羟乙基淀粉沉淀(HES)法分离脐血造血干细胞的临床优势分析

目的:探讨羟乙基淀粉沉淀(HES)法分离脐血单个核细胞(MNCs)的效果。方法:采集脐血31份,应用羟乙基淀粉(HES)沉淀飞和密度离心(Ficoll)法分离脐血MNCs,、纯化CD3+细胞、CD14+细胞、CD34+细胞,以台盼蓝拒染法检测细胞活力。结果:HES法MNCs回收率、CFU-GM计数高于Ficoll法(85.29±3.79 VS 47.06±4.61,t=35.67,P0.05;67.31±11.57/l×105MNCs VS28.54±6.39/l×105MNCs,t=16.33,P0.05);HES法分离的MNCs中CD3+、CD14+、CD34+细胞数均高于Ficoll法(t=5.76,t=2.51,t=6.67,P0.05)。结论:HES法分离MNCs可获得较好的回收率,是人脐血干细胞理想的分离方法。     

转基因骨髓基质细胞系对人脐血CD34+细胞的体外扩增作用

为探讨转染FL和/或TPO基因骨髓基质细胞系对脐血CD34+细胞的体外扩增效应, 建立了转基因骨髓基质细胞系共培养体系. 采用免疫磁珠法分离人脐血CD34+细胞, 在CD34+细胞不同体外培养体系中取样测试细胞总数、CD34+细胞百分率和CFC(包括CFU-GM 和BFU-E). 结果表明, 在8种不同组合的培养体系中, 转基因基质细胞共培养体系较无基质液体培养体系对细胞总数, CFC, CD34+细胞均具有明显的扩增效应, 其中以SCF + IL-3 + HFT扩增效果最好, 分别扩增了(893.3±52.1), (74.5±5.2)和15.7倍. CFU-GM和BFU-E在第2周时达扩增高峰, 扩增倍数分别为(78.1±5.5)和(57.0±19.7). LTC-IC测定结果显示, 只有SCF + IL-3 + FL + TPO和SCF + IL-3 + HFT组有LTC-IC的存在, 统计学检验无显著性差异. 上述结果提示, 转基因骨髓基质细胞系可通过细胞间的接触协同其他细胞因子增强对脐血CD34+细胞的体外扩增作用.     

人脐血CD133细胞的分离及其体外扩增的研究

目的:探讨免疫磁性纳米粒子分离人脐血CD133细胞的方法,了解分离出的CD133细胞在体外短期培养中的变化及其在体外扩增的可能性。方法:通过化学沉淀法制备具有超顺磁性的r-Fe_2O_3纳米粒子,在其表面包裹具有生物亲合性的二氧化硅,并在其表面通过化学修饰使其成为生物功能化的磁性纳米粒子。再通过一定的化学连接方法将单克隆抗体CD133连接到生物功能化的磁性纳米粒子表面使其成为免疫磁性纳米粒子,然后利用自制的免疫磁性纳米粒子从单个核细胞中分离出CD133细胞,并分别对单个核细胞和CD133细胞在体外短期培养中的动态变化进行了初步观察和比较。结果:经免疫磁性纳米粒子分离的脐血中CD133细胞平均数为(5±1．4)×10~7/ml,占单个核细胞数的(3±0．3)%;单个核细胞(对照组)和CD133细胞(实验组)分别进行红、粒系集落扩增培养14天、21天,实验组中两种造血祖细胞集落扩增倍数都明显高于对照组(P＜0．01)。结论:使用自制的免疫磁性纳米粒子能较好的分离脐血中的CD133细胞,分离与纯化出来的CD133细胞不仅细胞活力不受影响,而且与单个核细胞相比具有更强的增殖能力。     

CD34+和CD34-细胞在NOD/SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型, 比较了新鲜及培养后的CD34+和CD34-细胞在体内植入及重建造血能力。从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34+和CD34-细胞, 经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内, 6周后处死存活的小鼠, 取其骨髓、脾脏和外周血细胞, 分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测。经检测, 输注CD34+细胞和混合细胞的小鼠, 其体内CD45+细胞及人源各系血细胞的含量相近, 两者均远远高于输注CD34-细胞的小鼠。输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34+细胞的小鼠存活率约为66.7%, 而输注培养后混合细胞的小鼠全部存活, 且在两组存活的小鼠体内均能检测到CD45+细胞及人源各系血细胞。结果表明: 无论是新鲜还是培养后的CD34+细胞均具有在NOD/SCID小鼠体内植入和重建造血能力, 而CD34-细胞不具有该能力, 但CD34-细胞与CD34+细胞同时输注有助于提高小鼠的存活率, 说明其对CD34+细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用。     

脐带间充质干细胞联合UM171对脐血源CD34~+细胞的扩增效果研究

目的研究脐带间充质干细胞联合UM171对脐血来源CD34~+细胞的扩增效果。方法脐血来源CD34~+细胞及脐带来源间充质干细胞分为以下4组进行体外扩增培养10 d:对照组、UM171培养组、间充质干细胞共培养组、UM171联合间充质干细胞共培养组,采用方差分析比较不同组别间细胞扩增倍数及流式表型和集落培养情况。结果脐带间充质干细胞CD105,CD73,CD90,不表达CD14,CD34,CD19,CD45,HLA-DR,经过诱导可以向成骨细胞、脂肪细胞、软骨细胞分化。CD34~+细胞在不同条件下体外培养10 d后,UM171培养组总有核细胞数扩增14倍,CD34~+细胞扩增13.5倍;MSCs共培养组总有核细胞数扩增11倍,CD34~+细胞扩增10倍;联合培养组总有核细胞数扩增达22倍,CD34~+细胞扩增21倍。联合培养组扩增后细胞CD34~+CD38~-比例达(91.49±2.67)﹪,较间充质干细胞培养组(78.11±2.35)﹪及UM171培养组(91.49±2.68)﹪相比差异具有统计学意义(P均0.01)。扩增后细胞集落培养14 d后,各系集落形成良好,UM171扩增组细胞较MSCs扩增组在红系及粒系形成能力方面存在优势。结论脐带血间充质干细胞作为细胞滋养层可提高CD34~+细胞体外扩增效果,UM171在扩增过程中可较好的保持细胞干性,二者联合应用扩增效果最佳,建立的脐带间充质干细胞联合UM171对脐血源CD34~+细胞的扩增方法可用于CD34~+细胞体外扩增培养。     

低氧对CD34~ 造血干/祖细胞增殖分化及其对细胞因子依赖性的影响

采用免疫磁珠法分离脐血CD34 造血干 /祖细胞 ,进行低氧和常氧条件下单个核细胞 (MNC)及CD34 细胞的半固体及液体培养 ,计细胞总数和集落产率 ,并通过流式细胞仪检测细胞表型和细胞周期 ,以探讨造血干/祖细胞在低氧环境下增殖分化性能的改变及其对细胞因子反应性的变化。结果显示 :CD34 细胞在低氧条件下生成的BFU E集落数 ( 32 4 8± 41 4/10 4 细胞 )明显增多 (对照为 191 2± 34 5 /10 4 细胞 ,P <0 0 1) ;在无细胞因子存在的液体培养体系中 ,低氧组的BFU E产率 ( 15 2 4± 2 2 6 /10 4 细胞 )明显高于常氧组 ( 74 2± 9 3/10 4 细胞 ,P <0 0 1) ;低氧培养细胞中CD34 细胞的比例高于对照 2 5± 1 2倍 (P <0 0 5 )。但MNC生成的BFU E在常氧和低氧条件下无显著差异。这些结果表明 :体外低氧环境能显著增加CD34 造血干 /祖细胞形成红系祖细胞的产率 ,且使其对细胞因子的依赖性降低 ,并对早期红系祖细胞的维持有增强作用 ,但对粒系祖细胞的增殖则有抑制作用     

免疫磁珠分离淋巴细胞在流式交叉配型试验中的应用及研究

目的探讨免疫磁珠快速分离法及密度梯度离心(Ficoll)分离法分离外周血单个核细胞(PBMC)在流式细胞交叉配型中的应用比较。方法取10例交叉配型供体外周抗凝血各10 ml,受体非抗凝血5 ml,分别采用密度梯度离心与免疫磁珠阴性选择试剂盒从相同体积的抗凝血中分离PBMC。用血细胞计数比较两种方法分离的WBC、PLT、RBC粒度分布和PBMC中淋巴细胞的纯度;用细胞流式和淋巴细胞表面标记荧光抗体检测两种方法分离得到的PBMC中T、B和NK细胞的数量质量;将免疫磁珠阴性分离和密度梯度离心法分离的供者PBMC与受者血清共孵育,加入羊抗人IgG-FITC,洗涤后加入抗人CD3-PE、抗人CD19-APC单克隆抗体,再用流式细胞仪检测细胞表面荧光强度。采用方差分析和t检验进行统计学分析。结果免疫磁珠阴性分离的PBMC数量是Ficoll分离PBMC的0.42倍,但淋巴细胞的比例[(99.2±0.08)﹪]高于PBMC分离的淋巴细胞比例[(82.5±5.27)﹪(t=9.91,P0.01)],且两种方法分离的PBMC中RBC分别为(0.001±0.001)×10~6/μl和(0.02±0.009)×10~6/μl(t=6.64,P0.001);血小板的数量分别为(1.00±0.05)×10~3/μl和(196.00±4.21)×10~3/μl差异均有统计学意义(t=146.46,P0.01)。流式细胞检测免疫磁珠分离的PBMC中CD3~+T细胞为(81.33±4.60)﹪,CD19~+B细胞为(8.41±0.87)﹪,CD3-CD56~+NK细胞为(9.35±0.67)﹪和CD3~+CD56~+NKT细胞为(2.47±0.07)﹪。而Ficoll分离的PBMC中CD3~+T细胞为(37.36±3.27)﹪,CD19~+B细胞为(5.79±0.94)﹪,CD56~+NK细胞为(6.60±0.91)﹪,且差异均有统计学意义(t=24.64、6.470、7.70、51.31,P均0.01)。T和B淋巴细胞流式交叉配型试验中,设门定量读取T和B淋巴细胞与受者血清中抗体结合情况,密度梯度离心获得的淋巴细胞中混有血小板等,使流式检测结果中会混有假阴性,而免疫磁珠分离法没有出现假阴性结果。证明免疫磁珠分离的PBMC可应用于临床交叉配型试验。结论免疫磁珠阴性选择分离全血PBMC,用时短,纯度高,去除了99﹪的血小板,不混有红细胞、血小板,是一种优于Ficoll分离PBMC的新方法。     

人CD34^＋和CD34^-细胞在NOD／SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型,比较了新鲜及培养后的CD34 和CD34-细胞在体内植入及重建造血能力.从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34 和CD34-细胞,经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内,6周后处死存活的小鼠,取其骨髓、脾脏和外周血细胞,分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测.经检测,输注CD34' 细胞和混合细胞的小鼠,其体内CD45 细胞及人源各系血细胞的含量相近,两者均远远高于输注CD34-细胞的小鼠.输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34 细胞的小鼠存活率约为66.7%,而输注培养后混合细胞的小鼠全部存活,且在两组存活的小鼠体内均能检测到CD45 细胞及人源各系血细胞.结果表明:无论是新鲜还是培养后的CD34 细胞均具有在NOD/SCID小鼠体内植入和重建造血能力,而CD34-细胞不具有该能力,但CD34-细胞与CD34 细胞同时输注有助于提高小鼠的存活率,说明其对CD34 细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用.     

鼠骨髓源性内皮祖细胞的分离培养与鉴定

目的探讨大鼠骨髓源性内皮祖细胞(endothelial progenitor cells,EPCs)的分离培养鉴定的方法.方法 Percoll(1.077 g/ml)分离液分离大鼠骨髓单个核细胞,血管内皮生长因子(Vascular Endothelial Growth Factors, VEGF)和碱性成纤维细胞生长因子(basic Fibroblast Growth Factors, bFGF),对其进行诱导培养,光镜观察EPCs形态,免疫荧光检测血小板内皮细胞粘附分子-1(PECAM-1/CD31)、血管内皮钙粘蛋白(VE-cadherin/CD144)、荆豆凝集素-1(FITC-UEA-1)的表达和摄取Dil荧光标记的乙酰化-低密度脂蛋白(Dil-ac-LDL).结果 诱导培养7 d后,可见集落和铺路石样结构,激光扫描共聚焦显微镜(Laser Scanning Confocal Microscope, LSCM)显示表型为CD31+VE-cadherin+双阳性细胞以及具有内皮细胞功能的Dil-ac-LDL和FITC-UEA-1双染色细胞.结论 采用Percoll(1.077 g/ml)密度梯度离心结合VEGF、bFGF诱导培养可以获得EPCs,说明该培养方法可行.     

人脐血CD34+细胞来源的树突状细胞疫苗在SCID小鼠体内的抗肿瘤免疫作用

目的观察脐血CD34 干细胞来源的树突状细胞(dendritic cells, DC)疫苗在严重联合免疫缺陷(severecombined immunity deficiency,SCID)小鼠体内对人肝癌细胞的免疫治疗和免疫保护作用.方法采用微磁珠分选系统(Mini MACS)从脐血单个核细胞中分离CD34 干细胞.重组人粒细胞-巨噬细胞集落刺激因子(recombined human granulocyte-macrophage colony-stimulating factor, rhGM-CSF)、重组人肿瘤坏死因子(recombined human tumor necrosis factor,TNF)-α诱导脐血CD34 细胞向DC分化,相差显微镜下观察DC分化过程中细胞的形态及数量变化.用人肝癌细胞BEL-7402裂解物冲击DC制备DC疫苗.在SCID小鼠体内观察DC疫苗致敏的淋巴细胞对人肝癌细胞的免疫治疗和免疫保护作用.结果脐血CD34 细胞在细胞因子诱导下,细胞形态由小变大、由圆形逐渐变为不规则形;细胞分裂扩增过程中,数量逐渐增多,形成细胞集落.经过细胞因子联合诱导2周的DC胞质突起丰富,具有典型的树枝状形态.在体内的抗肿瘤实验中,经DC疫苗致敏的人外周血淋巴细胞治疗荷瘤小鼠,肿瘤生长速度、瘤体积和重量明显小于未致敏淋巴细胞治疗组(P<0.05).与未致敏淋巴细胞免疫组和DC疫苗致敏的淋巴细胞治疗组比较,经DC疫苗致敏的淋巴细胞免疫SCID小鼠,肝癌细胞攻击后,肿瘤的发病率降低(P<0.05),发病潜伏期延长(P<0.01),肿瘤体积和重量减小(P<0.05).结论人脐血CD34 细胞来源的DC疫苗能在一定程度上抑制人肝癌细胞在小鼠体内的生长,抵抗肿瘤细胞的攻击,对机体具有相应的抗肿瘤免疫治疗和免疫保护作用.     

胚胎干细胞起源的探讨

目前胚胎干细胞(ESCs)建系的取材来源包括桑椹胚的卵裂球、囊胚的内细胞团(ICM)、上胚层细胞和原始生殖细胞(PGCs),甚至从新生鼠睾丸细胞也分离得到类ES样细胞系。这就提出了一个问题,什么是ESCs最接近的体内细胞来源。传统观念常常把ESCs等同于ICM细胞,也有学者认为ESCs更象上胚层细胞,而在已知的分子标记基因方面,ESCs所具有的特征更接近体内早期生殖细胞。不清楚ESCs最接近的体内细胞来源,可能是制约许多品系小鼠和大多哺乳类动物建系成功率提高的原因之一。ESCs系与EG细胞系的分离条件不同表明,加强对ESCs多能性维持基因调控研究具有重要意义。本文从ESCs的经典概念及其发展,早期胚胎细胞和生殖细胞发育规律,早期胚胎细胞、早期生殖细胞和ESCs的关系等方面进行综合分析,认为ESCs可能有多种接近的体内细胞来源。进一步应通过对ESCs建系不同的取材细胞和不同品系的ESCs间进行比较研究,以便弄清ESCs的来源和转化机制,为提高不同物种ESCs建系效率提供理论支持。     

Fine needle aspiration (FNA) cytology of pilomatrixoma

FNA smears from five histologically confirmed cases of pilomatrixoma were reviewed to delineate the cytological features helpful in diagnosis. A combination of basaloid cells, ghost cells and foreign body giant cells appeared to be necessary in FNA smear for a confident cytodiagnosis of pilomatrixoma. Presence of naked nuclei, nucleated squamous cells and calcification were additional features in favour of the diagnosis. Another 10 cases with initial cytodiagnosis of pilomatrixoma or benign skin appendage tumour were reviewed. Using the above criteria, diagnosis of pilomatrixoma was easy in five cases. One case was problematical due to presence of atypical squamous cells. Initially the cytological features were most commonly confused with epidermal inclusion cyst, giant cell lesion or a squamous cell carcinoma. The main reasons for erroneous diagnosis were lack of awareness of cytological features, predominance of one component over the others, and non‐representative FNA smears. Atypia in nucleated squamous cells, and misinterpretation of basaloid cells as malignant can lead to diagnostic dilemma. Adequate clinical data are also necessary.     

树突状细胞与CIK细胞共培养诱生的DCCIK细胞群对肿瘤的杀伤作用

由树突状细胞(DC)与细胞因子诱导的同源杀伤细胞(CIK)的共培养诱生的细胞群(DCCIK)对肿瘤细胞的细胞毒活性的研究。DCCIK细胞体外杀伤肿瘤靶细胞A549(MTT法),效靶比为10∶1、5∶1时杀伤率分别为61%、52%。DCCIK细胞诱导培养3周后,效靶比为10∶1、5∶1时杀伤率分别为64%和56%。数据亦表明DCCIK细胞对靶细胞的杀伤优于CIK细胞。动物体内实验分荷瘤A549、BEL7404和A375三组,每组分(A)DCCIK 化疗、(B)单用化疗。治疗20天、35天后测量各组肿瘤消失率。结果显示:DCCIK 化疗的抑瘤效果明显好于单纯化疗。提示DCCIK细胞有临床应用前景。     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.     

Ultrastructure of the surface of the iris in the rat eye

Summary The surface structure of the iris in the rat eye was studied by light and electron microscopy.The anterior surface of the rat iris is covered with a discontinuous layer of large, polygonal endothelial cells with microvilli on their surface. Crypts and holes between adjacent endothelial cells extend into the stroma and form there a complicated network of interconnected spaces occupying about one half or more of the volume of the pupillary part of the stroma. The crypts are occasionally partly covered with endothelial cells. The posterior surface is covered with a continuous layer of polyhedronal epithelial cells. These are covered with many folds and processes, partly masked by an amorphous coat. The sphincter pupillae and dilatator muscles are possible to recognize on the scanning electron micrographs as well as blood vessels and nerve fibers in the iris stroma.The endothelial cells show many structural similarities with the endothelial cells on the cornea, probably reflecting their common origin. The results obtained, especially those from the scanning electron microscopic studies, are discussed and interpreted in relation to previous studies. The advantages in using different light and electron microscopic techniques are stressed.Supported by grants from Magnus Bergwall's Stiftelse and the Swedish Medical Research Council (B71-12X-2543-03).     

Enhancement of BDNF production by co-cultivation of human neuroblastoma and fibroblast cells

It has been proved that co-cultivation of human neuroblastoma cells and human fibrolast cells can enhance nerve cell growth and the production of BDNF in perfusion cultivation. In batch co-cultivation, maximum cell density was increased up to 1.76×10⁶ viable cells/mL from 9×10⁵ viable cells/mL of only neuroblastoma cell culture. The growth of neuroblastoma cells was greatly improved by culturing both nerve and fibroblast cells in a perfusion process, maintaining 1.5×10⁶ viable cells/mL, which was much higher than that from fed-batch cultivation. The nerve cell growth was greatly enhanced in both fed-batch and perfusion cultivations while the growth of fibroblast cells was not. It strongly implies that the factors secreted from, human fibroblast cells and/or the environments of co-culture system can enhance both cell growth and BDNF secretion. Specific BDNF production rate was not enhanced in co-cultures; however, the production period was increased as the cell growth was lengthened in the co-culture case. Competitive growth between nerve cells and fibroblast cells was not observed in all cases, showing no changes of fibroblast cell growth and only enhancement of the neuroblastoma cell growth and overall BDNF production. It was also found that the perfusion cultivation was the most appropriate process for cultivating two cell lines simultaneously in a bioreactor.     

间充质干细胞在动脉粥样硬化治疗中的研究进展

动脉粥样硬化是一种病因复杂的血管壁慢性炎症性疾病。动脉粥样硬化及其相关并发症已成为人类死亡的主要原因,然而,其病因和发病机制尚未完全阐明,治疗效果还不满意。目前已经证实,动脉内皮细胞功能发生障碍是动脉粥样硬化的始动过程,内皮细胞功能失调和内皮细胞丢失是动脉粥样硬化症的主要特点;而血管平滑肌细胞的异常增生在动脉粥样硬化的发生发展中也扮演着重要角色。因此,探索有效措施促进有益的内皮细胞再生并抑制平滑肌细胞增生是血管损伤防治的关键。近年来有研究发现,体外输注的间充质干细胞能够向受损部位募集,并进一步分化为内皮细胞,修复损伤血管。然而,也有研究显示体外输注的间充质干细胞还可以分化为血管平滑肌细胞进而在血管局部增生,参与血管再狭窄的发生。文中综述了间充质干细胞输注对动脉粥样硬化发展的最新研究进展,希望为后续开展的用间充质干细胞治疗动脉粥样硬化的研究提供一定的参考。     

Resistance and augmentation of innate immunity in mice exposed to starvation

Mice were exposed to starvation for 3 days. Body temperature and various parameters were examined. By starvation, body temperature, blood glucose and ACTH decreased, especially on days 2 and 3. The level of corticosterone increased at this time. On the other hand, the number of lymphocytes yielded by the liver, spleen and thymus decreased from day 1 to 3. The change of the distribution of lymphocyte subsets was unique because NK, NKT and extrathymic T cells were stress-resistant in the liver. Conventional T and B cells were stress-sensitive. Reflecting the increased proportion of NK and NKT cells, NK and NKT activities were augmented. The increased proportion of NKT cells produced both IFNγ and IL-4 (Th0-type profile). The proportion and some functions of granulocytes and macrophages increased on Day 1 after starvation. These results suggest that starvation has a potential to increase the functions of unconventional lymphocytes and myeloid cells.