【作者】 胡嘉波；

【导师】 许化溪；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2008， 博士

【摘要】 研究目的:探讨密度分离脐血造血干/祖细胞体外扩增和体内重建造血的潜能;胎儿骨髓间质干细胞对CD34+细胞体外扩增的造血支持作用;体外诱导脐血造血干/祖细胞向B细胞分化发育的规律。研究内容:（1）利用造血干/祖细胞培养、体外扩增、移植动物模型等技术,研究不同比密Ficoll-泛影葡胺分离液分离脐血造血干/祖细胞的造血活性。（2）体外分离、纯化胎儿骨髓间质干细胞;Mini-MACS免疫磁珠分离脐血CD34+细胞;建立胎儿骨髓间质干细胞、细胞因子与CD34+细胞共培养体系,用液体培养方法扩增细胞;实时定量RT-PCR技术分析培养细胞的nucleostemin基因表达。（3）体外免疫磁珠分离纯化脐血造血干/祖细胞;在小鼠S-17基质细胞支持下,脐血造血干/祖细胞、T3、IL7共培养建立体外B细胞分化发育培养体系,诱导脐血造血干/祖细胞B细胞分化;用流式细胞仪、PCR、RT-PCR技术检测不同培养时间分化的B细胞。研究方法:（1）密度分离脐血细胞配制不同密度Ficoll-泛影葡胺细胞分离液,分别应用密度为1.054 g/ml、1.064g/ml、1.072g/ml、1.077g/ml、1.084g/mlFicoll-泛影葡胺不连续密度梯度离心分离脐血有核细胞成分。（2）造血祖细胞集落培养五种不同比密Ficoll-泛影葡胺分离液分离的脐血细胞,在甲基纤维素体系中培养测定其集落形成能力。CFU-GM体系含有细胞2×10⁵/ml,20%FBS,20ng/ml GM-CSF,0.9%甲基纤维素。BFU-E体系含有细胞2×10⁵/ml,10^-5M 2-巯基乙醇,3mM L-谷氨酰胺,马血清25%,白细胞条件培养液20%,EPO 2U/ml,0.9%甲基纤维素。充分混匀以每孔0.25ml加入24孔培养板中,重复2孔,置37℃,5%CO₂培养箱培养,CFU-GM培养7天,BFU-E培养14天,倒置显微镜下观察计数CFU-GM,BFU-E。鉴别标准:CFU-GM每个集落至少含有40个细胞;BFU-E集落呈多中心状,橘红色,至少含有100个细胞。（3）密度分离细胞的生物学特性分析BALB/c照射小鼠体内输注不同比密Ficoll-泛影葡胺分离液分离的脐血细胞,观察其造血潜能。Ficoll-泛影葡胺分离液分离的细胞,用无菌生理盐水洗涤二次后,计数并调整细胞浓度为2×10⁶/ml,小鼠经致死剂量（8.5Gy）照射后随机分为3组,照射后2小时完成输注。Ⅰ组6只,输注无菌生理盐水0.2ml;Ⅱ组6只,输注经1.077g/ml Ficoll-泛影葡胺分离液分离洗涤后的脐血MNC（1.077细胞）0.2ml;Ⅲ组6只,输注经1.064g/ml Ficoll-泛影葡胺分离液分离洗涤后的脐血MNC（1.064细胞）0.2ml。所有小鼠均采用尾静脉输注。（4）脐血造血干/祖细胞体外扩增取经鉴定的MSCs接种于24孔板中,当细胞生长达80%汇合度时,接种MiniMACS分选的CD34+细胞（4000/孔）,总体积为1ml,共分4组（每组3孔）:①CD34+细胞组;②胎儿骨髓MSCs+CD34+细胞组;③细胞因子+CD34+细胞组,细胞因子浓度为50ng/ml SCF、50ng/ml IL-3、50ng/ml FL、50ng/ml TPO;④胎儿骨髓MSCs+细胞因子+CD34+细胞组。培养体系为含10%FBS的DMEM,置5%CO₂、饱和湿度37℃培养。每7天半量换液1次并补充新的细胞因子,浓度同前。（5）脐血造血干/祖细胞B细胞分化培养接种Dynal beads分选的CD34+CD19-细胞或细胞仪分选的CD34+CD19-CD38-细胞于预铺S-17基质细胞的96孔培养板中。培养体系为α-MEM培养液,含3%FBS,50μM2-巯基乙醇,1%L-谷氨酰胺、T3及细胞因子组合,每孔总体积100μl。置5%CO₂,饱和湿度,37℃培养。每7天半量换液1次并补充新的细胞因子,浓度同前。（6）流式细胞分析术分选或培养的1×10⁶细胞标记前用含1%牛血清白蛋白（HAS）的PBS洗涤1次,用荧光标记小鼠抗人单克隆抗体标记,置4℃条件下30 min,用含1%HAS的PBS洗涤1次,加0.4 ml含0.1%叠氮钠的PBS,流式细胞仪检测。分析软件为Cellquest。（7） CD34+细胞nucleostemin基因表达的分析定量PCR反应用于nucleostemin基因分析。反应体系为25μl,包括:1μlcDNA模板和24μl反应混合液[其中含2.5mM MgCl₂,20pmol引物,0.1μl Syber greenⅠ（1:1000）]。PCR循环参数为:95℃预变性2min,（94℃30s;58℃30s,72℃30s）,40个循环。以GAPDH为内参照,扩增长度为205 bp,GAPDH基因连于pMD-18T载体定量稀释,作为标准曲线的模板。每个DNA样品做2个平行管,重复3次实验,反应结束后使用定量PCR仪的分析软件对实验结果进行分析,计算各样本基因的原始拷贝数,单位为拷贝/μlcDNA。Nucleostemin PCR扩增产物与相应GAPDH的扩增产物原始拷贝数的比值能反映转录水平的差异。此外,采取上述方法（PCR或RT-PCR）分析T3Rα1、T3Rα2、hT3Rβ1、Iμ、Cμ、IgLL、DQ52（D7-27）、DXP1（D3-9）、DQ52/DXP1等基因的表达、重组情况。研究结果:（1） 1.064g/ml Ficoll-泛影葡胺分离液分离脐血细胞中,CFU-GM集落数为373±289/1×10⁵MNC,BFU-E为121±70/1×10⁵MNC;在细胞因子刺激下,1.064g/ml Ficoll-泛影葡胺分离液分离脐血MNC在体外扩增14天时CFU-GM的扩增倍数达52.2倍;1.064g/ml Ficoll-泛影葡胺分离液分离的造血干/祖细胞可在8.5Gy致死剂量照射小鼠体内植入,输注1.064g/ml Ficoll分离脐血MNC产生的脾结节数是输注1.077g/ml Ficoll-泛影葡胺分离液分离脐血MNC的2.2倍。（2）胎儿骨髓间质干细胞表达CD29、CD44;免疫磁珠分选CD34+细胞的平均纯度为97.4%;胎儿骨髓间质干细胞+CD34+细胞共培养28天,CD34+细胞仍占有核细胞的6.43%;CD34+细胞在胎儿骨髓间质干细胞、细胞因子作用下培养28天,有核细胞总数、CD34+细胞数分别被扩增1.65×10⁵倍、788倍。（3） T3诱导脐血造血干/祖细胞B细胞分化,在我们选用的实验条件下,T3、IL-7与小鼠S-17基质细胞共培养是诱导CD34+CD19-造血干/祖细胞向B细胞分化的最佳条件,诱导的CD19+B部分细胞表达CD21,还表达CD10、CD20、CD24抗原,弱表达CD23抗原、HSL11、HSL96,不表达CD3、CD33、CD34、IgM抗原,B细胞向成熟方向发育;在小鼠S-17基质细胞支持下,单纯T3诱导的CD19+B细胞大多数体积较小,CD19+B细胞几乎不表达CD21,B细胞发育停留在较原始阶段;34+CD19-CD38-造血干/祖细胞B细胞分化的扩增倍数明显高于34+CD19-造血干/祖细胞,持续扩增的时间也延长;新鲜脐血与冻存脐血CD34+CD19-造血干/祖细胞B细胞分化的纯度、扩增倍数无明显差异;小鼠S-17基质细胞高表达T3Rα1,传代20代以上小鼠S-17基质细胞,失去支持脐血CD34+CD19-造血干/祖细胞B细胞分化的能力。（4）纯化的脐血CD34+CD19-造血干/祖细胞检测到胚系DQ52基因,未发生DH-JH基因重排;诱导分化的B细胞优先选择DQ52-JH4、DQ52-JH3、DQ52-JH5基因重排,未检测到DQ52-JH1、DQ52-JH2、DQ52-JH6基因重排;在整个B细胞分化发育过程中,我们检测到持续的Iμ、Cμ、IgLL转录。研究结论:（1） 1.064g/ml Ficoll-泛影葡胺分离的造血干/祖细胞在体外具有较高的增殖、持续造血的能力及较强的体内造血重建潜能。（2）胎儿骨髓间质干细胞可有效扩增脐血造血干/祖细胞。（3） T3、IL7与小鼠S-17基质细胞体外能诱导脐血造血干/祖细胞B细胞分化,免疫球蛋白重链基因优势选择DH-JH4、DH-JH3、DH-JH5重排。脐血冻存三年对造血干/祖细胞活性、B细胞分化能力无影响。更多还原

【Abstract】 ObjectiveThe aim of this study is to investigate the expansion of hematopoietic stem /progenitor cell separated by Ficoll in vitro and the potential of hematopoietic reconstitution in vivo,the role of expansion of CD34+ cells supported by fetal mesenchymal stem cells(MSCs) in vitro and the B cell development of hematopoietic stem/progenitor cells from cord blood in vitro.MethodsBy using a series of techniques,i.e.,stem/progenitor cells culture,expansion in vitro and animal transplantation model,the hematopoietic activity of different Ficoll-Urografin gradient density separated cells was evaluated.Fetal MSCs were isolated and purified in vitro.Cord blood CD34+ cells were isolated by using Mini-MACS separation system.Co-culture of CD34+ cells with fetal MSCs and cytokines was established.CD34+ cells was expanded in liquid culture.Nucleostemin gene in cultured cells was analyzed by Real-Time RT-PCR.Hematopoietic stem/progenitor cells from cord blood were isolated and purified by using immunomagnetic beads separation system in vitro.Differentiation of B cells derived from cord blood hematopoietic stem/progenitor cells supported by murine S-17 stromal cells were stimulated in co-culture with T3 and IL-7.The differentiation and development culture system of B cells were established in vitro.Differentiated B cells were detected by using FACS,PCR and RT-PCR techniques at different culture time.ResultsAfter separation of 1.064g/ml Ficoll-Urografin gradient,the number of CFU-GM were 373±289/1×10~5 MNCs and BFU-E were 121±70/1×10~5 MNCs.Following treated with cytokines for 14 days,the number of CFU-GM expanded 52.2-fold in vitro.The number of CFU-S in BALB/c mice which were irradiated by a lethal dose of 8.5Gy and transfused subsequently with the hematopoietic stem/progenitor cells separated by 1.064g/ml Ficoll-Urografin gradient was 2.2-fold as many as that of separated by 1.077g/ml Ficoll-Urografin gradient.Fetal MSCs expressed CD29 and CD44.The average purity of CD34+ cells was 97.4%.After co-culture of CD34+ cells with fetal MSCs for 28 days,6.43%of CD34+ cells remained in the cultured cells,the amplification of the total cells and CD34+ cells reached 1.65×10~5-fold,788-fold increase in the co-culture of CD34+ cells with fetal MSCs and cytokines,respectively.T3 stimulated differentiation of B cells derived from cord blood hematopoietic stem/progenitor cells,most of the induced CD19+ B cells were smaller,CD19+ B cells scarcely expressed CD21,development of B cells remained in a primitive stage. In experimental conditions we had selected,co-culture with T3,IL-7 and murine S-17 stromal cells was optimum condition to stimulated CD34+CD19- hematopoietic stem/progenitor cells to differentiate to B cells.Cultured CD19+ B cells expressed CD21 as well as CD10,CD20,CD24,weakly expressed CD23,HSL11,HSL96 and did not expressed CD3,CD33,CD34,IgM,B cells developed to be mature.Fold increase of B cells derived from CD34+CD19-CD38- hematopoietic stem/progenitor cells was significantly higher than that of CD34+CD19-hematopoietic stem/progenitor cells,the time of lasting amplification also extended.There was no significant difference in differentiation purity and amplification folds of B cells derived from fresh or cryopreserved cord blood.Murine S-17 stromal cells passaged more than 20 generations lost the ability of supporting the differentiation of B cells derived from cord blood CD34+CD19-stem/progenitor cells.The germline configuration of the DQ52 gene segment could be detected as indicated by its expected size of 2216bp.We found preferential rearrangements with a predicted fragment size of DH-JH4,DH-JH3,and DH-JH5 joined segments,whereas rearrangements to JH1,JH2 or JH6 gene segments were not detectable.Our primer design showed continuous transcription of Iμ、Cμ、IgLL during B cell development throughout the course of our in vitro cell culture system. ConclusionThe hematopoietic stem/progenitor cells after 1.064g/ml Ficoll-Urografin density separation acquired higher ability of expansion and sustaining hematopoiesis in vitro and the potential of hematopoietic reconstitution in vivo.Fetal MSCs could support efficiently proliferation of hematopoietic stem/progenitor cells derived from cord blood.In experimental conditions we had selected,supported by murine S-17 stromal cells,T3 and IL-7 could stimulate cord blood hematopoietic stem/progenitor cells to differentiate to B cells in vitro.There were preferential rearrangements of heavy chain immunoglobulin genes with DH-JH4,DH-JH3 and DH-JH5 joined segments.The activity of hematopoietic stem/progenitor cells and the ability of differentiation of B cells derived from hematopoietic stem/progenitor cells had no significant difference between fresh cord blood and cryopreserved cord blood.更多还原