CCR7等表面分子的表达及细胞因子IL-4和IFN-γ的产生。结果 CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞的平均频率分别为0.35%和0.33%,二者无显著差别。CBMCs中CD4+NKT细胞频率(67.39%)高于PBMCs中CD4+NKT细胞频率(54.08%),但CD8+NKT细胞,PBMCs明显高于CBMCs(22.35%对5.86%),CD4-CD8-NKT二者相差无统计学意义。CBMCs中CD3+TCRvβ11+CD45RA+NKT细胞的频率(88.37%)高于PBMCs(61.32%)。CBMCs中CD62L(56.66%对49.60%)和CCR7(22.64%对20.03%)的比例稍高于PBMCs,但其差别无显著性。未刺激CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞均不产生IL-4和IFN-γ,PMA+Ionomycin刺激后,CBMCs中CD3+TCRvβ11+NKT细胞仍不能产生IL-4和IFN-γ,但PBMCs中CD3+TCRvβ11+NKT细胞却有6.74%产生IL-4、26.96%产生IFN-γ、1.26%同时分泌IL-4和IFN-γ,产生细胞因子的CD3+TCRvβ11+NKT细胞主要为记忆性NKT细胞。结论 CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞的亚群、表型和功能均存在一定差别,记忆性NKT细胞可能与其再次免疫应答相关。     

Impaired T-lymphocyte colony formation by cord blood mononuclear cells

When compared to adult mononuclear cells, cord blood mononuclear cells demonstrated significantly decreased T-lymphocyte colony formation (1351±643 vs 592±862,P<0.01). This diminished colony-forming activity did not appear to be associated with impaired responsiveness to the stimulant phytohemagglutinin or with excessive suppressor-cell activity. Irradiation reduced the colony-forming capacity of cord blood mononuclear cells more than it did that of adult mononuclear cells. Depletion of adherent cells reduced cord blood mononuclear-cell colony-forming capacity by 40%, while similar treatment reduced adult colony formation by 10%. Lymphocyte proliferation in liquid culture of cord and adult cells was minimally affected by these procedures. The colony-forming capacity of cord blood could be enhanced by the addition of irradiated adult cells (284±72 vs 752±78,P<0.01). This enhancement was demonstrated to be due to a soluble factor produced by a population of irradiated adult cells depleted of the OKT8⁺ subpopulation of lymphocytes. These results indicate that the progenitor cells of T-lymphocyte colonies in cord blood have distinct biologic characteristics when compared to colony progenitors present in adult blood. This assay may prove to be useful in our efforts to understand the differentiation of T-cell function in man.     

脐血单个核细胞和脐带间充质干细胞治疗儿童孤独症的安全性与有效性

背景：目前国内外尚没有治疗儿童孤独症的金标准,康复治疗效果不佳。 目的：评价脐血单个核细胞和脐带间充质干细胞治疗儿童孤独症的临床安全性和有效性。 方法：37例儿童孤独症患者非随机分为脐血组、混合组和对照组。脐血组应用脐血单个核细胞加康复训练治疗;混合组联合应用脐血单个核细胞和脐带间充质干细胞加康复训练治疗;对照组单纯行康复训练治疗。脐血组和混合组患者在干细胞治疗前和首次治疗后1,2,6个月分别行相关指标实验室检查,并观察有无不良反应发生。3组患者在治疗前和首次治疗后1,2,6个月分别行儿童孤独症评定量表(CARS)和异常行为量表(ABC)评估。 结果与结论：脐血组和混合组患者在干细胞治疗前和首次治疗后1,2,6个月相关指标实验室检查未发现有意义异常变化,干细胞治疗后无严重不良反应发生;根据CARS和ABC评分,3组治疗均有效,其疗效比较：混合组优于脐血组,脐血组优于对照组。     

离心管贴壁细胞对人脐血单个核细胞体外培养成功率的影响

背景：在常规的脐血间充质干细胞密度梯度离心分离过程中,离心管壁有大量单个核贴壁细胞,这些早期的贴壁细胞是否与脐血间充质干细胞的体外培养成功率低存在一定的联系呢？ 目的：比较4种方法分离人脐血单个核细胞的体外培养成功率,观察离心管贴壁细胞对人脐血单个核细胞体外培养成功率的影响。 方法：取足月健康顺产新生儿脐血36份,随机分为4组,每组9份,于采集后6 h内分别采用甲基纤维素沉降法(A组)、密度梯度离心分离法(B组)、甲基纤维素联合密度梯度离心分离法(C组)、甲基纤维素联合改良密度梯度离心分离法(D组)分离出单个核细胞,锥虫蓝染色检测细胞活力,用细胞计数板进行细胞计数,调整细胞浓度为1×10⁹ L-1~2×10⁹ L^-1,接种于含体积分数为10%胎牛血清的低糖DMEM培养基中培养和传代,相差显微镜下观察脐血单个核细胞的形态。收集第3代脐血单个核细胞,采用细胞计数板进行计数,计算扩增倍数,采用流式细胞仪鉴定细胞免疫表型。 结果与结论：36份脐血中,成功分离33份脐血单个核细胞,B组有3份分离失败。33份中共22份原代培养中出现大量贴壁细胞,其中A组8份,B组2份,C组5份,D组7份。9份(27%)培养出能融合且可稳定传代的成纤维样细胞,其中A组3份,B组0份,C组1份,D组5份。4组脐血单个核细胞在原代培养5~7 d后均可见数量不等的贴壁细胞,呈梭形成纤维样细胞或(和)圆形巨核样细胞。A组与D组于原代培养三四周可见成纤维样细胞集落形成,细胞形态与骨髓间充质干细胞相似,呈较均一的长梭形,可稳定培养至60~90 d形态无明显变化。流式细胞仪免疫检测,第3代脐血单个核细胞不表达或弱表达CD34、CD45和CD106等造血干细胞和内皮细胞标志,但显著表达CD29、CD105等间充质干细胞表面标志。结果显示离心管贴壁细胞可显著提高脐血单个核细胞的体外培养成功率,而甲基纤维素联合改良的密度梯度离心分离法,可充分回收离心管贴壁细胞,利于脐血单个核细胞的体外扩增和稳定传代。     

CpG oligodeoxynucleotides stimulate cord blood mononuclear cells to produce immunoglobulins

CpG oligodeoxynucleotides (ODNs) stimulate adult B cells leading to cellular proliferation and immunoglobulin production. It is unknown if CpG-ODNs similarly stimulate neonatal human B cells. Neonates have immature immune responses and are poorly responsive to thymus independent antigens such as polysaccharides. We determined umbilical cord cells' response to CpG-ODNs. Adult and umbilical cord B cells produced similar amounts of IgM (adult 1371 +/- 352 vs. cord 1873 +/- 1084 ng/ml) in response to CpG-ODN stimulation. Although CpG-ODN was able to stimulate adult IgG and IgA production, cord cells produced less IgG (153 +/- 58 vs. 10 +/- 2.5 ng/ml) and no detectable IgA upon CpG-ODN stimulation. CpG ODN stimulated IgM production from adult CD27-negative B cells which may account for their ability to stimulate the mostly na?ve cord B cells. The polyclonal IgM response included pneumococcal polysaccharide antigen-specific antibodies. CpG-ODNs may be useful as neonatal vaccine adjuvants for polysaccharide antigens that are otherwise non-immunogenic.     

Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds

Cord blood expansion ex vivo can be achieved in liquid suspension through the addition of cytokines at the expense of often undesirable cell differentiation. In order to derive a cytokine-free dynamic culture system, we hypothesised that a three-dimensional (3D) environment in the form of highly porous scaffolds made of poly (D,L-lactide-co-glycolide) (PLGA) or polyurethane (PU) for the biomimetic growth of cord blood mononuclear cells (CBMNCs), would facilitate expansion of hematopoietic cells without exogenous cytokines. Both scaffolds supported cellular expansion ex vivo. Cytokine-free, long-term culture was best in PU coated with collagen type I (54-fold expansion). In contrast, traditional 2D well-plate cultures collapsed within 4 days in the absence of cytokines. CBMNCs cultured in the scaffolds were visualised by scanning electron microscopy and immunophenotypic/immunostaining analysis and the studies validated the presence of a dynamic culture containing erythroid precursors (CD45(-)/CD71(+)/CD235a(+)), hematopoietic stem/progenitor cells (CD38(-)CD34(+), CD117(+)), maturing myeloid cells (CD38(+), MPO(+)), CD4(+) and CD8(+) T-lymphocytes and megakaryocytes (FVIII(+)). Colony forming unit (CFU) assays indicated that BFU-E and CFU-GM increased (p < 0.05) whereas CFU-GEMM were maintained at week 4. In conclusion, this 3D culture system is capable of long-term, cytokine-free expansion of CBMNCs, enabling the study of hematopoiesis and providing a potential platform for drug discovery and therapeutic applications ex vivo.     

Effects of recombinant interferon-gamma on HLA-DR antigen shedding by human peripheral blood adherent mononuclear cells

Two reproducible and sensitive assays have been developed for the detection of cell-free HLA-DR antigen, an antibody-mediated complement-dependent cytotoxicity inhibition assay (CIA) and a competition enzyme-linked immunosorbent assay (CELISA). One unit of cell-free HLA-DR antigen has been quantitated to be the equivalent of 27.5 ng pure DR antigen/ml. Human peripheral blood adherent mononuclear cells (PBAMC), as well as DR+ monocytes and B lymphoblastoid cell lines but not T lymphocytes, were observed to shed DR-bearing vesicles in vitro. Human recombinant IFN-gamma induces the expression of Ia/DR antigen by PBAMC by increasing both the number of cells expressing DR antigen and the density per cell. After incubation with IFN-gamma, PBAMC also shed significantly more DR antigen. The degree of DR expression and shedding is dependent on the dose of IFN-gamma. The shedding of DR antigen occurred concomitantly with the inductive phase of cell membrane antigen expression, and very little DR antigen appeared in culture supernatants subsequent to the removal of IFN-gamma. The possible physiologic significance of shed DR antigen is discussed.     

Use of Histopaque for isolating mononuclear cells from rabbit blood

This paper describes the use of Histopaque (Hp) density gradient medium and its advantage for separating mononuclear cells (mnc) from rabbit blood. Hp solutions of density (d) = 1.083 g/ml, 1.119 g/ml and 1:1 mixture of these solutions (final d = 1.103 g/ml) were compared to Ficoll-Paque (Fp, d = 1.077 g/ml) and Lymphopaque (d = 1.086 g/ml). The average leukocyte recovery was: Fp = 26% and Lp = 17%, while that with Hp was: Hp d = 1.083: 41%; Hp d = 1.119: 43%; Hp d = 1.103: 57%. Optimum mnc recovery (76%) was obtained with Hp d = 1.103. Average mnc purity for the various media was: Fp = 88% mnc; Lp = 91%mnc; Hp d = 1.083: 94% mnc; Hp d = 1.119: 93%; Hp d = 1.103: 94% mnc. Contamination was mainly from basophils. Viability was greater than 95% in all cases. Thus, Hp density gradient media provide increased recoveries of mnc from rabbit blood compared to Fp and Lp, while purity is not affected. Rabbit mnc appear to be denser than human mnc, which are recovered in greater numbers using Fp.     

新生儿脐血单核细胞在脂多糖刺激下分泌IL-6及出现IL-6mRNA表达的实验研究

目的 :通过观察LPS对新生儿脐血单个核细胞 (MC)分泌IL 6及表达IL 6mRNA基因的影响 ,探讨严重细菌感染时新生儿机体防御反应机制。方法 :取肝素抗凝剂脐血 ,用密度离心分离法分离MNC ,以RPMI16 40培养液调整细胞浓度为1× 10 6 ml- 1 ,将细胞悬液铺于 2 4孔培养板上 ,依次加入不同浓度脂多糖 (LPS)培养 36h或同一浓度LPS(1μg ml)培养不同时间 ,收集培养上清液及细胞 ,分别用ELISA和RT PCR方法测定IL 6及IL 6mRNA表达情况。结果 :①脐血MNC在LPS刺激 3、6、12、18、2 4、36h后IL 6分泌水平逐步增高 ,6h以后增加尤为明显 ,与其他各时间点比较有非常显著的差异 (P <0 0 0 1)。LPS刺激组与无LPS对照组相同时间点比较 ,6h内IL 6变化水平无差异 ,6h以上各点有显著差异 (P <0 0 1)。RT PCR方法检测显示LPS刺激后 3h即可见IL 6mRNA基因表达。②脐血MNC受不同浓度LPS刺激时 ,IL 6分泌水平随LPS浓度递增。③全部脐血MNC均检测到IL 6mRNA基因表达。结论 :LPS能诱导新生儿脐血MNCIL 6mRNA基因转录 ,从而促使IL 6合成、分泌 ,该作用呈时间、剂量依赖性变化。     

Interaction of respiratory syncytial virus with human cord blood mononuclear cells.

This study on the interaction between respiratory syncytial virus (RSV) and human cord blood mononuclear cells shows that RSV replication can occur in neonatal macrophages. Although neonatal lymphocytes were not supportive of RSV replication, exposure to RSV resulted in significant inhibition of mitogen-induced transformation. Both adult and neonatal NK cell cytotoxicity were unaffected by exposure to RSV. These results suggest that RSV has preferential effects on human cord blood mononuclear cell subpopulations.     

人脐血多种细胞因子的含量检测及植物血凝素和脂多糖的影响

目的:通过检测人脐血血清多种细胞因子含量,观察植物血凝素(PHA)和脂多糖(LPS)的诱生影响,探讨这些细胞因子的免疫功能及移植物抗宿主病(GVHD)的发病机制。方法:采用双抗体夹心酶联免疫吸附法(ELISA),测定26份正常足月新生儿脐血及16例正常儿童外周血血清中白细胞介素4(IL-4)、IL-10、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)、IL-12、IL-15、IL-18水平,以及PHA和LPS刺激后单个核细胞分泌上述细胞因子的诱生水平。结果:人脐血血清中IL-4的水平与正常外周血血清水平无显著差异(P＞0.05),脐血血清中IL-12的水平显著高于外周血血清(P＜0.05),其余5种细胞因子脐血血清水平均明显低于正常外周血血清水平(P＜0.05);PHA和LPS刺激后脐血单个核细胞分泌IL-4、IL-10、TNF-α、IFN-γ、IL-12、IL-15和IL-18的水平明显低于正常外周血(P＜0.05),尤以IL-4、TNF-α、IFN-γ、IL-15和IL-18非常明显。结论:脐血血清中上述细胞因子水平普遍低于正常外周血,以及脐血单个核细胞刺激后产生IL-4、IL-10、TNF-α、IFN-γ、IL-12、IL-15和IL-18等细胞因子水平的不足,表明脐血细胞免疫功能不成熟,是脐血移植后GVHD发生率低及程度轻的主要原因之一。     

Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10⁶ cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10⁶ cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.     

Human cord blood mononuclear cells decrease cytokines and inflammatory cells in acute myocardial infarction

We investigated whether human umbilical cord blood mononuclear cells (HUCBC), which contain hematopoietic and mesenchymal progenitor cells, can limit myocardial cytokine expression and inflammatory cell infiltration in acute myocardial infarction. We permanently ligated the left coronary artery of rats and injected into the myocardium either Isolyte or 4 x 10(6) HUCBC in Isolyte and measured myocardial cytokines with antibody arrays at 2, 6, 12, 24, and 72 hours after infarction. We then measured with flow cytometry myocardial macrophages, neutrophils and lymphocytes at 12, 24, and 72 hours after infarctions in rats treated with either intramyocardial Isolyte or 4 x 10(6) HUCBC. In the Isolyte-treated hearts, between 2 and 12 hours after myocardial infarction, tumor necrosis factor-alpha increased from 6.7 +/- 0.9% to 52.3 +/- 4.7%, monocyte chemoattract protein increased from 9.5 +/- 1.2% to 39.8 +/- 2.1%, fractalkine increased from 11 +/- 1.5% to 28.1 +/- 1.3%, ciliary neurotrophic factor increased from 12.1 +/- 0.02% to 25.9 +/- 1.1%, macrophage inflammatory protein increased from 10.3 +/- 1.5% to 23.9.0 +/- 1.4%, interferon-gamma increased from 8.7 +/- 0.4% to 26.0 +/- 1.6%, interleukin-1beta increased from 6.1 +/- 0.04% to 19.0 +/- 1.2%, and IL-4 increased from 5.9 +/- 0.03% to 15 +/- 1.5% (all p < 0.001 compared with controls). The concentrations of fractalkine remained significantly increased at 72 hours after acute infarction. In contrast, the myocardial concentrations of these cytokines did not significantly change in HUCBC treated hearts at 2, 6, 12, 24, or 72 hours after infarction. The percentage of neutrophils increased from 0.04 +/- 0.2%/50,000 heart cells in the controls to 5.3 +/- 1.2%/50,000 heart cells 12 hours after infarction in Isolyte-treated hearts but averaged only 1.3 +/- 0.7%/50,000 heart cells in HUCBC treated hearts (p < 0.02). Thereafter, the percentages of neutrophils rapidly decreased at 24 and at 72 hours after infarction and averaged 0.6 +/- 0.2%/50,000 heart cells at 72 hours after infarction in Isolyte-treated hearts in contrast to 0.2 +/- 0.1%/50,000 cells in HUCBC hearts (p < 0.05). Moreover, the percentages of neutrophils at 24 and 72 hours in HUCBC hearts were not significantly different from controls. At 24 hours post infarction, the percentage of CD3 and CD4 lymphocytes were 10.7 +/- 1.4% and 6.3 +/- 1.1%/50,000 cells in Isolyte hearts in comparison with only 4.9 +/- 0.8% and 2.9 +/- 0.5% in HUCBC hearts (p < 0.005 for Isolyte versus HUCBC). The percentage of CD11b macrophages was 2.8 +/- 0.3% in Isolyte hearts and 1.9 +/- 0.2% in HUCBC treated hearts (p < 0.05). At 72 hours after infarction, the percentage of CD3 and CD4 lymphocytes averaged 8.0 +/- 1.1% and 5.1 +/- 0.8%/50,000 heart cells in Isolyte hearts in comparison with only 4.1 +/- 0.5% and 2.3 +/- 0.4%/50,000 heart cells in the HUCBC treated infarctions (p < 0.005). Left ventricular infarct sizes in Isolyte-treated hearts at 72 hours post infarction averaged 15.7 +/- 1.4% of the left ventricular muscle area in contrast to HUCBC treated infarctions that averaged 6.9 +/- 1.4% of the left ventricular muscle area (p < 0.02). Moreover in rats followed for 2 months post infarction, the LV ejection fractions decreased to 65.4 +/- 1.9% and 69.1 +/- 1.9% at 1 and 2 months after infarction in Isolyte-treated hearts and were significantly different from HUCBC treated hearts that averaged 72.1 +/- 1.3% and 75.7 +/- 1.4% (both p < 0.02). The present experiments suggest that an important mechanism whereby HUCBC limit infarct size and improve left ventricular ejection fraction is by significantly limiting inflammatory cytokines and inflammatory cells in infarcted myocardium.     

影响脐血间充质干细胞分离培养的关键环节

背景：脐血中存在间充质干细胞,目前国内外尚未见到对脐血间充质干细胞体外分离、培养及扩增较统一且有效的方法。 目的：探讨影响人脐血间充质干细胞成功分离培养的相关因素。 方法：分别从不同胎龄(≥40周,37周和≤32周),脐血中单个核细胞数量(≥2.5× 10⁹ L^-1,< 2.5×10⁹ L^-1), 不同细胞接种浓度(1×10⁷,1×10⁹,1×10¹¹ L^-1),不同体积分数胎牛血清(5%,10%,15%,20%)以及培养瓶是否被胎牛血清包被等方面对人脐血间充质干细胞分离培养成功率进行比较。 结果与结论：脐血间充质干细胞的分离培养成功率为58.3%,且随胎龄的增高而培养成功率降低(P < 0.01);脐血中单个核细胞浓度≥2.5×10⁹ L^-1组培养成功率高于< 2.5×10⁹ L^-1组(P < 0.01);相同容量脐血中单个核细胞数量与胎龄呈负相关(r = -0.95,P < 0.01);1×10¹¹ L^-1组原代及传代培养的间充质干细胞生长及扩增情况高于1×10⁷,1×10⁹ L^-1;体积分数5%FBS组间充质干细胞贴壁速度较其他3组略慢,但细胞纯度较高,且细胞传代速度与其他3组无明显差别;胎牛血清包被组脐血间充质干细胞原代和传代后的纯度及扩增能力均高于未包被组。提示脐血间充质干细胞分离培养成功率受多种因素的影响：通过选择较低胎龄的胎儿,采集足够量的脐血,以较高的细胞密度接种,培养基中添加较低浓度的胎牛血清,并将培养瓶预先用胎牛血清进行包被,能在体外建立稳定的脐血间充质干细胞培养体系。     

Identification of nonadherent mononuclear cells in human cord blood that differentiate into macrophages

We describe a population of nonadherent cells in neonatal cord blood that, upon in vitro cultivation, develop into monocyte-macrophages. These cells initially are negative for nonspecific esterase cytoplasmic activity, lack the monocyte marker MO.2, fall into smaller, nonmonocytic cell size areas, as determined by fluorescence-activated cell sorter (FACS)-assisted size analysis, and differentiate into macrophages under nonstimulatory culture conditions (in the absence of exogenous colony stimulating factors, less than 0.1 ng/ml endotoxin, and growth in suspension). In contrast to the adherent, committed macrophage precursors in cord blood, which differentiate into macrophages after 2-3 days of culture, the nonadherent precursor does not acquire monocyte-macrophage characteristics until day 14 of culture. Earlier induction is achieved by adding the monocyte-activating agents lipopolysaccharide or 1,25 dihydroxyvitamin D3 to cultures.     

A population of human cord blood mononuclear cells with surface alpha fetoprotein.

Suspensions of mononuclear cells from adult peripheral blood (PBL) and mononuclear cells from cord blood (CBL) were examined for the presence of surface alpha-fetoprotein (AFP) using a fluoresceinated F(ab')2 fragment of rabbit IgG anti-human AFP. The mean proportion of CBL with AFP was increased (10%) when compared with PBL (1%) although some CBL specimens did not demonstrate such an increase (range 0--15%). The presence of AFP on CBL could be either due to cytophilic AFP attached to a unique surface receptor or intrinsic AFP synthesis. The following observations could not distinguish between these two possibilities: (1) After treatment with trypsin, only minor reappearance of surface AFP could be observed in AFP-free medium in contrast to the larger numbers observed in medium containing AFP. Such selective reappearance depending on the media could be related to either cytophilic attachment of heterologous or homologous AFP or preferential stimulation of intrinsic AFP synthesis. (2) The reappearance of AFP positive CBL following trypsin treatment and incubation in media with or without AFP containing sera was inhibited by cyclohexamide. Such inhibition could be due to inhibition of synthesis of an AFP surface receptor or intrinsic AFP. (3) The shedding of surface AFP observed at 2--4 degrees C could be due to release of exogenous cytophilic AFP or the continued "turnover" of intrinsic AFP without concomitant AFP synthesis due to the cold temperature. Finally, the removal of AFP positive cells via selective depletion of B cells using bead columns coated with IgG-anti-IgG and the absence of depletion of AFP positive cells after successive gradient centrifugation of E-rosettes and cells with IgG-Fc receptors are consistent with the identity of AFP positive CBL as cells without IgG-Fc receptors or lymphocytes without conventional T-cell markers as defined by E-rosettes.