利用诱导性多能干细胞技术体外扩增造血干/祖细胞的初步研究

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.     

AMD3100动员造血干/祖细胞的实验研究

目的观察AMD3100对造血干/祖细胞(HSC/HPC)动员作用。方法 C57BL/6小鼠分为两组:实验组皮下注射AMD3100 5mg/kg;对照组皮下注射等体积的生理盐水。分别于0、0.5、1、2、4、8及12h取血,检测外周血白细胞计数、流式细胞术检测KSL细胞及KL细胞。结果用药30min后,实验组白细胞、KSL细胞及KL细胞明显高于用药前及对照组(P<0.05),2h达到高峰,随后逐渐下降,到24h下降到用药前水平。结论 AMD3100对HSC/HPC有一定的动员作用,动员速度快,作用时间短。     

巴西蘑菇多糖对造血干、祖细胞生成的影响

目的:探讨国产巴西蘑菇多糖对造血干、祖细胞生成的影响及其作用机制。方法:应用造血细胞培养技术、流式细胞仪(FACS)、逆转录多聚酶链反应(RT-PCR)观察药物对小鼠骨髓细胞及人脐血单个核细胞的影响。结果:经过巴西蘑菇多糖处理的动物,造血干细胞(CFU-S)、粒-单祖细胞(CFU-GM)、红系祖细胞(CFU-E)和纤维母细胞(CFU-F)各组数值明显上升,与对照组比较,P均<0.01;FACS检测显示,实验组CD33+细胞明显上升,在诱导的d 7达高峰;经RT-PCR检测结果表明,巴西蘑菇多糖诱导人脐血单个核细胞48 h后,粒单细胞克隆刺激因子(GM-CSF)mRNA的表达量是对照组的2.72倍。结论:巴西蘑菇多糖可促进造血干、祖细胞生成并可促进人脐血单个核细胞GM-CSF基因表达。     

氨磷汀与峰龄多糖对外周血造血干细胞的刺激增殖及协同作用

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.     

三氧化二砷与顺铂联合诱导人卵巢癌HO8910细胞凋亡的初步研究

目的探讨三氧化二砷与顺铂联合对体外培养卵巢癌细胞株HO8910细胞增殖、细胞周期调控及细胞凋亡影响。方法用三氧化二砷与顺铂联合处理HO8910细胞,采用MTT法检测药物对细胞的抑制作用,倒置显微镜观察细胞形态学改变;琼脂糖凝胶电泳观察DNA降解,以及应用流式细胞仪观察细胞凋亡过程中细胞周期的变化。结果在三氧化二砷与顺铂联合作用下,HO8910细胞呈凋亡改变,DNA琼脂糖凝胶电泳呈典型的凋亡特征。细胞凋亡的同时,细胞周期发生特定的改变。结论三氧化二砷与顺铂联合能抑制卵巢癌细胞增殖,能诱导卵巢癌细胞凋亡。     

造血干/祖细胞移行能力及影响因素的实验研究

目的：研究纯化脐血CD34^ 细胞移行穿越血管内皮细胞能力及影响因素。方法：免疫微磁珠阳性选择法（MACs)纯化脐血CD34^ 细胞，流式细胞仪测定基质细胞衍生因子（SDF－1α）受体（CXCR4）表达率；与干细胞因子（SCF）、白介素（IL）－6，IL－3及flt3配体（FL）共同孵育12小时，静脉内皮细胞株（ECV）接种于transwell滤膜上层，研究CD34^ 细胞在SDF－1α作用下移行穿越ECV的能力，transwell滤膜孔径为8μm，并观察阻断其表面粘附分子（CD62L、CD62E和VLA－4）后对迁移的影响。结果：CD34^ 细胞在自然状态下也能少量通过ECV细胞层，SDF－1α可显提高CD34^ 细胞移行能力，通过率与CD34^ 细胞表面CXCR4表达相关；CD34^ 细胞与SCF与IL－6共同孵育后，可明显提高其穿越ECV细胞的能力（P＜0．01）；单独或联合应用抗粘附分子抗体显减少CD34^ 细胞的穿内皮细胞移行（P＜0．01）。结论：CD34^ 细胞可穿过ECV内皮细胞层向SDF－1α浓度高的一侧移行，与IL－6和SCF共同培养后可增强CD34^ 细胞的移行能力，抗粘附分子单抗显减少CD34^ 细胞的移行。     

青蒿琥酯对小鼠骨髓造血干/祖细胞分化的抑制与毒性作用

目的:观察青蒿琥酯(artesunate,AS)对小鼠骨髓造血干/祖细胞分化的影响。方法:取小鼠骨髓干细胞进行体外液体或半固体定向培养并加入不同浓度的AS,光镜下计数半固体培养的红系集落形成单位(CFU-E)与红系爆式集落形成单位(BFU-E)数量,流式细胞术检测液体定向培养的细胞凋亡和线粒体膜电位变化,同时进行DNAladder凝胶电泳实验。结果:AS可显著抑制CFU-E与BFU-E集落的生成(P<0.05)并具有一定的量效关系;DNAladder凝胶电泳结果显示0.01～1.30mmol/L的AS可明显诱导细胞凋亡,流式细胞仪检测细胞晚期凋亡/死亡率具有统计学意义(P<0.05);线粒体膜电位的下降程度也与AS浓度正相关(r=0.546,P=0.018)。结论:AS对小鼠骨髓造血干/祖细胞的增殖和分化具有明显抑制作用,小剂量AS可诱导小鼠骨髓造血干/祖细胞发生凋亡,大剂量可直接引起细胞坏死。     

Exposure of Tg.AC transgenic mice to benzene suppresses hematopoietic progenitor cells and alters gene expression in critical signaling pathways

The effects of acute benzene (BZ) exposure on hematopoietic progenitor cells (HPCs) derived from bone marrow cells were studied using homozygous male v-Ha-ras Tg.AC mice at 8-10 weeks of age. The mice were given 0.02% BZ in their drinking water for 28 days with the dose rate estimated to be 34 mg benzene/kg BW/day. Analysis of cultured HPCs indicated that BZ suppressed the proliferation of the multilineage colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM); colony forming unit-granulocyte, macrophage (CFU-GM); and blast forming unit erythrocyte/colony forming unit erythrocyte (BFUE/CFUE). A gene expression profile was generated using nylon arrays spotted with 23 cDNAs involved in selected signal pathways involved in cell distress, inflammation, DNA damage, cell cycle arrest, and apoptosis. Of the 23 marker genes, 6 (bax, c-fos, E124, hsf1, ikBa, and p57) were significantly (Mann-Whitney U tests, P < 0.05) overexpressed in BZ-exposed mice. Two genes (c-myc and IL-2) approached significance (at P = 0.053). The pattern of gene expression was consistent with BZ toxicity and the suppression of HPCs.     

三氧化二砷对三阴性乳腺癌细胞增殖及凋亡的抑制作用

目的探讨三氧化二砷对人三阴性乳腺癌细胞HCC1937的抑制作用。方法体外培养人乳腺癌HCC1937细胞,利用MTT法及倒置荧光显微镜观察三氧化二砷对人乳腺癌HCC1937细胞生长的抑制作用。结果不同浓度的三氧化二砷均可抑制人乳腺癌HCC1937细胞的增殖,促进其凋亡。三氧化二砷20μg/m1对肿瘤细胞作用72h,其生长抑制率达80.51%。结论三氧化二砷可有效抑制人乳腺癌HCC1937细胞生长,且呈时间、剂量依赖关系。     

重组白介素1α对粒单系祖细胞的辐射防护作用(英文)

高剂量(0.5或1.62 mg·kg~(-1))重组人白介素1α(IL-1α)在照射前给药时能抗辐射对C_3H小鼠骨髓粒单系祖细胞的损伤,照射前4h给药时GM-CFC的数目是照射前20h给药的1.70倍.实验进一步观察了IL-1α对血清粒单系集落刺激因子(GM-CSF)的产生以及GM-CFC在细胞周期中分布的变化,结果表明,IL-1α给药3.5 h后,使血清GM-CSF的产生达到最高水平,12 h后,恢复至给药前水平;相反,给药4h后对GM-CFC的细胞周期分布无影响,而20 h后,能诱导GM-CFC进入细胞周期的S期,提示IL-1α在照射前不同时间给药对GM-CFC的辐射防护作用的差异可能与GM-CFS产生及细胞周期的重分布有关。     

三氧化二砷与氟尿嘧啶、吉西他滨对胰腺癌细胞作用的对照研究

目的 研究三氧化二砷(As2O3)对人胰腺癌细胞的抑制作用.方法 应用Cell Counting Kit-8法(CCK-8法)测定As2O3对人胰腺癌PC-3细胞株的抑制率,并与氟尿嘧啶(5-Fu)、吉西他滨(GEM)进行比较.结果 与5-Fu、GEM相比,As2O3抑瘤作用最强(P＜0.01或P＜0.05).结论 As2O3体外试验能有效抑制人胰腺癌PC-3细胞株的生长;As2O3对人胰腺癌PC-3细胞株生长的抑制作用强于5-Fu、GEM.As2O3较强的抗胰腺癌细胞能力可能与其广泛而独特的抗癌机制有关.     

改良预处理方案在单倍体移植治疗恶性血液病中的应

目的 探讨采用改良白消安-环磷酰胺预处理方案行单倍体移植治疗恶性血液病的疗效.方法 采用阿糖胞苷、白消安、环磷酰胺、甲环己亚硝脲及抗胸腺细胞球蛋白联合作为改良白消安-环磷酰胺预处理方案行单倍体移植治疗恶性血液病.结果 6例患者完全植入,＋12 d～＋20 d（回输干细胞前为一,回输干细胞后为＋WBC＞1.0×10^9/L,＋20 d～＋51 d PLT＞20×10^9/L,4例出现急性移植物抗宿主病,其中Ⅰ度1例,Ⅱ度2例,Ⅳ度1例,4例出现慢性移植物抗宿主病,无致命性感染发生,2例出现迟发性出血性膀胱炎,目前无恶性血液病存活13～63个月.结论 采用改良白消安-环磷酰胺顶处理方案行单倍体移植治疗恶性血液病是安全有效的方法.     

不同剂量137Csγ-射线辐射后造血干/祖细胞辐射敏感性差异研究

摘要：目的 探讨不同剂量137Csγ-射线照射后不同时间点C57 BL/6小鼠造血细胞放射敏感性的差异。方法 选取6~8周龄雄性C57BL/6小鼠72只,完全随机法分为对照组和照射组。照射组小鼠分别接受2、4、6 Gy137Csγ射线一次性全身照射,对照组小鼠接受假照射。小鼠分别于受照后14 d、35d和56d断颈处死,取外周血进行血象测定,取骨髓细胞测定有核细胞数目和造血干/祖细胞数目。结果 不同剂量照射后小鼠的外周血常规指标有明显变化,白细胞数目明显下降,其次是血小板数目,且具有剂量效应关系;在照射后14d,2Gy、4Gy和6Gy照射组小鼠骨髓有核细胞数目与对照组比较分别下降21.9%、39.9%和54.4%（t=4.311、6.401、8.007,P<0.05）;照射后35d和56d,6Gy照射组小鼠骨髓有核细胞和造血祖细胞数目显著低于对照组（t=4.185、3.596,P<0.05）。照射后14d、35d和56d,2Gy、4Gy和6Gy照射组小鼠骨髓造血干细胞数目持续低于对照组（t=9.706、3.427~7.465,P<0.05）。结论 不同剂量137Csγ-射线照射对小鼠造血系统造成不同程度的损伤,造血祖细胞较造血干细胞辐射敏感,且辐射对造血干细胞造成的损伤是持久性的。     

盐酸贝那普利对造血祖细胞体外扩增的影响

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。     

糖尿病患者血管内皮祖细胞研究进展

血管内皮祖细胞(EPC)来源于骨髓,是具有修复内皮和新生血管功能的干细胞。糖尿病患者外周血EPC数量和功能均出现下降,EPC已成为糖尿病及其并发症治疗的一个新靶点。本文综述EPC在糖尿病病理生理中的作用和药物干预机理的研究进展。     

三氧化二砷对人脐静脉内皮细胞作用的研究

目的以人脐静脉内皮细胞作为血管新生内皮细胞的模拟物,通过研究As2O3对人脐静脉内皮细胞的影响,探讨As2O3的抗血管新生作用机制。方法分离和培养人脐静脉内皮细胞,以不同浓度和时间的As2O3和VEGF作用于脐静脉内皮细胞,应用流式细胞技术测定As2O3和VEGF对脐静脉内皮细胞增殖、细胞凋亡和癌基因bcl-2表达的影响。结果As2O3以时间和剂量依赖的方式抑制内皮细胞增殖,促进细胞凋亡,抑制癌基因bcl-2表达,而VEGF可拮抗As2O3的部分作用。结论As2O3可以通过直接作用于内皮细胞而抑制血管新生。     

兔骨髓源血管内皮祖细胞的体外培养与鉴定

目的 体外分离、培养、扩增血管内皮祖细胞(EPCs),观察EPCs生长分化过程并对其生物学特性进行鉴定.方法 采用密度梯度离心法从兔骨髓中提取单个核细胞,贴壁筛选法分离EPCs,于添加了Singlequotes的EBM-2培养液中扩增培养,对培养10d的细胞进行免疫荧光及免疫组织化学分析.结果 培养4 d,光电显微镜可见细胞集落形成,梭形贴壁细胞从集落中央以放射状向外周生长;培养7～10 d,细胞集落相互连接,呈典型的"鹅卵石"样外观;2周左右可见细胞排列成条索状结构.贴壁细胞呈DIL-ac-LDL及FITC-UEA-1双荧光阳性,阳性率为(65.0±4.0)%;贴壁细胞表达vWF,VEGFR-2和VE-cadherin的阳性率分别为(90.0±2.1)%,(77.0±4.1)%和(78.1±8.2)%.结论 骨髓中富含EPCs,成功建立了一整套骨髓源EPCs的分离、体外扩增培养的方法,且操作简便具有较好的可重复性.     

Phenotype and differentiation of bone marrow-derived smooth muscle progenitor cells

1. Smooth muscle progenitor cells (SPC) are undifferentiated vascular smooth muscle cells implicated in many hyperplastic diseases of the blood vessels. However, few in vitro studies have investigated the characteristics of SPC. 2. In the present study, we constructed a recombinant plasmid with the enhanced green fluorescent protein (GFP) gene and a rat SM22α promoter, which was exclusively promoted in a smooth muscle cell lineage. Constructs were then transferred into adherent mononuclear cells derived from rat bone marrow. After 3 days, GFP-positive cells, which should be SPC, were isolated by flow cytometry. 3. Flow cytometric analysis and dual immunofluorescent staining showed that the GFP-positive cells expressed both α-smooth muscle actin (a specific marker for smooth muscle) and the chemokine receptor CXCR4 (abundant on precursor cells), but not calmodulin or CD31. After stimulation of SPC with 50 ng/mL platelet-derived growth factor-BB, CXCR4 levels decreased and calmodulin protein content increased, as determined by western blot analysis. 4. On the basis of these results, we conclude that SPC have dual characteristics of both precursor and smooth muscle cells, and might well differentiate into smooth muscle-like cells under certain conditions.     

The acute exposure effects of inhaled nickel nanoparticles on murine endothelial progenitor cells

Abstract

Introduction: The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures, such as increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species.

Methods: Following an acute whole body inhalation exposure to 500?µg/m³ of nickel nanoparticles for 5?h, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs) and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure.

Results and conclusions: Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.     

Effects of encapsulated rabbit mesenchymal stem cells on ex vivo expansion of human umbilical cord blood hematopoietic stem/progenitor cells

The expansion of umbilical cord blood mononuclear cells (UCB MNCs) was investigated in a novel co-culture system by means of encapsulation of rabbit bone marrow (BM) mesenchymal stem cells (MSCs) in alginate beads (Alg beads). Three kinds of media were applied and the experiments lasted for 7 days. The total nucleated cell density was measured every 24 h. Flow cytometric assay for CD34⁺ cells and methylcellulose colony assays were carried out at 0, 72 and 168 h. It was found that the encapsulated MSCs illustrated remarkable effects on UCB MNCs expansion regardless of whether serum is present in culture media or not. At the end of 168 h co-culture, the total nucleated cell number was multiplied by 15 ± 2.9 times, and CD34⁺ cells 5.3 ± 0.3 times and colony-forming units in culture (CFU-Cs) 5.6 ± 1.2 times in the serum-free media supplemented with conventional dose of cytokines, which was very similar to the results in the containing 20% serum media. While in the control, i.e. MNC expansion without encapsulated MSCs, however, total nucleated cells density changed mildly, CD34⁺ cells and CFU-Cs showed little effective expansion. It is demonstrated that the encapsulated stromal cells can support the expansion of UCB MNCs effectively under the experimental condition.