优化骨髓单个核细胞分离技术的分析

目的优化骨髓单个核细胞分离技术以更好地适用于不同需要的临床治疗。方法应用COBE Spectra血液成分分离系统,按选择确定的参数分离患者骨髓单个核细胞[34人,分离量(500±50)ml/人],对分离前后的单个核细胞做白细胞分类,细胞计数检测及CD34、CD133、CD271的检测,计算有核细胞回收率、单个核细胞回收率,应用统计方法分析CD271+和CD133+表达与细胞形态学分类的相关性。结果1)相关分析:分离前后CD271+表达率均与类单核样细胞数目相关(P均<0.01),相关系数分别为0.730和0.774;分离后CD34+表达率与类单核样细胞和类淋巴样细胞数目均相关(P均<0.01),相关系数分别为0.741和0.645。2)偏相关分析:CD271与类单核样细胞相关(P均<0.01),CD34与类淋巴样细胞相关(P均<0.01)。结论应用该优化分离骨髓单个核细胞技术,选择相应的采集参数,获得了高效率的单个核细胞;分离后的骨髓可用于患者回输治疗。     

治疗用骨髓间充质干细胞分离的相关因素分析

背景：骨髓间充质干细胞的分离、鉴定技术在移植治疗中非常关键。目的：应用Cobe Spectra6．1骨髓处理程序分析骨髓间充质干细胞的分离方法。设计、时间及地点：以细胞为对象的观察性实验，于2005—11／2007—11在北京军区总医院完成。材料：骨髓来自进行白体骨髓干细胞移植治疗的缺血性股骨头坏死患者。方法：应用COBE Spectra血液成分单采系统骨髓单个核细胞分离程序，在已建立的运行参数条件下分离骨髓间充质干细胞。主要观察指标：分离前后进行白细胞分类、细胞计数检测、流式细胞分析CD34、CD133和CD271的表达，计算有核细胞回收率、单个核细胞回收率；统计方法分析CD271^＋和CD133^＋表达与细胞形态学分类的相关性。结果：①骨髓分离后，有核细胞、单个核细胞明显高于分离前（P均〈0．01），回收率分别为26．6％、40．0％。②分离的细胞产品中，CD271^＋、CD133^＋和CD34^＋表达率分别为0．63％、0．51％和1．17％，回收率则依次为72．8％、61．6％和67．6％。③相关分析显示，分离前后CD271^＋表达率均与类单核样细胞数目相关（P均〈0．01）；分离后CD34^＋表达率与类单核样细胞和类淋巴样细胞数目均相关（P均〈0．01）。④偏相关分析显示，CD271与类单核样细胞相关（P均〈0．01），CD34与类淋巴样细胞相关（P均〈0．01）。结论：应用Cobe Spectra6．1骨懿处理程序分离骨髓干细胞用于临床治疗，可以根据治疗需求选择分离类单核样细胞或类淋巴样细胞，有效提高分离效率。     

围术期心血管危险因素对骨髓祖细胞的影响简

背景：移植自体骨髓干细胞治疗缺血性心脏病已进行了10余年的临床试验,但试验结果在不同的患者中存在差异。因此,有必要鉴定哪些心血管病患者的危险因素影响骨髓干细胞的水平和功能。目的：观察冠心病患者围术期危险因素对骨髓祖细胞数量及功能的影响。方法：选择44例拟行冠状动脉旁路移植的冠心病患者,采集实验室和临床资料;术中经胸骨穿刺采集骨髓,应用 Ficol 淋巴细胞分离液密度梯度离心法分离骨髓单个核细胞,计数并应用锥虫蓝拒染法检测其活性;应用流式细胞仪分析检测CD34＋、CD133＋和CD34＋CD133＋细胞的水平;应用集落形成试验和细胞迁移试验评价骨髓祖细胞功能。结果与结论：术中经胸骨抽取20 mL骨髓可获得（10-89）×106个骨髓单个核细胞,活性在95%以上,等量的骨髓血获得的骨髓单个核细胞的量与患者年龄之间存在明显负相关关系（n=44,r=-0.788,P=0.001）;流式细胞仪检测 CD34＋细胞占（0.94±0.39）%,CD133＋细胞占（0.46±0.28）%,CD34＋CD133＋细胞占（0.53±0.26）%;糖尿病患者骨髓 CD34＋和 CD133＋细胞水平明显低于非糖尿病患者;高龄、女性和心功能较差与骨髓祖细胞集落形成能力降低有关;CD34＋细胞水平与骨髓单个核细胞的迁移能力存在明显的正相关。结果表明经胸骨应用密度梯度离心法可获得足够数量的骨髓单个核细胞作为缺血性心脏病治疗的供体细胞,年龄、性别、糖尿病、心功能与骨髓单个核细胞数量和功能有关。     

流式细胞术联合检测骨髓细胞CD271、CD133、CD34的表达与分析

本研究检测骨髓细胞的CD271、CD133和CD34的表达,分析细胞表达CD271与CD133及CD133与CD34的相关性。根据不同设计组合用CIN5-PerCP、CD271-PITC、CD133-PE和CD34-FITC标记骨髓细胞,获得细胞后以FSC、SSC和CD45反复对细胞群进行定位和筛选,然后用流式细胞术检测骨髓细胞和骨髓单个核细胞（MNC）的上述表达。结果表明,骨髓细胞CD271^＋、CD133^＋和CD34^＋表达率分别为0．16％、0．20％和0．43％,骨髓单个核细胞分离后CD271^＋、CD133^＋和CD34^＋表达率分别0．49％、0．47％和1．07％;骨髓细胞的CD271^＋CD133^共表达率为0．02％,单个核细胞分离后的CD271^＋CD133^＋的共表达率为0．03％。90％CD133^＋细胞表达CD34,40％CD34^＋细胞表达CD133。结论：建立的三色荧光联合检测方法可作为检测骨髓中CD271^＋,CD133^＋和CD34^＋细胞的方法。CD271阳性细胞与CD133和／或CD34阳性细胞是不同的细胞群,CD271可能在评价和指导骨髓间充质干细胞的临床治疗中有重要意义。     

骨髓基质细胞联合细胞因子对脐血单个核细胞表面抗原表达的影响

背景:课题组已建立胎儿骨髓基质细胞联合细胞因子的造血细胞体外培养体系,该培养体系能否有效扩增各个发育阶段的造血细胞有待验证。目的:观察骨髓基质细胞联合细胞因子培养体系对脐血单个核细胞表面抗原CD133、CD34表达的影响。方法:将从脐血标本中分离出来的单个核细胞接种于无血清培养体系,实验分为3组:①F组:干细胞因子+Flt3配体+促血小板生成素+单个核细胞。②S组:基质细胞+单个核细胞。③SF组:基质细胞+干细胞因子+Flt3配体+促血小板生成素+单个核细胞。在第0,6,10,14天检测有核细胞总数、CD133+、CD34+、CD133+CD34+细胞数以及集落形成单位数。结果与结论:SF组有核细胞总数在各个检测时间点均比其他两组高;除了第14天外,第6、10天两个时间点SF组中CD133+、CD34+、CD133+CD34+细胞及集落形成单位数均高于其他组;含骨髓基质细胞的S组和SF组中CD133+细胞/有核细胞、CD34+细胞/有核细胞、CD133+CD34+细胞/有核细胞的比例保持在较高的水平。结果说明骨髓基质细胞联合细胞因子能有效的扩增脐血单个核细胞及其中的CD133+、CD34+、CD133+CD34+细胞,基质细胞对维持造血干细胞的原始性具有重要的作用。     

外周血造血干细胞采集方法的选择与分析

目的以Cobe Spectra细胞分离系统为基础,为外周血造血干细胞的采集选择效率高,经济、运行时间短,更切合临床实际工作的方法提供客观依据。方法分别用Cobe Spectra细胞分离系统的Auto PBSC(外周血干细胞自动采集)方法和MNC(单个核细胞采集)方法采集外周血干细胞,对采集产品的进行细胞计数,记录采集过程参数,计算有核细胞、单个核细胞采集数和采集时间,分析采集效率以及经济成本。应用统计学分析方法,对这2种不同方法进行比较分析。结果 1)2组间处理血量差异没有统计学习意义,Auto PBSC采集组的采集产品中有核细胞数浓度和单个核细胞百分比高于MNC采集组,但产品体积小于MNC采集组(T=-1.704,0.494,1.941,1.742,P>0.05);2)MNC采集组获得的产品中有核细胞总数、单个核细胞数及CD34+细胞浓度均显著高于AutoPBSC采集组,采集所用的时间差异无统计学意义(T=-3.596,-0.349,13.188,-2.554,-2.818,P<0.05);3)MNC采集组的有核细胞采集率(22%)和单个核细胞的采集率(97.7%)也高于Auto PBSC采集组的15%和85%(T=5.5754,.572,4.3842,.926,-0.044,3.229,P<0.05)。结论 MNC采集外周血干细胞的采集效率优于AutoPBSC采集,并且经济。     

不同血细胞分离机采集程序对健康供者造血干细胞采集物细胞成分的影响

目的:观察COBE Spectra血细胞分离机AutoPBSC程序以及Spectra Optia血细胞分离机MNC程序对采集的健康供者外周血造血干细胞成分的影响。方法:2015年1月至2016年8月期间对12例供者随机应用COBE Spectra血细胞分离机AutoPBSC程序以及Spectra Optia血细胞分离机MNC程序进行外周血造血干细胞采集,观察应用不同采集程序获得的采集物中单个核细胞、CD34~+细胞、粒细胞、淋巴细胞、血小板数的差异。结果:两种采集程序在循环血量、采集时间及抗凝剂使用量方面无显著差异。COBE Spectra血细胞分离机AutoPBSC程序采集的采集物体积及单个核细胞计数均低于Spectra Optia血细胞分离机MNC程序的采集物体积及单个核细胞计数。AutoPBSC程序采集的CD34~+细胞计数高于MNC程序采集的CD34~+细胞计数。AutoPBSC程序采集物中混入的淋巴细胞及血小板较MNC程序中多,其差异均有统计学意义(P0.05)。结论:与Spectra Optia血细胞分离机MNC程序相比,COBE Spectra血细胞分离机AutoPBSC程序采集的造血干细胞数量多,混入的淋巴细胞及血小板多。     

骨髓基质细胞联合细胞因子对脐血单个核细胞表面抗原表达的影响

背景：课题组已建立胎儿骨髓基质细胞联合细胞因子的造血细胞体外培养体系,该培养体系能否有效扩增各个发育阶段的造血细胞有待验证。目的：观察骨髓基质细胞联合细胞因子培养体系对脐血单个核细胞表面抗原CD133、CD34表达的影响。方法：将从脐血标本中分离出来的单个核细胞接种于无血清培养体系,实验分为3组：①F组：干细胞因子＋Flt3配体＋促血小板生成素＋单个核细胞。②S组：基质细胞＋单个核细胞。③SF组：基质细胞＋干细胞因子＋Flt3配体＋促血小板生成素＋单个核细胞。在第0,6,10,14天检测有核细胞总数、CD133^＋、CD34^＋、CD133^＋CD34^＋细胞数以及集落形成单位数。结果与结论：SF组有核细胞总数在各个检测时间点均比其他两组高;除了第14天外,第6、10天两个时间点SF组中CD133^＋、CD34^＋、CD133^＋CD34^＋细胞及集落形成单位数均高于其他组;含骨髓基质细胞的S组和SF组中CD133＋细胞/有核细胞、CD34＋细胞/有核细胞、CD133＋CD34＋细胞/有核细胞的比例保持在较高的水平。结果说明骨髓基质细胞联合细胞因子能有效的扩增脐血单个核细胞及其中的CD133^＋、CD34^＋、CD133^＋CD34^＋细胞,基质细胞对维持造血干细胞的原始性具有重要的作用。     

骨髓基质细胞对多细胞因子介导人脐血单个核细胞体外扩增的影响

背景:体外扩增脐血造血细胞的目的是促进脐血造血细胞的植入能力,细胞因子介导的脐血造血细胞能使细胞数有效扩增,但同时也耗竭了具有分化潜能的造血干细胞.目的:观察骨髓基质细胞对多细胞因子组合介导人脐血单个核细胞体外扩增及扩增后黏附分子和CXCR4表达的影响.方法:将分离的人脐血单个核细胞接种在预先建立的人骨髓基质细胞层上,分组培养:对照组仅含有脐血单个核细胞;多种细胞因子+脐血单个核细胞组;骨髓基质细胞+脐血单个核细胞组;骨髓基质细胞+多种细胞因子+脐血单个核细胞组.培养0,7 d检测有核细胞数, CD34+细胞数及CD34+CXCR4+,CD34+CD62L+,CD34+CD49d+的细胞数.结果与结论:单纯骨髓基质细胞和单用细胞因子组均可有效地扩增脐血造血细胞,并提高造血细胞上CD49d,CD62L及 CXCR4表达.而单用细胞因子组促进脐血造血细胞扩增能力较单纯骨髓基质细胞强,提高造血细胞CD49d,CD62L及 CXCR4表达能力较单纯骨髓基质细胞差.提示骨髓基质细胞虽可支持脐血造血细胞扩增,但难以实现造血细胞的大量扩增,但在骨髓基质细胞层的支持下,多细胞因子可有效地促进脐血造血细胞的扩增,并优于单用细胞因子及骨髓基质细胞,证实骨髓基质细胞可协同多细胞因子有效地促进脐血单个核细胞的扩增,促进造血干细胞的黏附和趋化能力.     

两种分离人骨髓单个核细胞方法的比较

目的:探讨外周动脉性疾病患者自体骨髓移植治疗骨髓单个核细胞最佳的分离方法.方法:行自体骨髓移植外周动脉性疾病患者39例,分别采取羟乙基淀粉沉淀法和密度梯度离心法分离骨髓单个核细胞,观察骨髓丢失的单个核计数及CD34+细胞比率的差异.结果:羟乙基淀粉沉淀法得到的分离物的单个核细胞及CD34+细胞丢失率低于密度梯度离心法[羟乙基淀粉沉淀法为(55.08±6.00)%,(54.71±7.58)%;密度梯度离心法为(82.38±4.79)%,(73.30±3.68)%,P<0.01].结论:经比较,羟乙基淀粉沉淀法明显优于密度梯度离心法,在外周动脉性疾病患者自体骨髓移植治疗中建议使用羟乙基淀粉沉淀法分离骨髓单个核细胞.     

Toward closed-system culture of blood origin endothelial cells

BACKGROUND: Blood outgrowth endothelial cells (BOECs) are thought to arise from very rare progenitors that are present in the mononuclear fraction of marrow or peripheral blood. Recently, BOECs have been expanded from progenitors present in buffy coat into confluent monolayers on fibronectin- or collagen-coated polystyrene surfaces. A method for sterile closed-system culture of these cells has not been described, however. Here, efforts are described toward developing closed-system culture of BOECs derived from progenitors present in a mononuclear apheresis unit by use of a cord blood filter, a sterile connection device, and a fibronectin-coated polycarbonate cassette. STUDY DESIGN AND METHODS: Strongly adherent cells from a mononuclear apheresis unit were eluted from a cord blood filter and resuspended in EGM-2 with 10 percent serum. Approximately 2 x 10(8) eluted cells were introduced into human fibronectin-coated polycarbonate cassettes. Medium was introduced and removed from cassettes with a sterile connection device and changed every 2 days. After expansion, cells were either cryopreserved or characterized by fluorescence-activated cell sorting analysis and ability to take up Dil-Ac-LDL. RESULTS: After 2 to 3 weeks of culture, 3 to 28 colonies with cobblestone morphology were observed in cassettes and passed to new cassettes within 3 to 4 weeks. By approximately 5 weeks of culture, 2 x 10(6) cells were typically obtained. BOECs uniformly took up Dil-Ac-LDL and were CD31+, CD105+, CD146+, CD45-, and CD14-. A population of BOECs was HLA-ABC+ or CD34+. CONCLUSION: BOEC progenitors can be isolated from mononuclear apheresis units with cord blood filters, expanded with fibronectin-coated polycarbonate cassettes, and cryopreserved.     

Technical and safety aspects of blood and marrow transplantation using G-CSF mobilized family donors

An allogeneic transplantation programme using immunoselected blood progenitor and bone marrow CD34+ cells has been established. Thirteen healthy HLA-matched, MLC negative sibling donors received two doses of 5 micrograms kg-1 G-CSF (s.c. daily) for 5 days. On days 4 and 5, large-volume mononuclear cell aphereses were performed (COBE Spectra) and on day 5 one unit of autologous blood was obtained. Mononuclear cells were pooled and cryopreserved after CD34+ cell-immunoselection on day 5. Bone marrow (BM) of the same donors was procured under routine conditions 10-45 days later (median: 27 days). The final graft consisted of blood CD34+ cells with either complete BM (n = 5) or immunoselected BM CD34+ cells (n = 8). The present paper describes the progenitor cell mobilization and apheresis protocol and analyzes the cell loss by BM and peripheral blood progenitor cell (PBPC) donation. Considerably larger amounts of mononuclear cells (CD45+), T-lymphocytes (CD3+) and platelets were lost by the apheresis as compared to bone marrow without apparent immediate clinical consequences for the donors. Owing to cross-cellular contamination of the apheresis concentrate, blood platelet count (PC) significantly decreased (mean PC after the second apheresis 116 x 10 microL-1); furthermore on average 3.04 x 10(10) CD3+ cells were removed by two apheresis sessions. This loss did not lead to long-term total lymphocyte count changes (2370 microL-1 versus 1889 microL-1) as observed during the long-term follow-up of 7/13 donors (mean 290 days). Subjectively, the PBPC collections were better accepted than BM donations in all but one family donor.     

The effect of G-CSF on lymphocyte subsets and CD34+ cells in allogeneic stem cell transplantation.

The effect of granulocyte colony-stimulating factor (G-CSF) on peripheral blood lymphocytes (PBL) and CD34+ cell frequency in the apheresis product has been determined in 25 healthy stem cell donors. Peripheral blood mononuclear cells (PBMNC) were collected after five days of G-CSF 10 microg/kg/day s.c., which was well tolerated. The median number of leukocytes increased eight-fold over that of pretreatment levels. Collection of PBMNC lasted a median of two (range, 1-3) days. The mean mononuclear cell (MNC) count and total lymphocyte percentage were 6.69 x 10(8)/kg and 59.08%, respectively, and the frequency of CD34+ cell expression was 2.1% in the apheresis product. The frequency of CD3+, CD4+, CD25+, NK and CD122+ cell expressions in mobilized PBMNC and PBL showed no significant difference. However, the frequency of CD8+, CD8+28+, CD3+DR+, CD19+, CD20+ and CD22+ B cells expression in the apheresis product increased significantly compared to steady-state PBL. In contrast, the frequency of the CD11 a+ and CD8+38+ cell expressions in the apheresis product was decreased compared to the steady-state PBL. The mean yield of CD34+ and CD3+ cells were 13.6 x 10(6) and 2.69 x 10(8)/kg of recipient body weight (RBW), respectively. Following allograft all patients engrafted with >0.5 x 10(9)/l neutrophil and < or = 20 x 10(9)/l platelets on a median of day 13 and 12, respectively. Nine patients had grade II-IV acute GVHD and chronic GVHD occurred in eight patients. Four patients died due to transplant-related complications. There was one late engraftment failure which occurred on the fifth month. Thirteen patients are still alive. In conclusion, these results indicate that administration of G-CSF at 10 microg/kg/day in normal donors alters the lymphocyte subsets and there are significant differences in the lymphocyte contents of the recipients before apheresis and in apheresis product.     

Replication of the human immunodeficiency virus 1 and impaired differentiation of T cells after in vitro infection of bone marrow immature T cells.

HIV-1 infection in vitro of normal bone marrow mononuclear cells (BMMC) depleted of mature T cells was studied. BMMC depleted of either CD3, CD2, or both could replicate HIV-1 irrespective of the presence of macrophages/monocytes. Infected bone marrow cells were shown to differentiate during the culture into CD3+, CD4+, CD8+, and CD1+ cells, whereas noninfected BMMC gave rise to CD3+, CD4+, and CD8+ cells. Moreover, 9-14% of the cells also expressed the viral proteins p24 and gp120 on their surface. Double staining studies revealed that 72 and 83% of the CD4+ cells expressed the gp120 and p24, respectively, suggesting that virus replication occurred in CD4+ cells. T cell colony growth from infected BMMC, either unfractionated or depleted of mature T cells, was impaired in a time-dependent manner, and the differentiation capacity of T cell precursors was abnormal. Colony cells displayed an immature cell phenotype (CD1+ cells) and the viral proteins gp120 and/or p24 could also be detected on CD1+ cells. In addition, pooled colony cells derived from infected CD2- and CD3-depleted BMMC could infect normal mitogen-activated lymphocytes in coculture experiments. These findings strongly suggest that HIV-1 can infect immature bone marrow T cells and be transmitted to the progeny, but the massive viral replication occurs only when the cells differentiate toward CD4+ cells.     

Multipotency of Flk1CD34 progenitors derived from human fetal bone marrow

We report that a cell population derived from human fetal bone marrow, termed Flk1+CD34- multipotent stem cells, can differentiate not only into osteogenic, adipogenic, and endothelial lineages but also into hepatocyte-like cells and neural and erythroid cells at the single-cell level. We depleted mononuclear cells from fetal bone marrow of CD45+, GlyA+, and CD34+ cells with the use of micromagnetic beads, then cultured them by limiting dilution. Three single colonies were harvested, expanded, and characterized. The clones have been expanded for more than 50 cell doublings, and cell-doubling time was about 30 hours. About 90% cells were in the G(0)/G(1) phase of the cell cycle, and the cells from the single colony maintained Flk1+ and CD34-. Because fetal bone marrow-derived Flk1+CD34-multipotent stem cells have the capacity for self-renewal and multilineage differentiation even after being expanded for more than 50 cell doublings, they may be an ideal source of stem cells for the treatment of inherited or degenerative diseases.     

化疗联合G-CSF动员的骨髓及外周血CD34+细胞表达粘附分子的变化

探讨化疗合G－CSF动员前，后患骺髓及外周血CD34^ 细胞表达CD44，CD49d，CD62L及趋化因子CXCR4的动态变化，用免疫荧光直接三角标记和流式细胞术测定G－CSF动员前，后骨髓及外周血CD34^ 细胞的CD44，CD49d，CD62L及趋化因子CXCR4的表达，观察输注各表达亚群细胞数与移植后造血重建的关系。结果发现，动员后骨髓中CD34^ CD44^ 和CD34^＋CD49d^ 细胞的百分比较动员前明显降低, 而外周血中二的比例则显升高；动员前，后骨髓中CD34^＋CD62L^＋和CD34^＋CXCR4^ 细胞的变化并不明显，而外周血中前明显增加，后则显减少。输入CD44^＋，CD49d^ ，CD62L^ 及CXCR4^ 的CD34^＋细胞的量与移植后血中中性粒细胞和血小板恢复的时间未表明有显的相关，结论：G－CSF动员可下调骨髓CD34^＋细胞的CD44，CD49d，CD62L及CXCR4的表达，从而进入外周血循环，输注这些细胞的临床意义有待累积更多病例的分析。     

Characterization of mononuclear cells remaining in the leukoreduction system chambers of apheresis instruments after routine platelet collection: a new source of viable human blood cells

BACKGROUND: The yield of white blood cells (WBCs) extracted from whole-blood leukoreduction filters can be affected by the storage conditions and delay before filtration. Platelets (PLTs) collected with apheresis instruments (Trima Accel, Gambro BCT) are leukoreduced during the procedure on a fluidized particle bed in a leukoreduction chamber (LRS chamber). In this report, the residual cell content of these LRS chambers was characterized to determine whether it would be a valuable source of viable human blood cells. STUDY DESIGN AND METHODS: The content of LRS chambers was eluted by gravity, and peripheral blood mononuclear cells (PBMNCs) were purified on a Ficoll-Paque gradient. Analyses were performed before and after freezing. Proportions of CD3+, CD14+, CD16+, CD19+, CD34+, and CD45+ cells were determined by flow cytometry. The frequency of T cells expressing CD4, CD8, and CD27 and of B cells expressing immunoglobulin G (IgG), IgM, and CD27 was also determined. RESULTS: LRS chambers held approximately 10(9) CD45+ cells representing the normal proportions of CD3+, CD14+, CD16+, and CD19+ cell populations of PBMNCs. A small fraction of these CD45+ cells were CD34+CD38+ cells (0.3 +/- 0.2%). The viability of these cells, measured before and after freezing, was more than 95 percent. CONCLUSION: The residual cell content of Trima Accel LRS chambers recovered after PLT collection is a good source of viable monocytes and lymphocytes. These PBMNCs, containing CD3+, CD14+, CD16+, CD19+, and CD34+ cells can be frozen to prepare cell banks, which opens new avenues for utilization in several physiologic studies or even in cellular therapy applications.     

骨髓增生异常综合征骨髓单个核细胞内负调控因子的研究

为研究MDS患者细胞凋亡与骨髓内不同T细胞亚群胞内负调控因子的关系，采用胞内细胞因子染色技术对骨髓内不同亚群T细胞胞内的TNF—α和IFN-γ水平进行检测和分析，用RT—PCR技术测定BMMNC的凋亡基因Fas的mRNA水平。结果表明，MDS患者骨髓内产生TNF-α增多的淋巴细胞为CD4^ CD45RO^ 、CD8^ CD45RO^ 细胞，产生IFN-γ增多的为CD4^ CD45RO^ 、CD8^ CD45RO^ 、CD8^ CD45RA^ 细胞，但均以CD8^ CD45RO^ 细胞为主；MDS患者T淋巴细胞产生的IFN-γ与Fas mRNA存在相关性，而TNF—α与Fas mRNA相关性不强。结论：MDS患者造血微环境中不仅T淋巴细胞亚群失调，而且各亚群分泌负调控因子增多；MDS患者髓内IFN-γ主要来自于T淋巴细胞。     

Predictive value of the original content of CD34(+) cells for enrichment of hematopoietic progenitor cells from bone marrow harvests by the apheresis procedure

We retrospectively investigated the feasibility of the apheresis procedure for red blood cell (RBC) reduction with a closed-bag system. We also sought to determine the optimal processing volume for the maximal recovery of hematopoietic progenitor cells (HPC). Twelve bone marrow (BM) harvests were processed for major ABO-incompatible allogeneic transplantation and one BM harvest was processed for autologous transplantation. The processing was performed through seven apheresis cycles with a two-bag system using COBE Spectra Version 6.1. The mean recovery rates were compared in the products after four cycles and seven cycles of BM processing. Mean cell recovery rates were 79.2% (67.6-97.5%) and 87.3% (68.9-111.9%) for the mononuclear cells (MNC) and 84.5% (69.4-109.5%) and 92.0% (79.0-107.7%) for the CD34(+) cells after four and seven cycles, respectively. A mean of 96.3% (93.0-98.1%) of the RBCs were finally removed. The yield of CD34(+) cells after seven cycles of processing (median: 10.35 x 10(7) cells) was 7.9% greater than that after four cycles of processing (median: 9.65 x 10(7) cells), exhibiting a less-than-significant enhancement in yield. The CD34(+) cell contents recovered in the concentrates up to four cycles (r = 0.989) and up to seven cycles (r = 0.993) were strongly correlated with the original content of the CD34(+) cells. Engraftment was obtained in all patients except one patient infused with purified CD34(+) cells. This latter result confirmed the hematopoietic potential of the cell populations recovered. Granulocyte recovery (defined as an absolute neutrophil cell count > or = 500/microL for a period of three consecutive days) ranged from 8 to 25 days (median: 16 days) post-transplantation. No hemolytic reaction was observed in any of the patients. Our results confirmed the efficacy of BM processing cycles with the COBE Spectra device. However, we could not conclude that the large-volume apheresis for BM processing significantly enhanced the yields of HPC. The final recovery of CD34(+) cells after processing could be predicted from the CD34(+) cell content of the original collected marrow.