类风湿关节炎患者造血干/祖细胞核因子κB1、κB2基因表达的研究

目的检测类风湿关节炎(rheumatoid arthritis,RA)患者外周血CD34+造血干细胞/祖细胞(hematopoietic stem/progenitor cell,HSC/HPC)的核因子(nuclear factorκB,NFκB)κB1、κB2信号分子的基因表达水平,了解NFκB信号途径分子基因表达的异常与临床指标的关联性。方法收集24例RA患者,9例正常对照组,分别抽取50 ml抗凝外周血,淋巴细胞分离液分离单个核细胞,免疫磁珠分选CD34+HSC/HPC,Trizol法提取总RNA,逆转录为cDNA,实时荧光定量聚合酶反应方法检测患者组和对照组NFκB1、NFκB2 mRNA表达水平的差异,并分析RA患者组甲氨蝶呤(Methotrexate,MTX)治疗组和未治疗组NFκB1、NFκB2mRNA表达水平变化,结合其它临床资料进行相关性分析。结果 RA患者组NFκB1 mRNA表达相对量高于正常对照组[6.57(1.08~76.71),2.14(0.68~7.09),P=0.013];NFκB2 mRNA表达与正常对照组无统计学差异。RA患者外周血CD34+HSC/HPCNFκB1、NFκB2 mRNA表达与有无MTX的治疗无统计学差异,与疾病活动关节评分28(disease activity score 28,DAS28评分)、血沉、C反应蛋白等临床指标无相关性。结论 RA患者外周血CD34+HSC/HPC NFκB1基因表达水平比正常对照增高,可能是RA患者CD34+细胞重要缺陷。     

CD133两种亚型分子在急性白血病中的表达及意义

目的探讨CD133两种亚型分子在急性白血病的表达及意义。方法通过直接荧光标记流式细胞术检测42例急性白血病患者骨髓白血病细胞上CD133-1、CD133-2抗原的表达。结果 CD133-1、CD133-2在对照组均无阳性表达,在急性白血病的阳性表达率为33.3%、52.4%(P0.05);急性髓系白血病组和急性淋巴细胞白血病组中CD133-1的阳性率(37.9%vs23.1%)、CD133-2的阳性率(48.3%vs 61.5%)比较差异均无统计学意义(P0.05);在急性白血病中CD133-2的阳性率高于CD133-1的阳性率(52.4%vs 33.3%,P0.05);化疗1个疗程后,CD133阳性组与CD133阴性组的CR率比较无统计学意义(P0.05)。结论急性白血病患者CD133-1、CD133-2表达均高于对照,并且CD133-2的阳性率高于CD133-1。     

人胎盘CD133~+细胞具有高增殖潜能集落形成细胞特性

目的 通过对人胎盘CD133+细胞群中高增殖潜能集落形成细胞(HPP-CFC)检测与生物学特性的分析,证明人胎盘存在早期造血干/祖细胞(HSPC). 方法 采用机械法制备人胎盘组织(PT)单细胞悬液,用Histopaque-1007分离出单个核细胞(MNC),经磁式分选(MACS)富集CD133+细胞,培养28 d后观察HPP-CFC集落形成能力,用流式细胞仪(FCM)对分选的细胞组份和HPP-CFC进行表型分析,实验全程用脐带血(UCB)作平行比较分析. 结果 培养28 d后,PT-CD133+与UCB-CD133+细胞组份分别扩增了266和362倍,前者低于后者(P<0.01);PT-CD133+与UCB-CD133+细胞中HPP-CFC分别为(32.4±11.2)/5×103、(17.7±5.7)/5×103,前者形成的HPP-CFC数量明显高于后者(P<0.01);PT.CD133+、UCB-CD133+细胞培养至28 d时,除UCB-CD133+组的CD133+CD34-亚群比例无明显改变外,CD133+CD34+、CD133-CD34+和CD133+CD34-(PT-CD133+组)亚型均比培养前减少. 结论 人胎盘组织CD133+细胞中存在HPP-CFC,说明胎盘CD133+细胞群中存在早期HSPC.     

CD133和GW112在胃癌中的表达及其与预后的关系

目的 研究胃癌组织CD133 mRNA和GW112 mRNA的表达水平及其与患者临床病理参数、无瘤生存率和生存率的关系.方法 采用实时定量PCR技术检测25例手术切除的胃癌及相应癌外正常胃黏膜组织中CD133mRNA和GW112mRNA的表达,随访患者进行生存分析.结果 (1)胃癌组织CD133 mRNA和GW112 mRNA表达量[0.07(0.01～0.13),0.12(0.04～0.36)]均显著高于正常胃黏膜组织[0.01(0.00～0.07),0.02(0.01～0.05)](P＜0.05).(2)GW112 mRNA表达量随患者年龄增高(＞60岁),合并淋巴结转移,TNM分期Ⅲ期或Ⅳ期而升高,差异均有统计学意义(P＜0.05).CD133 mRNA则和临床病理参数无显著关系.(3)随访所有病例,5年生存率为(28.0+9.0)%,无瘤生存率为(24.6±9.5)%.分别按照癌组织CD133和GW112表达水平分为高、低表达组.CD133高、低表达组5年生存率(7.7%比50.0%)和无瘤生存率(0%比50.5%)差异均有统计学意义(P＜0.05);GW112高、低表达组5年生存率(11.1%比71.4%)和无瘤生存率(20%比29%)差异也均有统计学意义(P＜0.05).Cox多因素分析表明癌组织CD133和GW112的表达水平是患者无瘤生存期的独立影响因子(相对危险度分别为3.792和5.155);GW112同时也是患者生存期的独立影响因子(RR=4.978).(4)ROC曲线分析发现,联合检测CD133和CEA对患者预后有一定的预测价值.结论 胃癌组织CD133 mRNA和GW112 mRNA 表达水平与胃癌恶性潜能有关,可预测胃癌患者的预后.     

白细胞介素8参与CD133^+肝癌细胞的侵袭与转移北大核心

背景:白细胞介素8是一种重要的炎性趋化因子,其在调节肿瘤细胞增殖、血管新生方面发挥了重要作用。目的:探讨白细胞介素8对CD133^+肝癌细胞侵袭转移能力的影响。方法:分离培养人肝癌MHCC97-H细胞株,免疫磁珠分选出CD133^+/CD133-MHCC97-H细胞,流式细胞仪检测CD133表达,ELISA检测上清液白细胞介素8水平。克隆形成实验、裸鼠成瘤实验、Transwell小室分别检测CD133^+/CD133-MHCC97-H细胞克隆形成率、小鼠成瘤能力、细胞迁移侵袭能力。另外,采用白细胞介素8中和抗体处理细胞,比较CD133^+/CD133-MHCC97-H细胞CD133表达、上清液中白细胞介素8水平及细胞克隆形成能力和迁移、侵袭能力。结果与结论:(1)CD133^+MHCC97-H细胞的CD133表达率、上清液白细胞介素8水平及克隆形成率均显著大于CD133-MHCC97-H细胞(P<0.05);(2)细胞浓度为1×10~6 L^(-1)和1×10~7 L^(-1)的CD133^+MHCC97-H细胞接种后有部分小鼠出现皮下移植瘤,但CD133-MHCC97-H细胞在同样细胞浓度下,均未出现皮下移植瘤;(3)CD133^+MHCC97-H细胞的透膜细胞数量均显著大于CD133-MHCC97-H细胞(P<0.05);(4)经白细胞介素8中和抗体处理之后,CD133^+/CD133-MHCC97-H细胞的CD133表达率、上清液中白细胞介素8水平、克隆形成率均显著下降(P<0.05),且CD133^+MHCC97-H细胞下降程度更大(P<0.01);CD133^+MHCC97-H细胞的迁移、侵袭透膜细胞数显著减少(P<0.05),CD133-MHCC97-H细胞则未出现明显的改变(P>0.05);(5)结果表明,白细胞介素8可以特异性参与调节CD133^+MHCC97-H细胞侵袭、转移能力。     

干细胞表面标志CD133在肝癌中的表达及其阳性亚群增殖特性北大核心

背景:以CD133为标志物,利用其蛋白特异性特征可对不同的肿瘤干细胞进行分选和研究。目的:探讨干细胞表面标志CD133在肝癌中的表达及其阳性亚群的体外增殖特性。方法:对人肝癌MHCC97-H细胞株进行培养和分选,分别获得CD133^+与CD133^-MHCC97-H细胞,检测CD133表达及细胞迁移侵袭能力;培养14 d,检测细胞克隆形成率;培养7 d,检测细胞增殖及Notch基因蛋白表达。将CD133^+与CD133^-MHCC97-H细胞分别接种于裸鼠背部皮下,4周后,检测成瘤情况。结果与结论:(1)CD133表达与细胞克隆形成率:CD133^+MHCC97-H细胞CD133表达率、克隆形成率均显著大于CD133^-MHCC97-H细胞(P<0.05);(2)细胞增殖:CD133^+MHCC97^-H细胞培养3-7 d的吸光度值大于CD133^-MHCC97-H细胞(P<0.05);(3)细胞迁移侵袭:CD133^+MHCC97-H细胞迁移与侵袭实验的透膜细胞数均显著多于CD133^-MHCC97-H细胞(P<0.05);(4)Notch基因蛋白:CD133^+MHCC97-H细胞Notch基因蛋白表达多于CD133^-MHCC97-H细胞(P<0.05);(5)成瘤实验:CD133^-MHCC97-H细胞组成瘤体积大于CD133^-MHCC97-H细胞组(P<0.05);(6)结果表明:CD133阳性肝癌细胞亚群具有较强的体外增殖特性,具有一定的肿瘤干细胞特性,并有较强的侵袭、转移及致瘤能力。     

人胎盘组织细胞表型特征及其意义

目的:研究足月分娩胎盘组织细胞的表型特征并探讨其意义。方法:采用流式细胞术检测人胎盘组织单个核细胞(MNC)中CD133 细胞及其亚群含量;免疫组织化学法分析胎盘组织CD133、CD34、KDR、vWF和CD144的表达分布。结果:流式细胞仪检测结果表明,在胎盘MNC中,CD133 细胞所占百分率为(1.7±1.1)%,其中CD133 CD34 细胞亚群的比例为(0.5±0.5)%;胎盘CD133 细胞中表达CD34的细胞占(24.1±11.2)%。免疫组织化学结果显示,绒毛间质存在CD133 和CD34 细胞;胎盘绒毛合体滋养层和细胞滋养层细胞也表达CD133;而KDR、vWF和CD144的表达限于胎盘绒毛毛细血管内皮细胞,其表达CD34,但不表达CD133。结论:人足月胎盘组织间质中存在CD34 和CD133 细胞,晚期胎盘绒毛血管无内皮祖细胞存在。     

CD133和CD44在结直肠癌细胞中的表达及其与患者5年生存率的相关性分析

目的: 探讨不同CD133/CD44细胞亚群的成瘤能力及CD133、CD44的表达水平对根治性结直肠癌患者术后生存率的影响,明确CD133和CD44作为结直肠癌肿瘤干细胞表面标志物的意义。方法: 利用流式细胞术分选出SW480细胞系中CD133和CD44标记的不同细胞亚群,比较其在裸鼠皮下成瘤情况;并利用免疫组化的方法观察CD133和CD44在90例结直肠癌患者石蜡切片标本的表达情况,对CD133、CD44与患者临床病理资料及生存率进行分析。结果: CD133⁺CD44⁺细胞群成瘤能力明显优于其它各组。CD133和CD44均在细胞膜上表达,两者均与患者性别、年龄 、肿瘤位置、肿瘤大小、浸润深度、淋巴结转移、肝转移、肿瘤分化程度及UICC分期无关(P＞0.05)。Kaplan-Meier生存分析法显示,CD133高表达组5年生存率为45.2%,CD133低表达组5年生存率为83.8%,两者有明显差异(P＜0.01);而CD44高表达组5年生存率(75.6%)与低表达组(70.1%)无明显差异(P＞0.05);其中CD133/CD44同时高表达者5年生存率明显较差(P＜0.01)。Cox风险回归模型分析结果表明,CD133、肝转移、分化程度及淋巴结转移是影响结直肠癌患者预后的几个独立危险因素(P＜0.05)。结论: CD133可作为结直肠癌肿瘤干细胞的良好标志物。CD133是影响结直肠癌患者预后的独立危险因素,其表达水平越高,预后越差;尽管CD44与结直肠癌患者预后无明显相关性,但联合检测CD133/CD44却能更好地判断患者的预后情况。     

B细胞非霍奇金淋巴瘤中CD40和NF-κB表达及意义

目的 探讨B细胞非霍奇金淋巴瘤(non-hodgkinlymphoma,NHL)中CD40、NF-κB的表达及意义.方法 采用免疫组化SP法检测69例B细胞NHL及22例淋巴结反应性增生组织中CD40、NF-κB的表达.B细胞NHL患者应用CHOP(环磷酰胺、阿霉素、长春新碱、地塞米松)方案治疗6～8个周期.结果 CD40和NF-κB在B细胞NHL组织中阳性率分别为72.46%(50/69)(P<0.05)和59.42%(41/69)(P<0.001).CD40在Ⅰ+Ⅱ和Ⅲ+Ⅳ期B细胞NHL中的阳性率分别为87.88%(30/33)和58.33%(20/36)(P<0.05);NF-κB在Ⅰ+Ⅱ和Ⅲ+Ⅳ期B细胞NHL中的阳性率分别为36.36%(12/33)和80.56%(29/36)(P<0.05).B细胞NHL组织中CD40与NF-κB的表达呈负相关(r=-0.443,P<0.01).结论 CD40阳性提示B细胞NHL侵袭性低,NF-κB阳性则提示B细胞NHL侵袭性高;CD40是一种提示预后良好的因子,而CD40-NF-κB信号途径对B细胞NHL预后提示的作用尚需进一步研究.     

人胚胎脑室脉络丛上皮细胞表达神经干细胞分子标志

目的 探讨人12周龄胚胎脑脉络丛上皮细胞是否具有神经干细胞的生物学特性.方法制备室管膜/室管膜下区和纹状体脑切片和脉络丛组织铺片,采用免疫荧光染色,在激光扫描共焦显微镜下观察结果,采集图像数据.结果人12周龄胚胎脑室脉络丛上皮细胞表达神经干细胞分子标志CD133、Nestin和Sox2.神经干细胞的分子标志在脉络丛上皮...     

Regulation of alphabeta/gammadelta T cell lineage commitment and peripheral T cell responses by Notch/RBP-J signaling

RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells.     

Canonical notch signaling functions as a commitment switch in the epidermal lineage

Mammalian epidermis consists of a basal layer of proliferative progenitors that gives rise to multiple differentiating layers to provide a waterproof envelope covering the skin surface. To accomplish this, progenitor cells must detach from the basal layer, move upward, and execute a terminal differentiation program consisting of three distinct stages: spinous, granular layer, and stratum corneum. Notch signaling has been implicated in late stages of differentiation, but the commitment switch remains unknown. Here we show with loss and gain-of-function studies that active Notch intracellular domain (NICD) and its obligate canonical signaling partner RBP-J act at the basal/suprabasal juncture to induce spinous and down-regulate basal fate. Spinous layers are absent in RBP-J conditional null epidermis and expanded when Notch1 signaling is elevated transgenically in epidermis. We show that RBP-J is essential for mediating both spinous gene activation and basal gene repression. In contrast, the NICD/RBP-J target gene Hes1 is expressed in spinous layers and mediates spinous gene induction but not basal gene repression. These data uncover an early role for RBP-J and Notch in commitment of epidermal cells to terminally differentiate and reveal that spinous gene induction is mediated by a Hes1-dependent mechanism, while basal gene repression occurs independently of Hes1.     

Notch1/Hes1信号调控C/EBPα表达对AECII细胞增殖与分化的影响

目的:探讨Notch1/Hes1信号通路能否通过调控CCAAT/增强子结合蛋白α(C/EBPα)的表达从而影响肺泡Ⅱ型上皮细胞(AECⅡ)的增殖与分化功能。方法:体外培养人AECⅡ,将细胞随机分为对照组、激活剂组(加入Notch通路激活剂Jagged1蛋白500μg/L)和抑制剂组(加入Notch通路抑制剂DAPT 10μmol/L),于干预后24 h收获各组细胞。采用RT-qPCR和Western blot法分别检测Notch1、Hes1及C/EBPα的mRNA与蛋白表达水平;CCK-8法检测细胞活力;细胞计数检测细胞增殖;流式细胞术检测细胞周期及分化。结果:与对照组相比,激活剂组Notch1、Hes1和C/EBPα的mRNA和蛋白表达显著增加(P0.05),促进AECⅡ从S期进入G_2/M期,增殖增加而分化减少(P0.05);抑制剂组Notch1、Hes1和C/EBPαmRNA和蛋白表达水平明显降低(P0.05),AECⅡ被阻滞于G_0/G_1期,增殖减少而分化增加(P0.05)。结论:Notch1/Hes1信号可调控C/EBPα表达并能影响AECⅡ增殖与分化。     

Wnt和Notch信号通路在大鼠创面愈合模型中的表达作用

目的观察Wnt和Notch信号通路在大鼠创面愈合模型中的表达及作用。方法取25只SD大鼠幼鼠,早期用溴脱氧尿苷（BrdU）标记表皮干细胞,注射BrdU 60 d后建立全层皮肤缺损创面模型。于大鼠致伤后0 d、7 d、14 d、21 d、30 d五个时间点分别各断颈处死5只取标本,利用免疫印迹、免疫组化和免疫荧光双标法观察创面愈合过程中Wnt1、β-catenin、c-Myc、Jagged1、Notch1、Hes1和Brdu的表达情况。结果免疫印迹结果显示Wnt和Notch信号通路成员在伤后表达上调,于伤后7 d达高峰,持续表达至伤后30 d。免疫组化结果显示,β-catenin开始在细胞膜低表达,伤后逐渐转变为表皮全层表达,且部分呈现异常核表达;伤后,Notch1在表皮全层表达逐渐上调,且在基底层表达上调显著。免疫荧光提示,BrdU/c-Myc和BrdU/Hes1双染阳性细胞率在伤后上升,于伤后7 d达高峰,持续表达至伤后30 d。结论 Wnt和Notch双信号通路可能通过调控表皮干细胞的增殖分化参与大鼠创面愈合过程。     

The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke

Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a γ secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a γ secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.     

Effects of Delta1 and Jagged1 on early human hematopoiesis: correlation with expression of notch signaling-related genes in CD34+ cells

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34(+) CD38(+) cells. Jagged1 increases the number of bipotent colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34(+) cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1-CD34(+) cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34(+) CD38(+) cells as they develop in Jagged1- or Delta1-stromal cell environments, which appear to reflect sequential maturational stages of CD34(+) cells into distinct cell lineages.