黄芪体外作用对贫血小鼠骨髓基质细胞分泌SCF的影响

目的 研究黄芪注射液（AMI）对贫血小鼠骨髓基质细胞分泌干细胞因子（SCF）的影响。方法 在贫血小鼠骨髓基质细胞培养体系中，分别加入0.1mL不同浓度的AMI（终浓度分别为40mg/L,400mg/L)作为药物1组，药物2组，对照组加入等量的IMDM培养液，检测各实验组培养体系中骨髓成纤维细胞集落（CFU-F）数，同时，分别收集培养上清液，透析浓缩成冻干粉，经IMDM液稀释后作为条件培养液，分别加入到高增殖潜能的集落形成细胞（HPP-CFC）培养体系中，检测各实验组培养上清液中所含SCF的水平。结果 与对照组相比较，药物组的CFU-F数及骨髓基质细胞条件培养液（BMSCM）中SCF的水平均明显升高。结论 一定浓度的AMI可促进贫血小鼠骨髓CFU-F增殖。刺激其骨髓基质细胞分泌SCF。     

稳定表达狂犬病病毒CTN-1V株糖蛋白细胞系的建立

目的构建能稳定表达狂犬病病毒糖蛋白的CHO细胞系。方法用RT—PCR法扩增和分离CTN-1V株狂犬病毒糖蛋白基因,测序后克隆入pCDNA5．0FRT载体,构建重组质粒pCDNA5．0FRT—G,之后与POG44质粒共转染CHO细胞,采用潮霉素B抗性筛选,挑选阳性克隆,间接免疫荧光和WesternBlot进行稳定表达细胞系的鉴定。结果经酶切和测序鉴定,获得重组表达质粒pCDNA5．0FRT—G,共转染CHO细胞后,获得肉眼可见阳性细胞克隆,刮取阳性克隆进行传代扩大培养并定义为第2代,经过10代后免疫荧光和WesternBlot结果依然阳性。结论成功建立稳定表达狂犬病病毒糖蛋白的CHO细胞系,为深入研究糖蛋白的功能奠定基础。     

siRNA沉默AFP基因对肝癌细胞系 EGHC-9901 增殖的影响

 目的 建立稳定表达AFP-siRNA质粒的肝癌细胞系并探讨对其增殖的影响。方法 构建AFP-siRNA,用脂质体法转染肝癌细胞系,G418筛选4～5周,Western blot 及RT-PCR检测靶基因表达,分组：实验组,转染AFP-siRNA组;阳性对照组,转染空载体组;空白对照组,未处理组。MTT绘制生长曲线,平板克隆实验观察集落形成,流式细胞仪分析细胞周期。结果 成功建立稳定表达AFP-siRNA的肝癌细胞系,实验组表达AFP近乎完全抑制;实验组增殖能力显著低于空载体组;实验组＞75μm集落形成数19±2,低于空载体组62±6;实验组G1期细胞数较空载体组提高20％左右。 结论 成功建立稳定表达AFP-siRNA的肝癌细胞系,抑制AFP表达可使肝癌细胞发生G1期阻滞,明显抑制其增殖及集落形成能力。     

FOXO1质粒稳定转染人骨肉瘤细胞的筛选及意义

目的 建立稳定表达FOXO1-GFP的人骨肉瘤U2OS细胞系,并验证U2OS-FOXO1-GFP细胞模型对氧化应激损伤的指示作用.方法 将pcDNA3-GFP-FOXO1质粒转染到U2OS细胞中,利用G418和FCM筛选稳定细胞系.40mmol/LAAPH处理U2OS-FOXO1-GFP细胞4h,CCK-8检测细胞活力,荧光显微镜观察FOXO1-GFP的分布情况.结果 成功获得U2OS-FOXO1-GFP稳转细胞系.经G418和FCM筛选后,FOXO1-GFP阳性细胞占细胞总数的92％.CCK-8结果显示,AAPH处理后,U2OS-FOXO1-GFP细胞存活率下降至0.55 ±0.04明显低于对照组(P＜0.05).荧光显微镜观察发现,正常情况下,FOXO1-GFP位于细胞质;AAPH处理后,FOXO1-GFP易位到细胞核.结论 U2OS-FOXO1-GFP细胞系能很好的指示细胞氧化应激损伤,为筛选抗氧化应激药物提供了一种有利工具.     

OSKM诱导过表达c-Jun肝癌细胞重编程

目的构建OSKM诱导的过表达c-Jun肝癌细胞(C3A-c-Jun)重编程细胞系,探讨外源性c-Jun对肝癌细胞重编程的影响。方法通过C3A细胞稳定过表达c-Jun后进行OSKM诱导重编程,通过碱性磷酸酶染色,Real-time PCR,免疫印迹法,免疫荧光等技术对细胞进行鉴定,建立C3A-c-Jun诱导肿瘤干细胞系C3A-c-Jun-i CSCs。结果 C3A-c-Jun-i CSCs克隆呈圆顶状,边缘圆钝,克隆内细胞小且排列紧密,多层分布,碱性磷酸酶染色阳性,mRNA和蛋白水平均可表达多潜能性标记物OCT4、SOX2;C3A-c-Jun-i CSCs组外源性和内源性Sox2的表达量均明显高于C3A-i CSCs对照组;在重编程过程中c-Jun持续表达。结论 OSKM诱导C3A、C3A-c-Jun肝癌细胞重编程,分别建立C3A-i CSCs和C3A-c-Jun-i CSCs细胞系,发现外源性c-Jun通过上调Sox2基因的表达,启动下游一系列反应,对肝癌细胞重编程过程起促进作用。     

Gab2过表达对人结直肠癌SW480细胞株增殖和迁移的影响

目的:探讨Gab2过表达对人结直肠癌细胞株SW480增殖和迁移能力的影响。方法:用携带Gab2基因的重组慢病毒颗粒(LV-Gab2-GFP)感染人结直肠癌SW480细胞株,作为实验组(LV-Gab2-GFP组);以对照慢病毒颗粒(LV-GFP)感染人结直肠癌SW480细胞株,作为阴性对照组(LV-GFP组);空白对照组常规培养,不作任何处理。用RT-PCR和Western blot法分别检测细胞中Gab2 mRNA和蛋白表达的水平;CCK-8法和平板克隆实验检测细胞增殖能力的变化;划痕愈合实验检测细胞迁移能力的变化。结果:RT-PCR和Western blot均显示LV-Gab2-GFP组Gab2的表达均较对照组显著增高;与对照组比较,Gab2过表达显著增强人结直肠癌SW480细胞的增殖和迁移能力。结论:Gab2过表达可以促进SW480细胞增殖和迁移能力。     

Upregulation of microRNA-383 influences the PRDX3 expression on human medulloblastoma cell line D341

目的 探讨microRNA-383(miR-383)对人髓母细胞瘤D341细胞系中PRDX3基因的表达及细胞增殖活性与凋亡等细胞功能学的影响.方法 应用实时荧光定量PCR法检测人髓母细胞瘤D341细胞系、正常脑组织中miR-383及PRDX3 mRNA表达水平;采用Western blot法检测人髓母细胞瘤D341细胞系、正常脑组织中PRDX3基因、蛋白表达水平;人工合成miR-383模拟体(mimics)转染MBD341细胞系,采用cck-8法、流式细胞术等实验方法检测转染人髓母细胞瘤D341细胞系后各组D341细胞增殖与凋亡、细胞内活性氧水平、线粒体膜电位水平的变化.结果 人髓母细胞瘤BD341细胞系中miR-383表达水平为正常脑组织的0.524倍,明显下调.而PRDX3基因mRNA表达水平为正常脑组织的5.214倍,蛋白表达水平较正常脑组织明显上调.miR-383 mimics转染D341细胞系24、48 h,实验组miR-383表达水平均明显高于空白对照组,且48 h作用更加明显;而PRDX3基因mRNA、蛋白表达水平48 h较空白对照组降低.cck-8法检测提示实验组中细胞增殖率降低(P<0.05).AnnexinV-FITC法检测转染后24、48 h细胞凋亡结果显示miR-383 mimics实验组细胞早期凋亡率明显增高,且24 h作用较明显(P=0.000).细胞内活性氧水平和线粒体膜电位水平检测结果分别显示,转染后24、48 h实验组较对照组细胞内活性氧水平明显升高,而线粒体膜电位水平较对照组明显降低.结论 与正常脑组织相比,miR-383在人髓母细胞瘤D341细胞系中表达降低,PRDX3基因表达升高.上调miR-383可敲低D341细胞系中PRDX3基因表达,并可抑制D341细胞增殖活性,促进D341细胞凋亡,出现D341细胞内活性氧水平升高、线粒体膜电位水平降低的细胞功能学变化.     

稳定表达表皮生长因子受体293细胞系的建立及其生物学特性研究

目的 建立稳定表达野生型表皮生长因子受体(EGFR)全长的293细胞系.方法 通过脂质体瞬间转染的方法将含有EGFR全长的质粒转染293细胞系.利用抗生素筛选的方法使转染阳性的细胞扩增,单克隆化,进一步稳定传代.通过Western印迹的方法挑选高表达克隆,进而使用MTT法测细胞增殖活性.反复传代、冻存、复苏鉴定表达的稳定性.结果 获得1株稳定表达EGFR的293细胞系,命名为WT293,且转染前后细胞生长速度存在差异.结论 pcDNA3.1(一)载体能够作为稳定表达的载体用于筛选.稳定表达EGFR的293细胞系为配体受体相互作用及靶向EGFR的抗肿瘤药物作用机制的研究提供了实验素材和理论依据.     

上调microRNA-383对人髓母细胞瘤D341细胞系中PRDX3表达的影响

目的探讨microRNA-383(miR-383)对人髓母细胞瘤D341细胞系中PRDX3基因的表达及细胞增殖活性与凋亡等细胞功能学的影响。方法应用实时荧光定量PCR法检测人髓母细胞瘤D341细胞系、正常脑组织中miR-383及PRDX3 mRNA表达水平;采用Western blot法检测人髓母细胞瘤D341细胞系、正常脑组织中PRDX3基因、蛋白表达水平;人工合成miR-383模拟体(mimics)转染MBD341细胞系,采用cck-8法、流式细胞术等实验方法检测转染人髓母细胞瘤D341细胞系后各组D341细胞增殖与凋亡、细胞内活性氧水平、线粒体膜电位水平的变化。结果人髓母细胞瘤BD341细胞系中miR-383表达水平为正常脑组织的0.524倍,明显下调。而PRDX3基因mRNA表达水平为正常脑组织的5.214倍,蛋白表达水平较正常脑组织明显上调。miR-383 mimics转染D341细胞系24、48 h,实验组miR-383表达水平均明显高于空白对照组,且48 h作用更加明显;而PRDX3基因mRNA、蛋白表达水平48 h较空白对照组降低。cck-8法检测提示实验组中细胞增殖率降低(P<0.05)。An-nexinV-FITC法检测转染后24、48 h细胞凋亡结果显示miR-383 mimics实验组细胞早期凋亡率明显增高,且24 h作用较明显(P=0.000)。细胞内活性氧水平和线粒体膜电位水平检测结果分别显示,转染后24、48 h实验组较对照组细胞内活性氧水平明显升高,而线粒体膜电位水平较对照组明显降低。结论与正常脑组织相比,miR-383在人髓母细胞瘤D341细胞系中表达降低,PRDX3基因表达升高。上调miR-383可敲低D341细胞系中PRDX3基因表达,并可抑制D341细胞增殖活性,促进D341细胞凋亡,出现D341细胞内活性氧水平升高、线粒体膜电位水平降低的细胞功能学变化。     

Galecin-3在膜联蛋白A7表达抑制的HeLa细胞中的表达变化

目的探讨膜联蛋白A7(ANXA7)表达抑制的HeLa细胞galectin-3(gal-3)的表达改变。方法将细胞分为实验组、阴性对照组和空白对照组。采用RNA干扰技术将靶向ANXA7的siRNA和阴性对照siRNA脂质体2000转染法分别转染宫颈癌细胞系HeLa细胞,空白对照组不予任何处理。转染48h后采用Western blotting法和RT-PCR法分别在蛋白水平和mRNA水平进行抑制效果的鉴定。Western blotting和实时定量PCR法分别检测ANXA7表达抑制的HeLa细胞中galectin-3的表达改变。结果靶向ANXA7的siRNA可显著抑制ANXA7的表达;当ANXA7表达受抑后,galectin-3在HeLa细胞中的表达显著下降,与阴性对照组和空白对照组相比,差异有显著性(P0.05)。结论 ANXA7表达抑制的宫颈癌细胞系HeLa细胞中galectin-3的表达显著下调,这可能是ANXA7表达受抑的细胞行为学发生改变的机制之一。     

Bone morphogenetic protein 4 contributes to the maintenance of primitive cord blood hematopoietic progenitors in an ex vivo stroma-noncontact co-culture system

Establishment of conditions supporting hematopoietic stem cell (HSC) maintenance and expansion ex vivo is critical for wider clinical application of cord blood (CB) transplantation. AFT024 is a murine fetal liver cell line that expands primitive hematopoietic cells via a process that is not understood. Here we show that bone morphogenic protein 4 (BMP4) is produced by AFT024 and contributes significantly to the maintenance of co-cultured CB-derived primitive cells. Significant amounts of BMP4 mRNA are produced by the supportive AFT024 stromal cell line, and secreted BMP4 protein accumulates in AFT024 conditioned medium. Blockade of BMP4 activity in this coculture model using neutralizing BMP4 monoclonal antibody reduced expansion of primitive CB cells on the basis of phenotypic (CD34(+)CD38(-)) and functional criteria [long-term culture initiating cells (LTC-IC)] and significantly reduced the capacity of the cultured CB stem cells to support repopulation in the nonobese diabetic-severe combined immunodeficiency (NOD-SCID) xenograft model. Therefore, BMP4 is a key growth factor for maintenance of HSC and contributes to the unique properties of the AFT024 stromal noncontact culture.     

Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction

Cell-cell contact between stem cells and cellular determinants of the microenvironment plays an essential role in controlling cell division. Using human hematopoietic progenitor cells (CD34+/CD38-) and a stroma cell line (AFT024) as a model, we have studied the initial behavioral and molecular sequel of this interaction. Time-lapse microscopy showed that CD34+/CD38- cells actively migrated toward and sought contact with stroma cells and 30% of them adhered firmly to AFT024 stroma through the uropod. CD44 and CD34 are colocalized at the site of contact. Gene expression profiles of CD34+/CD38- cells upon cultivation with or without stroma for 16, 20, 48, or 72 hours were analyzed using our human genome cDNA microarray. Chk1, egr1, and cxcl2 were among the first genes upregulated within 16 hours. Genes with the highest upregulation throughout the time course included tubulin genes, ezrin, c1qr1, fos, pcna, mcm6, ung, and dnmt1, genes that play an essential role in reorganization of the cytoskeleton system, stabilization of DNA, and methylation patterns. Our results demonstrate directed migration of CD34+/CD38- cells toward AFT024 and adhesion through the uropod and that upon interaction with supportive stroma, reorganization of the cytoskeleton system, regulation of cell division, and maintenance of genetic stability represent the most essential steps.     

膀胱肿瘤细胞培养上清对树突状细胞的形成及功能的影响

目的: 探讨人膀胱肿瘤细胞BIU -87培养上清对树突状细胞(DC)的生成和功能的影响。方法: 分离培养外周血单个核细胞(PBMC)来源的DC, 实验组加入BIU -87细胞的培养上清液, 以正常培养的DC作为对照组, 应用流式细胞术检测在实验组和对照组不同条件下培养的DC上表面标志的表达, 并用改良的MTT比色法检测和对比两组所培养的DC激活同种异体T细胞的能力及其差别。结果: DC与BIU- 87细胞的上清液共培养后, CD1a、CD83及CD86均呈低表达。实验组与对照组相比较, DC的成熟受到抑制, 对同种异体T细胞激活的作用明显降低 (P<0. 05 )。结论: BIU- 87细胞的上清液可抑制DC的生成及相关表面标志的表达, 这可能是导致膀胱肿瘤患者DC数量减少和功能降低的原因。     

TH1/TH2平衡的体外诱导转换及其对嗜酸性粒细胞功能的影响

目的：探索TH1／TH2平衡的体外诱导作用及其对嗜酸性粒细胞（Eos)功能的影响。方法：取人肝素抗凝外周全血，加入结核菌素纯蛋白衍生物（purified proteinderivative,PPD)10μg/ml，培养3，5，7d，用ELISA方法检测上清IL－4和γ－IFN的含量，取3d上清洁培养液加入到肝素抗凝全血中，体外培养72h,IL-5作为阳性对照，用流式细胞术分析CD16阴性细胞群体中CD69的表达和碘化丙啶（PI）的染色情况。结果：应用PPD体外诱导全血产生较高水平的γ－IFN，IL－4含量未测出；培养体系中加人PPD诱导上清液明显抑制了Eos和IL－5活化的Eos CD69的表达并诱导其凋亡，结论：PPD体外成功诱导TH1／TH2平衡的转换。PPD诱导上清液具有活化Eos和诱导其凋亡的作用。     

Human Mast Cell Apoptosis Is Regulated Through Bcl-2 and Bcl-XL

It is well established that human mast cell proliferation and maturation are regulated by kit ligand (stem cell factor). Little is known, however, about how these two processes are negatively regulated and thus, how mast cell number is controlled in normal and pathologic conditions. We therefore first hypothesized that SCF-dependent human mast cells would undergo programmed cell death (apoptosis) on removal of SCF as has been shown for growth factor-dependent rodent mast cells. We then examined whether SCF acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. As hypothesized, elimination of SCF from primary peripheral blood-derived human mast cell cultures resulted in a significant apoptotic process. During apoptosis, down-regulation of the two apoptosis-regulatory proteins Bcl-2 and Bcl-X_Lwas observed. Moreover, a deregulated expression of these two proteins was found in two human mast cell lines which are SCF-independent. Thus, SCF functions as a survival factor by repressing apoptosis of human mast cells through Bcl-2 and Bcl-X_L. Deregulated expression of these antiapoptotic proteins may contribute to proliferation and accumulation of mast cells in certain forms of systemic mast cell disorders.     

Culture of colony-forming hematopoietic progenitor cells from human peripheral blood

Summary Methods for culturing hematopoietic colonies from human peripheral blood are described in this report. Granulocyte-macrophage progenitor cells (CFU-GM: colony forming units-granulocyte/macrophage) are grown in 35-mm dishes containing 4 ml of 0.35% agarose in Iscove's modified Dulbecco's medium (IMDM) with 20% prescreened fetal bovine serum (FBS). Colony-stimulating activity is provided by leukocyte-conditioned medium, and nonactivated autologous T lymphocytes or recombinant granulocyte- and granulocyte/macrophage-colony stimulating factor or both. This procedure minimize the growth inhibition caused by monocytes. Erythroid progenitor cells (BFUe: burst forming units-erythroid) are cultured in 1 ml of 0.35% agarose in IMDM with 30% FBS and conditioned medium from human bladder carcinoma cell line 5637 or recombinant interleukin 3. Both assays have proved useful in studying the enrichment of peripheral blood hematopoietic cells.     

乙型肝炎患者骨髓造血干细胞来源的树突状细胞表型分析

目的 检测乙型肝炎患者骨髓造血干细胞（HSC）诱导的树突状细胞（DC）的表面分子的表达,评价其相关因素及临床意义.方法 收集慢性乙型肝炎患者的骨髓液9例,健康者7例后用磁珠分离仪分离纯化骨髓液CD34＋细胞,在含有干细胞生长因子（SCF）、酪氨酸激酶受体家族Ⅲ的配体（FLT3）、促血小板生成素（TPO）、IL-3和10%FBS的IMDM培养基中孵育并进行扩增,在干细胞扩增基础上,在GM-CSF和IL-4作用下诱生DC,通过流式细胞仪分析其表面分子的表达并对DC进行形态学观察.结果 乙型肝炎患者HSC经诱导为DC后,其免疫表型CD80、CD86、CD1a的表达均低于正常对照组,差异有统计学意义：CD1a（t=3.94,P＜0.05）,CD80（t=7.08,P＜0.01）,CD86（t=3.65,P＜0.05）,而HLA-DR表达与正常组比较差异无统计学意义（t=0.34,P＞0.05）.结论 慢性乙型肝炎患者HSC来源的部分DC的表面分子表达低下可能与HBV感染HSC后出现病态的免疫细胞分化有关.