全反式维甲酸对脐血红系祖细胞Hoxb2、Hoxb4基因表达的影响

本研究探讨全反式维甲酸（ATRA）对人脐血造血干细胞向红系定向增殖分化过程进行干预后hoxb2、hoxb4基因表达的影响。取12例正常足月顺产新生儿断脐后的胎盘段脐血,采用造血干细胞体外培养技术及FQ—RT—PCR方法．以6×10μmol／L的ATRA干预人脐血造血干细胞向红系定向增殖分化的过程,进而检测空白对照组、ATRA组在培养的第3天、第7天和第10天红系祖细胞hoxb2、hoxb4基因表达情况。结果表明：本实验各组的hoxb2、hoxb4基因在第3天时开始少量表达,在第7天表达明显升高,而在第10天表达最强烈。在空白对照组中,第3天、第7天、第10天hoxb4基因相对表达量较hoxb2高。与空白对照组相比较,ATRA组Hoxb2、Hoxb4基因表达量明显增加。结论：hoxb2、hoxb4基因在脐血HSC向红系定向增殖分化过程中（体外）均有表达,说明hoxb2、hoxb4均与红系造血有相关性,且与hoxb2相比,可能hoxb4与红系造血相关性较大;6×10μmol／L的ATRA能显著上调正常红系祖细胞hoxb2、hoxb4基因的表达。     

人类巨细胞病毒感染对淋巴系祖细胞增殖过程中Hoxc4及Hoxc6基因表达的影响

本研究探讨人类脐血造血干细胞（HSC）向淋巴系祖细胞（CFU—TL）分化过程中hoxc4、hoxc6基因表达的情况,并且用人类巨细胞病毒（HCMV）和／或全反式维甲酸（ATRA）进行干扰,在基因水平探讨HCMV感染导致脐血CFU—TL损伤的机制。采用体外定向培养CFU—TL,经MTT法检测后,应用荧光定量RT-PCR技术检测经人HCMV和／或ATRA处理的人脐血CFU—TL中hoxc4、hoxc6基因的mRNA表达水平。HCMV—AD169滴度为10^6蚀斑形成单位（PFU）／ml,按0．1ml 10^5 PFU／ml接种培养体系。结果表明：正常对照组、病毒处理组、维甲酸处理组hox04、hoxc6基因均在增殖分化的第3天少量表达,第7天表达最强烈（P〈0．05）,第12天表达明显下降。各组中hoxc4基因表达量高于hoxc6组（P〈0．05）。与正常对照组比较,维甲酸组（6×10^-8 mol/L）的hoxc4、hoxc6基因表达上调（P〈0．05）,而病毒处理组hoxc4、hoxc6基因表达下调（P〈0．05）。结论：hoxc4与hoxc6基因在脐血CFU—TL中各组呈现规律的表达,提示hoxc4、hoxc6均与淋巴系造血有密切的相关性。HCMV能使hoxc4、hoxc6基因表达下调．提示HCMV致造血细胞损害可能与hoxc4、hoxc6基因表达异常有关。ATRA能显著上调hoxc4、hoxc6基因的表达。     

造血干细胞向淋巴系祖细胞增殖HOXB4基因的表达

背景:HOXB4基因不仅能促进造血干细胞的扩增及其功能的活化与表达,而且体内试验表明它不会诱发白血病.因此更好地研究HOXB4在造血细胞增殖分化中的变化及作用对进一步研究造血干细胞的扩增可以提供更多的理论基础.目的:观察人类脐血造血干细胞向淋巴系祖细胞增殖分化过程中HOXB4基因表达的情况及全反式维甲酸对HOXB4基因表达的影响.方法:将培养的淋巴系造血祖细胞按干预方式不同分为2组,全反式维甲酸组:在培养体系中加入全反式维甲酸,终浓度6×10-8mol/L.正常组:不加全反式维甲酸,代之以等量的1640培养液.观察人类脐血造血干细胞经植物血凝素诱导后,在培养第3,7,12天的淋巴细胞集落形成单位生成情况.采用实时荧光定量PCR技术检测脐血造血干细胞向淋巴系祖细胞增殖分化过程中HOXB4基因的表达水平.结果与结论:人脐血造血干细胞向淋巴系祖细胞增殖分化过程中,HOXB4基因晕规律的表达.随培养时间推移,正常组和全反式维甲酸组HOXB4基因的表达均逐渐降低.与正常组比较,全反式维甲酸可上调HOXB4基因的表达.     

巨细胞病毒对人骨髓粒-巨噬系祖细胞生长的影响

目的探讨人巨细胞病毒（ＨＣＭＶ）对人骨髓粒巨噬系祖细胞集落（ＣＦＵＧＭ）是否有直接抑制作用。方法采用造血祖细胞体外培养技术,研究病毒株ＨＣＭＶＡＤ１６９对ＣＦＵＧＭ生长的影响,用原位聚合酶链反应（ＩＳＰＣＲ）技术检测ＣＦＵＧＭ的细胞内ＨＣＭＶＡＤ１６９ＤＮＡ。结果高浓度ＨＣＭＶＡＤ１６９（２×１０６ｐｆｕ／ｍｌ和２×１０５ｐｆｕ／ｍｌ）在体外能明显抑制ＣＦＵＧＭ的生长（Ｐ＜０．０１）,而低浓度（２×１０４ｐｆｕ／ｍｌ）ＨＣＭＶＡＤ１６９则无此作用,ＨＣＭＶＡＤ１６９对骨髓ＣＦＵＧＭ的抑制作用因其浓度的不同而有差异;经ＩＳＰＣＲ检测发现在ＣＦＵＧＭ的细胞核中存在ＨＣＭＶＡＤ１６９ＤＮＡ。结论ＣＦＵＧＭ是ＨＣＭＶＡＤ１６９的宿主细胞之一,ＨＣＭＶＡＤ１６９对骨髓ＣＦＵＧＭ生长的抑制作用与其对ＣＦＵＧＭ的直接感染有关。     

人巨细胞病毒抑制人巨核系祖细胞增殖的体外研究

目的 探讨人巨细胞病毒（HCMV）对人巨核系祖细胞增殖的影响。方法 收集20份脐血标本,采用巨核系祖细胞体外半固体培养技术,观察HCMV AD169株对巨核细胞集落形成单位（CFU－MK）生长的影响,分别用原位聚合酶链反应（IS－PCR）和RT－PCR检测集落细胞中的HCMV DNA与抑刻早期抗原（IEA）mRNA。结果 HCMV AD169株在体外能明显抑制CFU－MK生长,抑制程度与病毒感染滴度呈剂量依赖关系,经IS－PCR检测发现病毒感染组CFU－MK细胞中有HCMV DNA存在;RT－PCR检测发现病毒感染组CFU－MK细胞中有IEAmRNA的表达。结论 HCMV AD169株可直接感染巨核系祖细胞,并抑制其增殖与分化;HCMV对巨核系祖细胞的直接抑制作用可能是该病毒感染后引起血小板减少的原因之一。     

应用FQ-PCR和ELISA方法检测人巨细胞病毒感染的临床价值

目的 探讨检测巨细胞病毒(CMV)的方法学及在临床诊断与治疗中的应用价值 方法 对456例临床疑似HCMV感染的患者分别应用FQ-PCR、ELISA法检测其尿液、血液HCMV-DNA及血清HCMV-IgM抗体水平.结果 尿液HCMV-DNA 阳性281例,血HCMV-DNA阳性238例,HCMV-IgM抗体阳性144例；HCMV-DNA阳性者中,血清IgM抗体阳性HCMVDNA拷贝数对数均值高于阴性(P＜0.01).结论 尿液、血液HCMV-DNA定量和血清HCMV-IgM抗体联合检测有助于HCMV感染的诊断.     

应用FQ—PCR和ELISA方法检测人巨细胞病毒感染的临床价值

目的探讨检测巨细胞病毒（CMV）的方法学及在临床诊断与治疗中的应用价值方法对456例临床疑似HCMV感染的患者分别应用FQ-PCR、ELISA法检测其尿液、血液HCMV-DNA及血清HCMV-IgM抗体水平。结果尿液HCMV-DNA阳性281例,血HCMV-DNA阳性238例,HCMV-IgM抗体阳性144例 HCMV-DNA阳性者中,血清IgM抗体阳性HCMV-DNA拷贝数对数均值高于阴性（P〈0.01）。结论尿液、血液HCMV-DNA定量和血清HCMV-IgM抗体联合检测有助于HCMV感染的诊断。     

肾移植术后尿液中巨细胞病毒DNA的定量测定

人类巨细胞病毒 (ＣＭＶ)感染能引起移植物的慢性排异 ,正确诊断及治疗ＣＭＶ感染可有效提高移植物的存活率 ,减少医疗费用。然而在众多ＣＭＶ感染的实验室检查中 ,究竟哪一种方法最可靠一直存有争议。本项目采用荧光定量聚合酶链反应 (ＦＱ ＰＣＲ)检测ＣＭＶＤＮＡ ,报道如下。1　材料和方法1 1　 92名 1997年 1月至 2 0 0 0年 5月在我院接受肾移植的患者 ,移植时间 2个月～ 4 0个月。收集中段晨尿 ,临床上有感染症状的患者在抗病毒治疗后反复留取尿标本 ,共收集标本 112份。1 2　ＴａｇＭａｎ和ＣＭＶＤＮＡ荧光定量试剂由达安基因诊断中…     

荧光定量聚合酶链反应技术检测儿童患者人巨细胞病毒结果分析

目的 了解本院儿童患者人巨细胞病毒(HCMV)感染现状.方法 采用实时荧光定量聚合酶链反应(PCR)技术对243例住院疑似患儿进行HCMV-DNA定量检测.结果 HCMV总阳性率为16.05%;男性和女性阳性率分别为17.83%和12.79%;1个月至1岁年龄组(婴儿期)患者最高为17.41%.临床诊断主要以肝损害、腹泻和黄疸为主.结论 加强儿童患者HCMV的检测,有助于临床诊断与治疗方案的选择.     

全反式维甲酸对人脐血CD133+细胞体外短期扩增中生物学特性的影响

维甲酸 (RA)对许多细胞的增殖和分化具有调节作用。全反式维甲酸 (ATRA)在体内外能诱导急性早幼粒细胞白血病 (APL)细胞向成熟粒细胞分化 ,对造血细胞的调节作用尚未完全明了 ,有报道认为RA能促进粒系和红系祖细胞的增殖 ,也有报道认为 ,RA对造血干 /祖细胞 (HSPC)的增殖和分化具有抑制作用[1,2 ] 。近来有研究显示 ,RA在体外能促进小鼠HSPC的自我更新 ,并能延迟HSPC的分化[3 ,4] 。为了解ATRA对人脐血HSPC体外增殖和分化的影响 ,我们观察了ATRA对人脐血CD133 细胞体外增殖和分化的影响。材料和方法1　标本来源和细胞制备…     

人巨细胞病毒感染致造血祖细胞增殖抑制与更昔洛韦的影响

背景:临床研究显示,骨髓移植后人巨细胞病毒感染可阻碍血小板植入、出现表型异常的外周血白细胞,导致骨髓移植失败,这可能是由于人巨细胞病毒感染直接影响了造血祖细胞所致.目的:观察更昔洛韦对人巨细胞病毒感染所致脐血粒-巨噬系祖细胞、红系祖细胞、淋巴系祖细胞、多向造血祖细胞及巨核系祖细胞体外增殖抑制的影响及更昔洛韦对此的保护作用. 设计:对比观察性实验.单位:泸州医学院附属医院分子生物学实验室.对象:正常足月顺产新生儿脐血标本20例,每份10 mL,由泸州医学院附属医院妇产科提供,产妇对本实验知情同意.实验经医院伦理委员会批准.方法:实验于2004-06/2006-12在泸州医学院附属医院分子生物学实验室完成.采用脐血标本造血祖细胞培养技术,观察、计数100 TCID50人巨细胞病毒-AD169感染粒-巨噬系祖细胞、红系祖细胞、淋巴系祖细胞、多向造血祖细胞及巨核系祖细胞后各祖细胞集落数,记录集落维持时间.用PCR和荧光定量PCR技术检测集落细胞内人巨细胞病毒-AD169DNA.将7.5 mg/L更昔洛韦作用于人巨细胞病毒感染的造血祖细胞,再分别计集落数、集落维持时间及集落细胞内人巨细胞病毒-AD169DNA.设正常培养的造血祖细胞为空白对照组,设与含灭活性人巨细胞病毒的正常造血祖细胞培养系统为灭活对照组.主要观察指标:①祖细胞集落数、祖细胞集落维持时间.②祖细胞集落细胞内人巨细胞病毒-AD169DNA. 结果:①集落数和集落维持时间:人巨细胞病毒感染的祖细胞集落数较对照组明显减少(P < 0.01),集落维持时间较对照组明显缩短(P < 0.01).在人巨细胞病毒感染的集落细胞内检测到人巨细胞病毒-DNA的存在;更昔洛韦作用于人巨细胞病毒感染的祖细胞后,与人巨细胞病毒感染的祖细胞比较集落数明显提高,差异有非常显著性意义(P < 0.01) ,集落维持时间明显延长,差异有非常显著性意义(P < 0.05).②集落增殖提高率: 粒-巨噬系祖细胞、红系祖细胞、淋巴系祖细胞、多向造血祖细胞、巨核系祖细胞分别为37.4%,74.2%,40.1%,67.4%,38.9%.③集落细胞内人巨细胞病毒-AD169DNA:荧光定量PCR显示更昔洛韦作用于人巨细胞病毒感染的祖细胞后核酸载量较人巨细胞病毒感染的祖细胞降低,差异有非常显著性意义(P < 0.01).结论:人巨细胞病毒-AD169株在体外能明显抑制粒-巨噬系祖细胞、红系祖细胞、淋巴系祖细胞、多向造血祖细胞及巨核系祖细胞集落细胞的增殖;在体外更昔洛韦有抗人巨细胞病毒的活性,能促进人巨细胞病毒感染的造血祖细胞增殖.     

Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK- Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU- granulocyte erythroid macrophage megakaryocyte colonies from mock- infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.     

人脐带血内皮祖细胞体外培养和鉴定

背景:传统免疫磁珠法分选内皮祖细胞的方法操作复杂、费用大,细胞获得率较低,且内皮祖细胞的增生及分化受限.目的:尝试改良人脐带血内皮祖细胞体外诱导分化及鉴定的方法,为实现内皮祖细胞移植、改善血管功能提供足量细胞来源.方法:采用密度梯度离心方法获得人带血单个核细胞,体外进行诱导、分化和扩增,通过免疫组织化学、免疫荧光和流式细胞仪等技术对脐血来源的内皮祖细胞进行鉴定.结果与结论:脐带血单个核细胞在培养过程中出现梭形贴壁和铺路石样等形态;在细胞培养至第7天,免疫组织化学显示:CD31、vWF在体外培养过程中的表达阳性;免疫荧光染色表明:(83.0±4.3)%的贴壁细胞双染呈阳性,并且DiI-acLDL标记的内皮祖细胞红色荧光在体外可以持续6周以上;第7天贴壁细胞流式细胞仪分析显示:CD34、CD133和KDR分别为(17.8±3.7)%、(22.1±4.4)%、(81.5±5.0)%.结果提示,实验成功从脐带血单个核细胞中分离培养出内皮祖细胞,在其体外可扩增并向为内皮细胞方向分化.     

外周血红系祖细胞来源诱导多能干细胞的建立

目的建立和鉴定外周血中红系祖细胞来源的诱导多能干细胞(induced pluripotent stem cells,i PSCs)。方法从健康人外周血标本中分选红系祖细胞,将表达Oct4、Sox2、Lin28、L-Myc和Klf4转录因子的ori P/EBNAl附着体电转染红系祖细胞使其重编程获得i PSCs,并通过核型鉴定、碱性磷酸酶染色、RT-PCR反应、畸胎瘤形成实验及类胚体形成实验检测其干细胞多能性特性。结果获得的i PSCs核型正常,碱性磷酸酶染色呈阳性,表达干细胞多能性基因Sox2、Oct4、Nanog和Klf4和Lin28,体内畸胎瘤形成实验可分化为内、中、外三胚层细胞,体外可形成类胚体。结论成功建立外周血红系祖细胞来源具有多向分化潜能的i PSCs。     

Direct demonstration of the human parvovirus in erythroid progenitor cells infected in vitro.

The human parvovirus (HPV), the cause of transient aplastic crisis of hereditary hemolytic anemia, has been shown to be cytotoxic for erythroid progenitor cells and its presence in these cells demonstrated by morphologic techniques. A relatively pure population of progenitors, isolated by removal of immature erythroid bursts from primary culture, was the target of the virus infection. Infected cells failed to proliferate in secondary culture. Using a monoclonal antibody to HPV, specific fluorescence was demonstrated in a minority of cells 24-48 h after infection with virus. Infected cells examined by electron microscopy showed marked toxic ultrastructural alterations and parvovirus-like particles in crystalline arrays in the nucleus.     

Association of cord blood platelet characteristics and hematopoietic progenitor cells

BACKGROUND: Total nucleated cell (TNC) dose is associated with neutrophil and platelet (PLT) engraftment after cord blood (CB) transplantation and thus is used for selection of CB for banking. The goal of this study was to evaluate the internal relationships of CB PLT characteristics, TNC, and the hematopoietic progenitor cell (HPC) content of CB units. STUDY DESIGN AND METHODS: HPC and TNC counts of 167 CB units processed with an automated cell separation system were compared with CB PLT count and mean PLT volume (MPV). Megakaryocyte progenitors (CFU-MK) were cultured from a subset of units (n = 24). RESULTS: PLT concentration correlated with MPV (r = -0.39), which was also associated with both TNC and total CD34+ cells before and after processing (r = 0.37 and 0.35 and r = 0.41 and 0.42, respectively). In addition, MPV was associated with HPC counts in the CB unit. The p value was less than 0.001 for all associations. PLT count was inversely associated with markers of hematopoietic potential. Median removal of PLTs during processing was 62 percent (range, 40%-84%). All 24 CB units of the subset exhibited CFU-MK growth. In multivariate linear regression analysis, MPV improved prediction of the HPC content of the CB unit compared to prediction with CB volume and nucleated cell concentration only. CONCLUSION: Mean PLT volume correlated with current markers of CB hematopoietic potential and is potentially useful for evaluating CB collections for banking. The question of the clinical significance of PLT characteristics in CB transplantation remains unanswered.     

Enhancing effects of co-transduction of both human erythropoietin receptor and c-kit cDNAs into hematopoietic stem/progenitor cells from cord blood on proliferation and differentiation of erythroid progenitors

Steel factor (SLF) and erythropoietin (Epo) play critical roles in erythropoiesis. To evaluate interactive effects of Epo and SLF receptors (R) in erythropoiesis, CD34+ and CD34 cord blood cells were transduced with human EpoR and c-kit cDNAs by retroviral mediated gene transfer. Erythroid (BFU-E) colonies derived from CD34+ or CD34 cells transduced with either the EpoR or c-kit gene were significantly increased in the presence of interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), Epo, and different concentrations of SLF compared with that from mock transduced cells. This number was further enhanced by co-transduction of both genes. Enhancement was more apparent in the absence of SLF. Cell numbers in individual erythroid colonies were also significantly increased in cells transduced with both genes compared with cells transduced with a single gene. Short-term liquid culture showed that ex vivo expansion for five days and numbers of CD34+CD71+ cells in expanded cells from single CD34 cells co-transduced with both EpoR and c-kit genes were increased compared with those of EpoR or c-kit-transduced cells. These results demonstrate that co-transduction of both c-kit and EpoR enhances the proliferative capacity of erythroid progenitors under cytokine stimulation above that of single-gene transduced cells.     

HCMV感染致内皮细胞粘附分子表达的变化

目的探讨HCMV感染对体外培养内皮细胞粘附分子表达的影响。方法应用双抗体夹心酶联免疫分析法及细胞ELISA方法检测体外培养人脐静脉血管内皮细胞在感染HCMV后,血管内皮细胞粘附因子(VCAM-1)及可溶性细胞间粘附因子1(sICAM-1)表达的变化。结果感染组细胞培养液sICAM-1 24小时和对照组相比明显升高达10.7±2.4μg/L,而正常对照为5.7±3.1μg/L,而后48小时、72小时均高于相应对照组(P<0.05)。VCAM-1在感染12小时开始表达升高,48小时和72小时和对照组相比明显增高(P<0.05)。结论体外情况下HCMV感染可上调内皮细胞粘附分子表达,这可能在HCMV致动脉粥样硬化的病理过程中发挥重要作用。