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1.
目的 利用诱导性多能干细胞技术体外扩增造血干/祖细胞.方法 利用非整合型载体,将四个转录因子:Sox2、Klf4、Oct4和c-Myc导入脐血来源的造血干/祖细胞(CD34+细胞)重编程获得诱导性多能干细胞(iPSCs),再利用与小鼠骨髓基质细胞OP9共培养法将其定向分化成CD34+造血干/祖细胞.借助iPSCs可以在体外无限传代、大量扩增的特点,实现体外保存及大量扩增造血干/祖细胞的目的.结果 脐血CD34+细胞可以在体外重编程为非整合型iPSCs,并能够高效定向分化成为CD34+细胞,其分化效率比胚胎干细胞(ESCs)有显著提高.结论 利用诱导性多能干细胞技术体外大量扩增脐血造血干/祖细胞是一个可行的方案,具有良好的应用前景.  相似文献   

2.
人脐血间充质干/祖细胞诱导为神经样细胞的实验研究   总被引:6,自引:0,他引:6  
目的探讨人脐血间充质干细胞用于神经细胞再生的可行性与人脐血间充质干/祖细胞最佳的诱导培养条件。寻找一种合适的细胞来源进行移植以代替受损的神经元、星形胶质细胞和少突胶质细胞来修复神经损伤。方法将脐血单个核细胞置于低血清(2%)达氏修正依氏培养基中培养,生成贴壁细胞层,依传代方法,用相同培养条件进行扩增,扩增后的贴壁细胞置入细胞培养液(加入新生大鼠神经细胞分离培养上清液)中,并添加表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)进行诱导分化。采用流式细胞术、免疫组织化学法分别检测被诱导的神经样细胞的表面标志及细胞特异性标志、结构,并进行分析。结果脐血间充质干/祖细胞克隆在脐血单个核细胞中出现频率为0.5×10-6,在传20代时,可有效扩增1.5×106倍,诱导后,70%的脐血间充质干/祖细胞分化为神经样细胞。结论采用上述扩增与诱导条件,脐血间充质干/祖细胞可得到有效扩增,并可高效向神经样细胞分化,从而证实脐血是一种细胞替代治疗前景光明的细胞来源。  相似文献   

3.
脐血造血干细胞分离方法的实验研究   总被引:1,自引:0,他引:1  
目的 优化脐血造血干细胞/祖细胞的分离方法.方法 脐血按体积比1:5与含有树脂海绵筛(筛孔直径约12μm)的6%羟乙基淀粉混合,静置60min,提取界面有核细胞.倒置显微镜下计数有核细胞数,集落培养记数CFU-GM数,流式细胞仪检测CD34 细胞数,并与按照标准的羟乙基淀粉沉淀法和淋巴细胞分离液密度梯度离心法分离的脐血相对照.结果 采用本实验方法分离脐血获得的有核细胞数、CFU-GM数和CD34 细胞数分别为(96.82±25.78)×105/ml,(66.70 ±12.47)×105,(1.33±0.57)×105/ml,明显高于由羟乙基淀粉沉淀法(70.72±20.93)×105/ml,(28.79±10.25)×105,(1.01±0.36)×105/ml和淋巴细胞分离液密度梯度离心法(25.33±7.27)X 105/ml,(20.13±7.55)×105,(0.34±0.01)×105/m1分离的脐血(P<0.05).结论 本实验方法可由脐血中分离出大量的造血干/祖细胞,是一种分离脐血造血干/祖细胞的理想方法.  相似文献   

4.
盐酸贝那普利对造血祖细胞体外扩增的影响   总被引:1,自引:0,他引:1  
目的探讨盐酸贝那普利(洛汀新,血管紧张素转换酶抑制剂)对不同系别造血祖细胞分化的影响及其分子机制。方法由正常脐血分离出单个核细胞,在含有不同浓度盐酸贝那普利与其他因子包括干细胞因子(SCF)、白细胞介素-3(IL-3)、粒-巨噬细胞集落刺激因子(GM-CSF)、红细胞生成素(EPO)、血管紧张素Ⅱ(AngⅡ)的培养基中悬浮培养,然后转到半固体培养基,对粒-巨噬细胞集落形成单位(CFU-GM)和红细胞样爆发形成单位(BFU-E)集落进行观察并计数,同时应用流式细胞仪检测红系和粒系所占比例;巢式反转录聚合酶链反应(RT-PCR)监测AngⅡ1型受体(AT1R)mRNA表达。结果①红系祖细胞在悬浮培养6、9d时均可检测到AT1R的表达,两时间点各组AT1R的表达量差异均无统计学意义;在上述两时间点各对应组之间AT1R的表达量差异无统计学意义;②半固体培养14d后,各组的粒系集落明显减少,而各组之间比较差异无统计学意义;③各组红系集落形成丰富,但各组间差异无统计学意义;④各组红系和粒系所占的百分比差异有统计学意义(85.85%vs13.15%,85.78%vs13.21%,85.77%vs13.20%,85.79%vs13.18%,85.81%vs13.23%,P<0.05)。结论在体外细胞培养过程中AngⅡ能促进红系祖细胞的扩增,而盐酸贝那普利不能阻断AngⅡ对红系造血祖细胞的优势分化作用。  相似文献   

5.
目的观察刺梨提取物CL1对胃癌SGC-7901细胞增殖和人脐血CD34+造血干/祖细胞(HSPC)增殖分化能力的影响.方法采用MTT法测定活细胞OD值,计算CL1对胃癌SGC-7901细胞增殖抑制率.免疫磁珠分选人脐血CD34+造血干/祖细胞,细胞记数、双色直接免疫荧光标记法和流式细胞仪分析细胞增殖分化能力变化.结果MTT检测法显示,0.01~10 μmol·L-1的CL1处理,剂量依赖性和时间依赖性地抑制胃癌SGC-7901细胞生长.经 14 d 的培养,与细胞对照组相比,10、1 μmol·L-1的CL1细胞数有下降趋势,但差异无显著性;表达粒系/单核巨噬系细胞表型CD15和CD11b的细胞百分数无显著差异.经 14 d 的培养,与培养 0 d 比较,CD15显著增高(P<0.05),CD11b有增高趋势,但无显著性差异.结论CL1对胃癌SGC-7901细胞的体外生长具有抑制作用,但对人脐带血CD34+ HSPC的增殖、和向粒细胞、单核巨噬细胞分化无明显影响,表明CL1在抗肿瘤作用剂量下,不会明显抑制造血干/祖细胞向粒-单细胞的增殖分化,提示CL1可能不会引起明显的造血抑制.  相似文献   

6.
目的 观察系统性红斑狼疮(SLE)患者血清中的白细胞介素6(IL-6)对脐血CD34 造血干细胞(HPC)体外诱导分化为树突状细胞(DCs)的影响.方法 淋巴细胞分离液分离获得脐血单个核细胞,免疫磁珠阳性分选CD34 HPC,采用粒-巨噬细胞集落刺激因子(GM-CSF) IL- 4 肿瘤坏死因子α(TNF-α)方案体外诱导分化形成DCs,加入高IL-6水平的SLE患者血清观察对细胞分化的影响.在培养的7、10和14d收集细胞,流式细胞术检测表型,细胞增殖/毒性(CCK-8)试剂盒分析DCs刺激同种异体淋巴细胞增殖的能力.结果 在高IL-6水平SLE血清的作用下,由HPC分化而来的DCs表达CD80、CD86上升,刺激同种异体淋巴细胞增殖的能力增强;IL-6中和抗体能够降低这种DCs的CD80、CD86表达水平和对同种异体淋巴细胞增殖的刺激能力.结论 SLE血清中的IL-6对造血干细胞向DCs分化、发育及DCs功能的活化可能起促进作用.  相似文献   

7.
目的通过细胞形态学观察以及计数法对分离得到的脐血CD34+造血干细胞进行研究,掌握脐血CD34+造血干细胞的生长规律和体外培养方法。方法采用Isolex50免疫磁珠法分离纯化脐血CD34+造血干细胞。在体外扩增培养中,将细胞因子SCF、IL-3、IL-6、G-CSF、EPO组合成不同实验组进行诱导,以检测单个核细胞数和集落形成单位(CFU)的形成率来找到细胞培养较好的细胞因子组合。结果本研究中使用细胞因子的细胞培养孔的单个核细胞数明显大于无细胞因子的细胞培养孔,使用此5种细胞因子联合对脐带造血干细胞培养效果最好。结论研究结果表明,不同的细胞生长因子组合会对培养CD34+造血干细胞产生不同的作用效果,对单个核细胞的增殖与扩增有明显的促进作用。  相似文献   

8.
范伟  张莹  郭江福  王金龙  柳杨 《贵州医药》2011,35(10):871-875
目的 证实脐血造血干/祖细胞移植重建SCID鼠的免疫功能对人肝癌有抑制及杀伤作用.方法 首先进行人肝癌高转移细胞株HCCLM6细胞培养,然后将HCCLM6细胞株分别在脐血移植免疫功能重建组(A组)及未移植脐血造血干/祖细胞组(B组)建立SCID鼠皮下荷瘤模型.接种肿瘤后5周处死,分别进行以下检测:(1)比较A、B两组间...  相似文献   

9.
目的:比较皮肌炎患与正常人的造血干/祖细胞体外增殖分化能力及从造血干/祖细胞水平探讨皮肌炎的发病机制。方法:用半固体甲基纤维素集落培养法观察22例皮肌炎患的粒-巨噬细胞系、红系、巨核细胞系的集落及丛落数。结果:皮肌炎患的粒-巨噬细胞系、红系、巨核细胞系的集落分别为119.4351.44、75.1236.26、9.183.76均明显低于正常对照组,丛落数分别为240.5392.11、60.6920.72、29.0113.54均高于正常对照组。结论:皮肌炎患的造血干/祖细胞集落数减少而丛落数不减少,提示皮肌炎患和造血干/祖细胞的增殖、分化功能异常,造血干/祖细胞的异常可能是皮肌炎的发病机制。  相似文献   

10.
目的研究氨磷汀(WR-2721)和峰龄多糖(FLPS)对人外周血造血干/祖细胞的刺激增殖及协同作用.方法分离单个核细胞(MNC),进行甲基纤维素半固体培养,观察氨磷汀和峰龄多糖对骨髓粒-巨噬细胞集落(CFU-GM)的作用.结果经0.01~5.0 mmol/L氨磷汀37℃作用30分钟的MNC以及峰龄多糖0.5~25μg/ml时CFU-GM集落数均显著高于对照组(P<0.05),且集落较大.CFU-GM平均集落数在对照组、WR-2721(1 mmol/L)组、FLPS(5μg/ml)组和AMF+FLPS组每105MNC分别为91.4±50.4、119.8±62.9、143.2±76.4和179.2±97.6,各组与对照组相比,差异有显著性(P<0.05);AMF+FLPS组CFU-GM集落数显著高于AMF和FLPS组(P<0.01和<0.05).结论氨磷汀和峰龄多糖对CFU-GM具有明显的刺激增殖及协同作用.  相似文献   

11.
张海玲  张永富  张君奎 《天津医药》2001,29(10):609-612
目的:检测小鼠原始造血细胞c-kit基因表达以评价体外模拟骨髓造血微环境。方法:建立微载体-基质细胞体外造血模型。根据已发表的小鼠c-kit基因序列设计引物,采用RT-PCR方法检测c-kit mRNA水平,并测定祖细胞集落产率。结果:小鼠骨髓造血细胞2周培养实验显示,粒系-巨噬系造血祖细胞集落产率(CFU-GM/10^5):模型组比液体悬浮培养和单纯微载体基质细胞培养对照组集落产率之总和高2.1倍(t=2.869,P<0.05);原始造血细胞的c-kit基因表达水平(OD比率):模型组比两个对照组之总和高3.1倍(t=2.858,P<0.05)。结论:在没有外加细胞因子的条件下,微载体-基质细胞造血模型可抑造血干、祖细胞过度分化与耗竭,维持其c-kit mRNA较高的表达水平。  相似文献   

12.
转HOXB4基因人骨髓MSC促进脐血CD34~+细胞体外扩增   总被引:1,自引:0,他引:1  
目的 研究转HOXB4基因的人骨髓间充质干细胞(MSC)对脐血CD34~+细胞体外扩增的支持作用.方法 用病毒载体质粒转染包装细胞293T,收获病毒上清(VCM)感染MSC后,用潮霉素筛选获得转HOXB4的MSC(MSC-HOX).分别将MSC(对照组)与MSC-HOX细胞(实验组)作为CD34~+细胞体外扩增的饲养层细胞,结合细胞因子Fh3/Flk3配体(FL)、促血小板生成素(TPO)、干细胞因子(SCF)和粒细胞-集落生长因子(G-CSF)体外扩增脐血CD34~+细胞10 d,收集所有脐血细胞,检测细胞总数、CD34~+细胞总数,集落细胞形成单位(CFU)数.结果 生产重组逆转录病毒可有效将HOXB4基因转入靶细胞内并表达.脐血CD34~+细胞经体外扩增10 d后,细胞总数和CFU数两组间差异无统计学意义(P>0.05),脐血CD34~+细胞总数和比例高于对照组(P<0.05).结论 转HOXB4基因的人骨髓MSC在脐血CD34~+细胞体外扩增中,可保持CD34~+细胞未分化状态,有潜在的应用价值.  相似文献   

13.
目的探讨中药鸡血藤Spatholobus suberectus Dunn活性成分儿茶素对小鼠骨髓细胞增殖周期及其脾细胞内造血生长因子IL-6和GM-CSF mRNA表达的影响,以初步阐明其造血调控作用机理。方法用流式细胞仪检测技术观察儿茶素对正常及骨髓抑制小鼠骨髓细胞增殖周期的影响,同时采用RT-PCR技术,检测在儿茶素刺激条件下,小鼠脾细胞内IL-6和GM-CSF mRNA表达的变化。结果儿茶素可促使正常及骨髓抑制小鼠骨髓细胞G0/G1期细胞比例下降,S+G2/M期细胞比例增加,并可使正常及骨髓抑制小鼠脾细胞内IL-6 mRNA和GM-CSF mRNA表达显著上调。结论儿茶素可通过诱导脾细胞内IL-6 mRNA和GM-CSF mRNA的表达,促进正常小鼠骨髓细胞进入增殖周期,并促使骨髓抑制小鼠的骨髓细胞跳出“G1期阻滞”,进入细胞增殖周期,从而加速造血干/祖细胞的增殖和分化。  相似文献   

14.
In this study, carboxylic acid functionalized single walled carbon nanotubes (f-SWCNT-COOH) was shown to support the viability and ex vivo expansion of freeze-thawed, non-enriched hematopoietic stem and progenitor cells (HSPC) in human umbilical cord blood–mononucleated cells (UCB-MNC). Our in vitro experiments showed that f-SWCNT-COOH increased the viability of the CD45+ cells even without cytokine stimulation. It also reduced mitochondrial superoxides and caspase activity in CD45+ cells. f-SWCNT-COOH drastically reduced the proportions of CD45? cells in the non-enriched UCB-MNC. Phenotypic expression analysis and functional colony forming units (CFU) showed significant ex vivo expansion of HSPC, particularly of CD45+CD34+CD38? population and granulocyte-macrophage (GM) colonies, in f-SWCNT-COOH augmented cultures supplemented with basal cytokines. In vivo data suggested that f-SWCNT-COOH expanded UCB-MNC could repopulate immunodeficient mice models with minimal acute or sub-acute symptoms of graft-versus-host disease (GVHD) and f-SWCNT-COOH dependent toxicity.From the Clinical EditorIn this paper a novel method is presented by using single wall functionalized carbon nanotubes to enhance viability and ex vivo expansion of freeze-thawed, non-enriched hematopoietic stem and progenitor cells in human umbilical cord blood -mononucleated cells. Detailed data is presented about enhanced viability, including improved repopulation of immunodeficient mice models with minimal acute or sub-acute symptoms of graft-versus-host disease.  相似文献   

15.
1. There are increased numbers of circulating CD34(+) progenitor cells for eosinophils in patients with atopic asthma, with a further increase following allergen exposure or spontaneous worsening of asthma. We investigated the expression of IL-5 and IL-5Ralpha receptor in circulating CD34(+) progenitor cells in allergic asthmatics and the effects of corticosteroids. 2. Using double-staining techniques, up to 50% of CD34(+) cells expressed intracellular IL-5, and by RT - PCR, there was significant expression of IL-5 mRNA. When cultured in a semi-liquid methylcellulose medium, there were more eosinophil colony-forming units grown from asthmatic non-adherent mononuclear cell depleted of T cells in the presence of the growth factors GM-CSF, SCF and IL-3, but not of IL-5. 3. An anti-IL-5Ralpha receptor antibody and an anti-sense IL-5 oligonucleotide reduced the number of eosinophil colony forming units. No IL-5 mRNA or protein expression on T cells was observed in asthmatics or normal subjects. In the presence of growth factors including IL-5, there were significantly greater colony numbers with eosinophilic lineage grown from either asthmatics or normal subjects. 4. Dexamethasone (10(-6) M) suppressed IL-5 mRNA and protein expression in CD34(+) cells, and reduced eosinophil colony-forming units in asthmatics, but not in normal subjects. Dexamethasone did not change the expression of IL-5Ralpha on CD34(+) cells. 5. We conclude that there is increased expression of IL-5 on blood CD34(+) cells of patients with asthma and that this expression may auto-regulate eosinophilic colony formation from these progenitor cells. Corticosteroids inhibit the expression of IL-5 in circulating CD34(+) progenitor cells.  相似文献   

16.
脐血造血细胞扩增后细胞成分的变化   总被引:1,自引:0,他引:1  
目的 探讨人脐血造血细胞体外扩增细胞数量和质量的变化及收获的最佳时机。方法 用重组人白细胞介素 - 3(rh IL- 3)、粒单集落刺激因子 (GM- CSF)和重组促红细胞生成素 (rh EPO)长期培养人脐血单个核细胞 ,动态观察其细胞构成和集落生成能力。结果 培养 1周后细胞总数逐渐增多 ,3周末细胞总数最多 ,扩增倍数为 (8± 4)倍 ,细胞构成则以髓系细胞为主 ;淋巴细胞 (CD3+ 、CD19+ )培养 1周明显下降 ,2周后低水平维持 ;培养 2周时 CD34+ 细胞百分比最高 ,为 (2 .4± 0 .7) % ,培养 3周时 CD34+ 细胞扩增倍数最大 ,为 (30± 7)倍 ,其中CD33+ CD34+ 细胞扩增倍数为 (4 5± 5 )倍。 CD71+ 细胞以培养 2周时最多 ,最高为 (5 1± 8) % ;CD42 a+ 细胞培养 3周时最高 ;单位数量细胞集落生成能力培养 ,1周时最大 ,但总的集落生成能力第 3周时最大。结论 含有上述生长因子的培养体系主要扩增脐血造血细胞的髓系细胞 ,次要扩增红系、巨核系细胞 ,最佳扩增时机为 3周。  相似文献   

17.
刘晓梅  张洪泉 《药学学报》2002,37(8):616-620
目的研究螺旋藻多糖(PSP)对肿瘤化疗后造血细胞增殖、凋亡及Bcl-2表达的影响。方法小鼠移植性实体瘤模型、用造血祖细胞体外培养、荧光和普通光学显微镜、ELISA及免疫组化方法,检测了造血细胞增殖、凋亡、Bcl-2表达及相关细胞因子含量。结果PSP明显改善了CTX引起的CFU-GM减少、造血细胞凋亡,并促进了IL-1,IL-3和GM-CSF分泌及造血细胞Bcl-2表达。结论PSP促进内源性细胞因子的分泌间接上调抗凋亡蛋白Bcl-2表达可能是其促进肿瘤化疗后造血细胞增殖并抑制其凋亡的分子机制之一。  相似文献   

18.
There is increasing evidence that neuronal factors can affect hematopoietic cell proliferation. Endogenous opioids with specificity for several opioid receptor classes were tested for their ability to inhibit murine and human hematopoietic progenitor cell proliferation. Tyr-MIF, an opioid tetrapeptide (H-Tyr-Pro-Leu-Gly-NH2), demonstrated a dose-dependent inhibition of colony formation at concentrations < 10 uM, inhibiting M-CSF and G-CSF-responsive progenitor cells equally. Tyr-MIF did not inhibit the number of colonies responsive to recombinant interleukin 3 (rmIL-3) or recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF), but significantly reduced colony size of GM-CSF responsive colonies. Colony formation by human low density and CD34+ marrow cells in response to G-CSF was also inhibited by Tyr-MIF and was more sensitive to inhibition than murine progenitor cells. Colony formation by single CD34+ cells was also inhibited by Tyr-MIF, indicating an effect directly on progenitor cells. Incubation of marrow cells in liquid culture and removal of Tyr-MIF prior to quantitating progenitor cell proliferation demonstrated that opioid-induced inhibition was reversible. The inhibitory effect of Tyr-MIF was not blocked by naloxone, a mu receptor specific antagonist, or diminished in mu opioid receptor deficient mice. HPLC analysis of cell-free culture medium containing Tyr-MIF showed no presence of the parent peptide after 24 h while progenitor cell inhibitory activity was retained. Analysis of potential degradation products of Tyr-MIF indicated that only H-Gly-NH9 or H-Gly-NH2 containing peptides inhibited colony forming unit (CFU) proliferation. These results indicate that Tyr-MIF is a reversible inhibitor of mature hematopoietic progenitor cell proliferation, and that this effect is most likely mediated by the degradation product H-Gly-NH2. Potential applications including protection of myeloid cells after cytosuppresive therapy are discussed.  相似文献   

19.
1. Theophylline possesses anti-inflammatory activities in asthma. We examined whether theophylline and agents that modulate cyclic AMP can determine the survival and proliferation of progenitor cells. 2. Progenitor cells from the blood of normal and asthmatic subjects were cultured for 14 days in methylcellulose with GM-CSF, stem cell factor, IL-3 and IL-5. Apoptosis was measured by flow cytometry of propidium-iodide-stained cells. 3. A greater number of colonies with a higher proportion of cells of eosinophil lineage from asthmatics compared to normal subjects were grown. Theophylline (at 5 and 20 micro g ml(-1)) significantly inhibited colony formation and increased apoptotic cells in asthmatics compared to control. Salbutamol (0.1, 1, 10 micro M), dibutyryl-cAMP (0.1, 1 mM) and rolipram (0.1, 1 mM), a phosphodiesterase IV inhibitor, also dose-dependently decreased colony numbers and increased apoptosis of progenitor cells from asthmatics. 4. There was no significant effect of theophylline, db-cAMP, salbutamol or rolipram on colony formation or the survival of progenitor cells from normal subjects. AMP did not affect the colony formation and apoptosis. Expression of Bcl-2 protein on progenitor cells of asthma was downregulated by theophylline, salbutamol, db-cAMP and rolipram. 5. Theophylline and rolipram decreased colony formation committed to the eosinophil lineage, together with an increase in apoptosis through an inhibition of Bcl-2 expression effects that may occur through cAMP. The anti-inflammatory properties of theophylline include an inhibition of circulating progenitor cells.  相似文献   

20.
地黄低聚糖对快速老化模型小鼠造血功能的影响   总被引:14,自引:1,他引:14  
目的:研究地黄低聚糖(RGOS)对快速老化模型小鼠(SAMP8)造血功能的影响。方法:采用造血祖细胞体外克隆培养等实验血液学方法。结果:RGOS10,20mg·kg-1可使SAMP8小鼠的CFU-S、CFU-GM、CFU-E和BFU-E明显增多,并可使外周血中降低的WBC数明显增加。提示RGOS可刺激SAMP8小鼠造血干细胞、祖细胞的增殖分化。集落刺激活性实验显示RGOS可使小鼠体内的集落刺激因子产生明显增多。小鼠骨髓组织学研究结果也进一步证实了RGOS增强SAMP8小鼠造血功能的作用。结论:RGOS可以增强SAMP8小鼠的造血功能。  相似文献   

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