干细胞因子对内皮细胞增殖、迁移、管状形成能力的影响及对CD133+细胞的趋化效应

目的 探讨干细胞因子(SCF)对脐静脉内皮细胞(HUVEC)增殖、迁移、管状形成能力的影响,以及对CD133+细胞的趋化效应.方法 应用MTT及CCK-8增殖分析法检测HUVEC在不同细胞因子[空白试剂、SCF、血管内皮生长因子(VEGF)、抗人SCF、人IgG]条件下增殖能力的差异性;采用细胞划痕法与Matrigel体外三维成型法分别检测内皮细胞的增殖、迁移和管状形成能力;并应用Transwell技术检测不同细胞因子诱导的CD133+细胞体外趋化效应.结果 MTT及CCK-8增殖分析结果显示SCF无HUVEC增殖刺激活性;SCF可显著提升HUVEC迁移能力;SCF呈剂量依赖性增强HUVEC 管状形成能力,在适宜浓度SCF(100 ng/ml)作用下,HUVEC完整小管形成数量[(30.0 ±3.4)/105HUVEC]显著高于空白试剂组[(5.0±2.6)/105HUVEC,P<0.01];SCF可高效诱导CDl33+细胞体外趋化,SCF组[(118.0±6.5)/104CD133+细胞]Transwell小室跨膜迁移细胞数显著高于空白试剂组[(47.0±4.7)/104CDl33+细胞,P<0.01].结论 SCF可显著增强HUVEC的迁移及管状形成能力,并有效诱导CD133+细胞体外趋化,提示SCF/c-kit信号转导在内皮细胞及其祖细胞的血管新生与血管发生过程中可能发挥重要作用.

Abstract：

Objective To explore the effects of stem cell factor (SCF) on proliferation, transmigration, capillary tube formation of human umbilical vein endothelial cells (HUVEC) and on the chemotaxis of CD133+ cells. Methods In the presence of blank control, SCF, vascular endothelial growth factor ( VEGF) , anti-human SCF (anti-SCF) or human IgG, the difference in proliferation capacity of HUVEC was analyzed by MTT and CCK-8 methods, and wound scratch assay and three-diamensional in vitro Matrigel assay were used for transmigration and capillary tube formation of HUVEC, respectively. In addition, the chemotaxis of CD133 + cells sorted from human umbilical cord blood by flow cytometry was investigated by Transwell migration assay. Results SCF didn't improve the proliferative capacity of HUVEC, but significantly enhanced the transmigration capacity, and increased capillary tube formation in a dose-dependent manner.The number of intact tubules [(30.0 ±3.4)/105 HUVEC] formed by HUVECs in the presence of the optimal concentration of SCF (100 ng/ml) was remarkably higher than that in blank control group [(5.0 ±2.6)/105HUVEC,P <0.01]. SCF also significantly induced a chemotactic response of CD 133+ cells, the transmembrane migration cell number into Transwell lower chamber was significantly higher in SCF group [(118.0 ±6.5)/104 CD133+ cells] than in blank control group [(47. 0 ±4. 7)/104 CD133 + cells,P <0.01]. Conclusions SCF significantly promotes the transmigration and capillary tube formation of HUVEC, and induces a chemotactic response of CD133 + cells. SCF/c-kit signaling possibly plays a critical role in regulating angiogenesis of vascular endothelial cells and vasculogenesis of endothelial progenitor cells.     

甾醇类新药NSC67657诱导THP-1细胞分化及ICAT蛋白在细胞分化中的作用研究

Objective To study the potential mechanism of the new steroidal drug NSC67657 induced leukemic cels differentiation.Methods Cell proliferation was assayed by MTT assay.Surface antigen CD14 on THP-1 cells treated by NSC67657 at different time different concentration,was detected by flow cytometry(FCM).The expression of beta-catenin-interacting protein 1(ICAT)gene and protein were detected by RT-PCR and Western blot.Eukaryotic expressing vector pDsRed-ICAT was constructed and transfeeted into HL60 cell line.FCM,Wright's staining and electronmicroscope were employed to analyse the difierentiation of transfected THP-1 cells after they were treated with NSC67657 for 24 hours.Results The proliferation of THP-1 cells was significantly inhibited by NSC67657 treatment.The level of CD14 expression was elerated in line with the increasing drug concentration and treatment time.10 μmol/L NSC67657 treatment for five days was the optimal condition for the induction of THP-1 cells ditierentiation,when the CD14+ THP-1cells were more than 90%.Morphological study indentified the THP-1 cells of monocvtic differentiation.The eukaryotic expressing vector pDSRed-ICAT was successfully constructed,and almost 90%positive clone could be obtained after G418 screening.Electro-transfection was employed for transfecting the vector into THP-1cells.After the transfection the expression of ICAT gene and protein was increased.On the NSC67657 treatment,there was not significant differenee in CD14 expression on transfected THP-1 cells compared to that on the control groups.After 24h treatment,the transfected THP-1 cells remained in early differentiated stage.Conclusion NSC67657 can induce THP-1 cell to monocytic differentiation and activate the expression of ICAT gene,but overexpression of ICAT itself is not sufficient to induce such differentiation.     

甾醇类新药NSC67657诱导THP-1细胞分化及ICAT蛋白在细胞分化中的作用研究

Objective To study the potential mechanism of the new steroidal drug NSC67657 induced leukemic cels differentiation.Methods Cell proliferation was assayed by MTT assay.Surface antigen CD14 on THP-1 cells treated by NSC67657 at different time different concentration,was detected by flow cytometry(FCM).The expression of beta-catenin-interacting protein 1(ICAT)gene and protein were detected by RT-PCR and Western blot.Eukaryotic expressing vector pDsRed-ICAT was constructed and transfeeted into HL60 cell line.FCM,Wright's staining and electronmicroscope were employed to analyse the difierentiation of transfected THP-1 cells after they were treated with NSC67657 for 24 hours.Results The proliferation of THP-1 cells was significantly inhibited by NSC67657 treatment.The level of CD14 expression was elerated in line with the increasing drug concentration and treatment time.10 μmol/L NSC67657 treatment for five days was the optimal condition for the induction of THP-1 cells ditierentiation,when the CD14+ THP-1cells were more than 90%.Morphological study indentified the THP-1 cells of monocvtic differentiation.The eukaryotic expressing vector pDSRed-ICAT was successfully constructed,and almost 90%positive clone could be obtained after G418 screening.Electro-transfection was employed for transfecting the vector into THP-1cells.After the transfection the expression of ICAT gene and protein was increased.On the NSC67657 treatment,there was not significant differenee in CD14 expression on transfected THP-1 cells compared to that on the control groups.After 24h treatment,the transfected THP-1 cells remained in early differentiated stage.Conclusion NSC67657 can induce THP-1 cell to monocytic differentiation and activate the expression of ICAT gene,but overexpression of ICAT itself is not sufficient to induce such differentiation.     

Extracorporeal regulation effect of cytokines combination on stem cell or progenitor of umbilical blood

AIM：To study the regulation effect of different cytokines combinations on stem cell or progemitor of umbilical blood when no blood serum and matrix exist.METHODS:Collect and analyze the 23 sample of umbilical blood.RESULTS:(1) The combination of SCF+Flk2/Flt3 ligand and (FL)&;#177;TPO can amplify CD34^+,CD34+Thy-1^+,CD34+CD33+and LTC-IC in umbilical blood effectively and rapidly.(2) After add solubility type (sIL-6R) of IL-6 and IL-6 receptor into this combination,the stem cell or progenitor pmliferated prominently,When add IL-6 alone(without sIL 6R),the content of CD34^+ cells and LTC IC didn‘‘‘‘‘‘‘‘t increased obviously in early stage,but the amplification of CFU-Mix and BFU-E was not prominent as CFU-GM.(3) Detect apoptosis rate of cells by FITC-Annexin-V labeled by membranous change in early stage of cellular apoptosis,After add Flt3L and /or IL-6+ sIL-6R,the Annexin-V positive cells decreased from 15.2%-19.1% to 2.8%-3.5%,CONCLUSION:In suspending system composed with SCF+TPO+Flt3L+IL-6+sIL-6R without matrix and blood serum,the umbilical blood can not only produce large amount of committed progenitor but also Keep certain quantitive hematopoietic cells of early stage.     

THE EFFECT OF STEM CELL FACTOR, INTERLEUKIN-6 AND ERYTHROPOIETIN ON EXPANSION OF CD34~ CELLS FROM HUMAN UMBILICAL CORD BLOOD

CD34 cells from human umbilical cord blood were purified by Dynal beads M-450 CD34 immunoselection system and cultured in the presence of various cytokines alone or in combination, including stem cell factor (SCF), interleukin-6 (IL-6) and erythropoietin (EPO). The results revealed that: (D In methylcellulose culture, the plating efficiencies of purified cord blood CD34 cells were much different when stimulated by various cytokines. IL-6 alone had the lowest colo-ny yield, while the combination of SCF, IL-6 and EPO had the highest yield. ② In the suspension culture, IL-6 alone or IL-6 EPO had little expanding effect on cord blood CD34 celis, the other cytokine combinations could expand cord blood CD34 celis at different Ievels. Among them, the combination of SCF, IL-6 and EPO had the maximal expanding effect on cord blood CD34 celis, the number of progenitor celis peaked at day 21, about 29-fold increase and nucleated celis increased approximately 3676-fold at day 28. The expanding effect of the     

细胞因子诱导的杀伤细胞体外培养的初步研究(英文)

Objective To investigate the culture method of cytokines induced killer cells using CD3 monoclonal antibody,IL-2,IFN-γ and IL-1α in vitro.Methods Peripheral blood mononuclear cells (PBMC) were collected by a blood cell separator from 8 patients with refractory lymphoma,then expanded by priming them with recombinant IFN-γ,monoclonal antibody (mAb) to CD3 and human recombinant IL-1α,followed by adding recombinant IL-2 the next day.The CIK cells were counted on d1,4,7,10,13 of incubation,respectively.The phenotypic patterns were characterized before culture and on d13 of culture.Results CIK cells colony formed on d3.On d13,the CIK cells counting reached up to (7-18)×109(mean 12.7×109),multiplying 44-140 times(mean 98 times).Cell survival rate was over 90%.The average percentage of cells expressing CD3+,CD4+,CD8+ and CD3+ CD56+ were also increased from (50.9±3.5)%,(29.9±1.7)%,(41.3±3.2)%,(1.6±0.2)% to (90.2±1.6)%,(40.6±5.5)%,(52.8±4.9)% and (33.1±4.0)%,respectively.Conclusions CIK cells developed by the culture method have high in vitro proliferate rate and tumor-killing capacity.The simplicity of the operation and autogenic source of the cells indicate the method could be applied clinically treating patients with refractory lymphoma.     

血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

脐血来源树突状细胞的体外诱导及扩增

本研究的目的是分析脐血的细胞组成 ,研究加入细胞因子培养前后脐血树突状细胞的变化 ,探索体外诱导、扩增树突状细胞的方法并进行表型鉴定。选择正常成人外周血 9份 ,脐血 12份 ,分离单个核细胞。在脐血单个核细胞中加入细胞因子GM CSF、IL 3、SCF和EPO ,培养 4周。应用流式细胞仪和CD4、CD8、CD19、CD34、CD38、CD1a、CD11c及CDw12 3单克隆抗体测定正常成人外周血、培养前后 1,2 ,3,4周脐血细胞表面抗原及树突状细胞情况。结果表明 :正常成人外周血CD34 细胞 0 .0 2× 10 5 ml,CD1a 细胞 0 .0 1× 10 5 ml,CD11c 细胞 4 .32×10 5 ml,CD83 细胞 1.31× 10 5 ml,CDw12 3 细胞 1.4 1× 10 5 ml。新鲜脐血中CD34 细胞 0 .2 2× 10 5 ml,CD1a 细胞 0 .2 7× 10 5 ml,CD11c 细胞 5 .87× 10 5 ml,CD83 细胞 1.94× 10 5 ml,CDw12 3 细胞 2 .73× 10 5 ml。加入细胞因子GM CSF ,IL 3,SCF ,EPO后培养 1- 4周的脐血单个核细胞分化为CD1a ,CD11c ,CD83 ,CDw12 3 树突状细胞 ,在培养的 2 - 4周 ,脐血树突状细胞数量明显增多 ,此后逐渐减少。通过培养 ,树突状细胞数量增加 ,CD1a 细胞达 11.0 2× 10 5 ml,CD11c 细胞 2 8.2 4× 10 5 ml,CD83 细胞 10 .5 7× 10 5 ml,CDw12 3 细胞 18.7× 10 5     

血管内皮生长因子对CD34+干/祖细胞来源的树突细胞分化及功能的影响

目的　探讨血管内皮生长因子 (VEGF)对人脐血CD34 干 /祖细胞来源的树突细胞(dendriticcell,DC)分化和功能的影响。方法　利用免疫磁珠分离法 (MACS)分离纯化脐血CD34 造血干 /祖细胞 ,并在体外将其诱导扩增为DC ,观察VEGF在培养早期和晚期对DC分化和功能的影响。观察培养过程中细胞增殖方式 ,用流式细胞术检测DC表面分化相关抗原CD1α、CD83、CD80、CD5 4、HLA DR等的表达 ,混合淋巴细胞反应法测定DC体外刺激同种异体T细胞增殖的能力 ,ELISA法检测DC培养上清中IL 12的含量。结果　在细胞增殖方面 ,培养第 1天加入VEGF(2 5ng/ml)可显著促进细胞增殖 ,第 14天收获的总细胞数量较对照组增高 (1.5 1± 0 .2 3)倍 (P =0 .0 0 1) ,而培养第 9天加入VEGF则未出现明显的促细胞增殖效应 (P >0 .0 5 ) ;在细胞分化和功能方面 ,培养第 1天加入VEGF明显抑制DC的分化和功能 ,第 1天加VEGF组和对照组DC分化抗原的表达CD1a分别为(33.0 0± 2 .12 ) %和 (81.2 0± 6 .93) % ,CD83分别为 (42 .2 3± 1.15 ) %和 (87.98± 9.79) % ,CD80分别为 (42 .93± 1.32 ) %和 (94 .5 3± 0 .87) % ,HLA DR分别为 (37.93± 5 .30 ) %和 (74 .15± 3.74 ) % (P值均 <0 .0 0 1) ,同时CD14的表达较对照组明显升高 ;刺激同种异体T淋巴     

人类ermap基因在脐血单个核细胞向红细胞系分化发育过程中表达的研究

为探讨人类红细胞膜相关蛋白（erythroid membrane associated protein，ERMAP）基因在红细胞分化发育过程中的作用，本研究以SCF、IL-3和EPO体外诱导脐血单个核细胞（mononuclear cells，MNCs）向红系细胞方向分化，在此过程中用光学显微镜观察细胞形态，用联苯胺染色计数细胞阳性率，流式细胞术检测CD36^＋／CD235a^-、CD36^＋／CD235a^＋、CD36^-／CD235a^＋细胞的比例，同时以荧光定量PCR（fluorescent quantitative PCR，FQ—PCR）检测人类ermap基因表达量的变化。结果表明，SCF、IL-3和EPO可诱导脐血MNCs向红系方向分化，在此过程中人类ermap基因的表达量不断增加。结论：人类ermap基因与红细胞分化发育密切相关     

人脐带静脉灌注液间充质干细胞的分离和鉴定

本研究旨在探讨人脐带静脉灌注液中是否存在间充质干细胞及其分离和扩增方法。从正常产妇分娩后的脐带静脉灌注液分离间充质干细胞,观察细胞形态并用流式细胞仪分析其细胞表型和细胞周期,在体外分别诱导其成骨、成脂肪和成软骨细胞分化。结果表明:脐带静脉灌注液来源的间充质干细胞为成纤维细胞样,高表达CD29、HLA-ABC、CD166、CD105、CD73、CD44;不表达造血和内皮标志(CD34、CD45、CD144和CD14)。它们可向骨、脂肪和软骨细胞分化,符合间充质干细胞的基本功能特征。结论:人脐带静脉灌注液存在间充质干细胞,能够在体外培养和扩增,可作为今后细胞治疗和组织工程的种子细胞。     

人胎盘源贴壁细胞支持脐血CD34+细胞体外扩增

从胎盘中分离培养人胎盘源贴壁细胞 (humanplacentaderivedadherentcells,hPDAC) ,研究其对脐血CD34+细胞体外扩增作用。以酶消化法自人胎盘组织中分离培养hPDAC ,并以流式细胞术对其进行鉴定。进一步 ,采用免疫磁珠法分离人脐血CD34+细胞 ,建立以hPDAC为滋养层的CD34+细胞体外扩增培养体系 ,并与无滋养层的液体培养体系相比 ,观察不同培养体系对有核细胞总数、CFC及CD34+细胞百分率的影响。结果表明 ,人胎盘组织中可分离培养出hPDAC ,并证实其为混合性细胞群体 ,主要含有间充质细胞。以hPDAC为滋养层的共培养体系较无滋养层液体培养体系对有核细胞总数、CFC及CD34+细胞均具有明显的扩增效应 ,其中以SCF +IL 3+IL 6 +FL +hPDAC组扩增效果最强 ,分别扩增了 ( 12 6 .0± 6 .7)倍 ,( 4 9.8± 1.7)倍和 ( 8.3± 1.6 5 )倍。上述结果提示 ,hPDAC具有体外支持造血作用 ,并提供了一与脐血CD34+细胞体外扩增相适应的新滋养层。     

SCF与bFGF联合诱导脐血CD133~+细胞分化及基因表达的变化

本研究探讨干细胞因子(SCF)与碱性成纤维细胞生长因子(bFGF)体外联合诱导脐血CD133+细胞分化及基因表达的变化,认识脐血CD133+细胞的生物学特性,为体外诱导分化和临床应用打下基础。利用MiniMACS免疫磁珠法从脐静脉血单个核细胞中分离出CD133+细胞,用含干细胞因子(SCF)、bFGF、B27的DMEM/F12细胞营养液培养10-15天,提取培养前后细胞总RNA,利用Oligo GEArray芯片和GEArray表达分析软件进行相关基因表达的芯片数据分析。结果表明:在所检测的263个基因中,发现21个基因培养后表达比培养前显著上调,7个基因表达显著下调。这些基因主要涉及干细胞特异性标志、细胞周期、干细胞分化标志以及与干细胞相关的信号转导、细胞因子及其受体等。结论:SCF和bFGF通过一些特定基因的变化影响脐血CD133+细胞增殖分化,这一结果有助于从基因水平理解SCF与bFGF联合作用对CD133+细胞增殖分化的影响,有利于细胞因子诱导的CD133+细胞在临床上的应用。     

基于磁性纳米粒子-抗体复合物分离CD34+细胞的新技术

本研究合成磁性纳米粒子-抗体复合物（magnetic nanoparticles-antibody,MNPs-Ab）,建立用此复合物从脐带血中分离CD34^＋细胞的方法并评价其效果。利用磁性纳米粒子-抗体复合物的超顺磁性和对CD34^＋细胞的识别及结合功能,在外加磁场的作用下,分离脐血中的CD34^＋细胞;用扫描电子显微镜（scanning electron microscopy,SEM）观察分离后CD34^＋细胞的形态变化;用流式细胞仪（flow cytometer,FCM）分析分离效率;用液体培养和集落培养鉴定CD34^＋细胞增殖能力和多向分化能力。结果显示：利用磁性纳米粒子-抗体复合物可以快速、高效地将CD34^＋细胞从脐血中分选出来,而且分离后的CD34^＋细胞仍然保持正常形态、高度的增殖能力和多向分化能力。结论：成功地研制了磁性纳米粒子-抗体复合物分离CD34^＋细胞技术,该技术能高效、快速地分离CD34^＋细胞。     

胎肺间充质干细胞对脐血来源的单个核细胞体外扩增为巨核细胞的影响

本研究探讨胎儿肺部组织来源的间充质干细胞在无血清条件下体外对脐血单个核细胞诱导扩增为巨核细胞的影响。取脐血单个核细胞,在TPO,IL-11,肝素的扩增体系中分为2组培养。第1组为脐血单个核细胞单独培养,第2组脐血单个核细胞与间充质干细胞共培养,在第7,10,14天时进行活细胞计数,用流式细胞仪分析CD41a和CD61的表达情况。采用免疫组织化学和透射电子显微镜观察第14天细胞的形态与结构,用流式细胞仪分析细胞DNA含量。结果表明,第2组在第10天时获得的CD41^＋细胞和CD61^＋细胞数量最多,分别为第1组的4．5倍和4．7倍;但免疫组织化学和电子显微镜显示,2个组的CD41a^＋和CD61^＋细胞的核发育不成熟。结论：胎肺间充质干细胞能有效刺激CD41a^＋和CD61^＋细胞生成,但不能促进幼巨核细胞核的发育。     

人脐血造血干/祖细胞体外定向诱导分化过程中端粒酶的表达

探讨脐血造血干/祖细胞体外诱导分化过程中端粒酶的表达,为造血干细胞产品的临床应用提供一个监测细胞增殖潜能和安全性的指标。用源自人脐血的CD34~+细胞及不同细胞因子组合(SCF+IL-3+IL-6+G-CSF,SCF+IL-3+IL-6+EPO)在体外进行诱导分化;用TRAP聚丙烯酰胺凝胶电泳法、TRAP-ELISA法检测CD34~+细胞及诱导分化细胞的端粒酶活性;用Western印迹法在蛋白水平检测端粒酶催化亚基hTERT的表达,用RT-PCR在细胞转录水平检测端粒酶催化亚基hTERT mRNA的表达。结果显示,在体外诱导分化14-21天为细胞生长峰值,细胞总数可增加(1006.4±103.2)倍,随着培养时间的延长,细胞数量不再增加。CD34~+细胞低度表达端粒酶活性和hTERT基因,在细胞诱导分化过程中端粒酶活性及hTERT表达逐渐升高,细胞诱导14天后端粒酶活性及hTERT表达下降,28天端粒酶活性及hTERT基因检测不出。结论:利用CD34~+细胞在体外定向诱导分化出的大量细胞,不具有无限增殖的特性,可安全地应用于临床,且利用端粒酶的检测可为临床应用诱导分化细胞的时机提供依据。     

细胞因子体外诱导脐血CD34+细胞增殖并向巨核细胞/血小板分化的研究

本研究旨在观察几种不同细胞因子组合通过体外培养以诱导造血干/祖细胞增殖和向巨核细胞/血小板分化.应用无血清培养基(StemSpan SFEM)体外扩增脐血CD34+细胞并向巨核细胞/血小板定向分化,将不同细胞因子组合分为3个阶段培养,并比较其培养效果.结果表明,在第一阶段的第14天时,SCF + TPO + FL + ...