肿瘤干细胞标记物CD_(133)与miR-429在大肠癌组织中的表达及其相关性研究

目的:检测CD133不同亚群大肠癌细胞HT-29的miR-429表达情况,探讨miR-429及CD133的表达与肿瘤的发生发展之间的关系。方法:采用荧光活化细胞分选法(FACS)分选出CD133不同亚群细胞,实时荧光定量PCR分别检测两组细胞miR-429的表达,合成miR-429寡核苷酸和阴性对照miRNA并分别转染CD133+和CD133-两个亚群细胞。再将细胞种植于非肥胖糖尿病/严重联合免疫缺陷(NOD/SCID)小鼠体内构建移植瘤模型,不同时间测量肿瘤体积和重量,RT-PCR及蛋白质印迹检测CD133+和CD133-两组肿瘤CD133mRNA和蛋白质表达。结果:血清检出CD133+细胞为67.9%,miR-429的表达量是CD133+细胞的(1.83±0.91)倍(P0.05),CD133+比例与miR-429表达呈负相关(r=0.591,P0.05);miR-429+/CD133+组的移植瘤体积及重量与对照组比较有统计学差异(P0.05),且miR-429+/CD133+组成瘤时间较对照组晚约2周,但miR-429+/CD133+组的移植瘤CD133表达量低,与阴性对照组比较无明显差异(P0.05)。结论:miR-429可能作为CD133的负性调控因子,具有抑制肿瘤生长的作用,但miR-429与CD133在肿瘤发生、发展过程中的作用机制有待进一步研究阐明。     

肿瘤干细胞标记分子CD133在胶质瘤细胞U87中的稳定表达

目的：在体外胶质瘤U87细胞中稳定表达肿瘤干细胞标记分子CD133。方法：通过脂质体介导将表达载体质粒CD133-1／pCR3．1-Uni转染U87细胞,G418筛选稳定表达抗性的细胞株;用细胞免疫荧光染色鉴定表达CD133分子的U87细胞。结果：转染CD133表达载体的U87细胞可以被CD133单抗识别,而转染空载体的U87细胞免疫染色结果为阴性,表明CD133分子在U87细胞中稳定表达。结论：U87细胞稳定表达CD133分子,为体内外分析CD133阳性U87细胞特性奠定了基础：U87CD133阳性细胞可以作为免疫组化或流式细胞术等检测其他肿瘤干细胞CD133表达的阳性对照细胞。     

miR-429与肿瘤

miR-429是miR-200家族成员之一。研究表明,miR-429异常表达与肿瘤的发生、发展、转移、凋亡和耐药等密切相关。但miR-429在肿瘤中所起的作用一直有争议,可作为肿瘤抑制剂或促进剂,具肿瘤细胞/组织特异性。其在骨肉瘤、肾癌、卵巢癌、乳腺癌、宫颈癌、胶质瘤、口腔鳞状细胞癌、胃癌、食管癌、胰腺癌中起抑癌作用,而在肺癌、前列腺癌和子宫内膜癌中起促癌作用,但在结肠癌、肝癌、膀胱癌中的作用尚不明确。本文综述了近年来miR-429在肿瘤发生、发展中的作用及潜在的调控机制,为其作为肿瘤诊断、治疗及预后的潜在生物标记分子提供新的启示和参考。     

CD133 蛋白在非小细胞肺癌组织中的表达及其与临床病理参数及预后的关系

目的:研究CD133蛋白在非小细胞肺癌(NSCLC)组织中的表达及其与临床病理参数及预后的关系。方法:选择2011年4月~2012年4月间我院收治的42例NSCLC的临床资料,采用免疫组织化学染色法检测癌组织和其中30例癌旁边正常组织CD133的表达,并分析其与肺癌临床病理参数及预后的关系。结果:癌症组织CD133蛋白阳性表达率为57.1%,高于癌旁边正常组织的16.7%,差异有统计学意义(P均0.05);CD133蛋白阳性表达与NSCLC患者年龄、性别以及肿瘤大小、肿瘤位置、组织学类型、组织分化程度以及不同临床分期无关(均P0.05),与淋巴结转移有关,差异有统计学意义(P0.05);CDD133阳性及阴性患者近3年生存率比较,1年生存率比较差异不显著(P0.05),2、3年生存率比较,差异显著有统计学意义(P0.05)。结论:CD133蛋白在NSCLC组织中的高表达,且与NSCLC转移及预后密切相关,对于患者临床特征及预后的关系具有重要的研究价值。     

CD133和maspin在肝癌中的表达及其临床病理意义

目的 探讨肿瘤干细胞标志物CD133和maspin在肝细胞性肝癌（hepatocellular carcinoma HCC）中的表达情况及其临床病理意义.方法 采用免疫组织化学ElivisionTM plus法检测100例HCC组织和20例癌旁肝组织中CD133和maspin蛋白的表达情况.结果 在癌旁肝组织中CD133和maspin蛋白的阳性表达率分别为5.0%和100%,而在HCC组中其阳性表达率分别为51.0%和48.0%,两组之间差异有显著性（P〈0.01）.CD133蛋白的表达水平与肿瘤的Edmondson分级、淋巴结转移、肿瘤的数目和有无血管侵犯有关（全部P〈0.05）;maspin蛋白的表达水平与肿瘤的Edmondson分级、肿瘤的数目和有无血管侵犯有关（全部P〈0.05）.且CD133蛋白的表达与maspin蛋白的表达呈负相关（P〈0.01）.结论 CD133和maspin蛋白的表达与HCC组织的Edmondson分级、肿瘤数目和血管侵犯有关;CD133和maspin的联合检测对HCC的进展及预后判断有重要意义.     

CD133在胰腺癌中的表达及临床意义

目的:探讨CD133在胰腺癌中的表达及其与临床病理特征和预后的关系.方法:采用免疫组织化学方法,检测71例胰腺癌和10例正常胰腺组织中CD133的表达.分析CD133在胰腺癌中的表达与临床病理特征和预后之间的相关性.结果:CD133在胰腺癌中的表达的阳性率64.79%明显高于正常胰腺组织中的10%.胰腺癌中CD133的表达率随着临床TNM分期的增加而明显增高(P＜0.05).胰腺癌中CD133的表达率在有淋巴结转移中为71.4%(20/28)明显高于无淋巴结转移中的23.3%(10/43)(P＜0.05).CD133的表达与年龄、性别、肿瘤部位、肿瘤大小、神经浸润、切缘、病理学分级无显著相关性(P＞0.05).CD133高表达者的中位生存期明显短于低表达者(9.1个月vs 23.9个月,P＜0.05).结论:胰腺癌中CD133的高表达与TNM分期高及淋巴结转移有关.CD133高表达与胰腺癌预后差有关.CD133在胰腺癌的发展、转移及预后中可能发挥重要作用,可作为临床评价胰腺癌生物学行为及评估预后的指标之一.     

miR-221在肝癌干细胞亚群中的差异表达

目的：分离肝癌细胞系MHCC97中肝癌干细胞并分析肝癌细胞高表达miR-221在肝癌干细胞和非干细胞亚群中的表达差异情况,探讨miR-221表达水平与肝癌干细胞分化之间的关系。方法：利用流式细胞荧光激活分选法从肝癌细胞系MHCC97中分选出肝癌干细胞（hepatocareinoma stem cells,HSCs）和非干细胞（non-hepatocareinoma stem cells,non-HSCs）两个亚群。采用实时荧光定量RT-PCR（Real-time RT-PCR）检测miR-221在两个不同肝癌细胞亚群中的表达。结果：HSC亚群肝癌细胞仅占细胞总体的2.59%;HSC亚群细胞中miR-221的表达明显高于non-HSC亚群（P〈0.01）。结论：miR-221在HSC亚群肝癌细胞中的明显高表达,提示miR-221可能在维持HSC亚群肝癌细胞的干细胞特性方面具有重要意义。通过调控肝癌干细胞中miR-221的表达,可以促进其分化成熟,从而为肝癌治疗提供新的思路。     

CD133分子与造血干/祖细胞和癌干细胞之间的关系

越来越多的实体瘤癌干细胞(CSCs)研究中鉴定分离出含有CD133+的CSCs亚群。深入了解CD133+分子的生物学特性及在细胞与肿瘤信号转导、药物耐受和放化疗抵抗等的相关性,将有助于靶向治疗CSCs。本文就CD133分子与造血干/祖细胞(HSPC)和CSCs之间的关系及其耐药机制的研究进展作一综述。     

CD68、CD133和TGF-β在肝癌及癌旁组织中的表达及其在临床转归中的意义

目的研究CD68^+肿瘤相关巨噬细胞(TAMs)、CD133^+肿瘤干细胞(CSCs)及TGF-β在肝癌及癌旁组织中的表达及其在临床转归中的意义。方法选取2014年1月至2016年1月在中国医学科学院肿瘤医院综合科进行手术治疗的89例肝癌患者为研究对象。收集患者手术标本,进行免疫组化染色(IHC),采用半定量积分法对免疫组化分析正常、癌及癌旁组织中CD68^+肿瘤相关巨噬细胞(TAMs)、CD133^+肿瘤干细胞(CSCs)及TGF-β表达情况,结合随访资料对其意义进行分析,其表达水平与临床病例资料之间关系采用c^2检验或Fisher确切概率法检验或Wilcoxon秩和检验,相关性分析采用Spearman检验。结果 CD68^+TAMs细胞在癌组织中阳性率为59.55%,癌旁组织为37.08%,差异具有统计学意义(Z=-3.182,P=0.001);CD133^+细胞在癌组织中阳性率为52.81%,癌旁组织中为57.30%,表达差异无统计学意义(Z=-0.558,P=0.557);TGF-β在癌组织阳性率为70.79%,癌旁组织中为56.18%,差异具有统计学意义(Z=-2.306,P=0.021)。CD68、CD133、TGF-β表达水平与患者肿瘤分化程度及淋巴结转移情况密切相关,差异具有统计学意义(P<0.05);TGF-β表达水平与Child-Pugh分级相关,差异具有统计学意义(c^2=10.930,P=0.001)。HCC癌组织中CD68与TGF-β表达,CD133与TGF-β表达之间存在正相关关系,相关系数分别为(r=0.579、0.611,P=0.026、0.020)。Kaplan-Meier生存曲线分析可知,3种分子均无表达的患者中位生存时间最长,为23.4个月,3种分子均有表达的患者中位生存期最短,仅为6.3个月(P=0.021)。结论 CD68^+TAMs细胞与CD133^+CSCs细胞之间存在相关性,二者之间具体机制有待进一步研究,以便为肝细胞癌(HCC)治疗提供有力依据。     

CD133在胃肠间质瘤的表达及其临床意义

目的 探讨胃肠间质瘤（gastrointestinal stromal tumor,GIST）中的CD133的表达及其与GIST临床病理特征的关系.方法 采用免疫组织化学法,检测122例胃肠间质瘤患者组织中的CD133、CD117、CD34、DOG-1、KI-67的表达情况.结果 CD133、CD117、CD34 、DOG-1、KI-67的阳性表达率分别为74.6%（91/122）、98.4%（120/122）、86.9%（106/122）、95.1%（116/122）、47.5%（58/122）.CD133的表达水平与胃肠间质瘤危险度分组高低、核分裂像数目、肿瘤部位有关（P〈0.05）,而与患者性别、年龄、肿瘤大小无明显相关性（P〉0.05）.CD133的表达水平与DOG-1的表达水平无明显相关性（P〉0.05）,而与CD117、CD34、KI-67的表达水平呈正相关.结论 CD133蛋白的表达可能与GIST的恶性行为与预后有关,与CD117、CD34和DOG-1联合检测对于判断GIST的病理性质可能具有重要价值.     

肝癌细胞系中SP及CD133+细胞的特性分析

近年的研究表明体外培养的肝癌细胞系中存在着少量具有肝癌干细胞功能的细胞群。有研究指出肝癌干细胞存在于侧群细胞(sidepopulation,SP)中,另有研究则发现CD133+细胞是肝癌干细胞的特征。本研究以三种肝癌细胞系(Hep3B、Huh-7和PLC/PRF/5)为对象,利用流式细胞术对其中的SP细胞与CD133+细胞进行了分析,并进一步检测了它们的增殖能力、表型及耐药性等特性。结果显示,肝癌细胞系中存在不同比例的SP细胞和CD133+细胞,且大部分SP细胞呈CD133阳性表达。表型特征分析显示SP细胞表达CK7和CK19,不表达AFP,而CD133+细胞则表达AFP和CK19,却不表达CK7。SP与CD133+细胞都具有较强的增殖能力。另外,相比于其它细胞,SP细胞具有最强的化疗药物抗性。结果表明,肝癌细胞系中SP细胞与CD133+细胞整体特征有一定的区别,提示了它们不同的分化途径。     

抗CD133胞外区骆驼多克隆抗体的制备及鉴定

目的:制备高效价、高特异性的抗CD133胞外区新疆双峰驼来源的多克隆抗体,为制备高亲和力的抗CD133纳米抗体做准备。方法:将CD133胞外区基因序列构建到原核表达载体pET28a中,诱导表达及纯化CD133蛋白,免疫新疆双峰驼及新西兰兔。通过酶联免疫吸附实验(ELISA)和Western blot检测多克隆抗体的效价及与CD133特异性结合活性。结果:ELISA测定抗-CD133骆驼源抗体的效价可达到百万以上,通过Western blot检测多克隆抗体可特异性结合CD133蛋白。结论:重组人CD133蛋白可以在骆驼体内激发高滴度抗体反应,为今后构建骆驼免疫单域抗体噬菌体展示文库奠定基础。     

Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G₂/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.     

Identification of cancer stem cells from human glioblastomas: growth and differentiation capabilities and CD133/prominin-1 expression

CD133 can be a marker of tumorigenic CSCs (cancer stem cells) in human GBM (glioblastoma multiforme), although tumorigenic CD133-negative CSCs have been also isolated. Additional evidence indicates that CSCs from GBM exhibit different phenotypes, with increasing interest in the potential significance of the different CSCs with respect to diagnosis, prognosis and the development of novel targets for treatment. We have analysed the expression of CD133 in freshly isolated cells from 15 human GBM specimens. Only 4 of them contained cells positive for AC133 by FACS analysis, and all of them yielded distinct CSC lines, whereas only 6 CSC lines were obtained from the other 11 GBMs. Of these 10 CSCs lines, we further characterized 6 CSC lines. Three CSCs grew as fast-growing neurospheres with higher clonogenic ability, whereas the remaining 3 grew as slow-growing semi-adherent spheres of lower clonogenicity. In addition, the former CSC lines displayed better differentiation capabilities than the latter ones. PCR and Western blot analysis showed that all 6 GBM CSC lines expressed CD133/prominin-1, suggesting that cells negative by FACS analysis may actually represent cells expressing low levels of CD133 undetected by FACS. Nevertheless, all the 6 CSC lines were tumorigenic in nude mice. In conclusion, CSCs from human primary GBMs show different phenotypes and variable levels of CD133 expression, but these parameters did not directly correlate with the tumorigenic potential.     

Isolation and characterization of the CD133+ precursors from the ventricular zone of human fetal brain by magnetic affinity cell sorting

A fast and effective method to enrich large number of neural precursors from the ventricular zone of human fetus by magnetic affinity cell sorting (MACS) is reported. After incubation with phycoerythrin (PE)-conjugated anti-CD133 antibodies and anti-PE magnetic beads followed by one cycle of MACS, CD133(+) cells were harvested at 85% purity as confirmed by flow-cytometry and immunocytochemistry. In contrast to CD133(-) cells, these CD133(+) cells initiated primary and secondary neurospheres in culture, and the progeny of sorted cells could be differentiated into both neurons and glia, indicating that these highly enriched cells are capable of self-renewal and multi-lineage potential.     

人脐血CD133细胞的分离及其体外扩增的研究

目的:探讨免疫磁性纳米粒子分离人脐血CD133细胞的方法,了解分离出的CD133细胞在体外短期培养中的变化及其在体外扩增的可能性。方法:通过化学沉淀法制备具有超顺磁性的r-Fe_2O_3纳米粒子,在其表面包裹具有生物亲合性的二氧化硅,并在其表面通过化学修饰使其成为生物功能化的磁性纳米粒子。再通过一定的化学连接方法将单克隆抗体CD133连接到生物功能化的磁性纳米粒子表面使其成为免疫磁性纳米粒子,然后利用自制的免疫磁性纳米粒子从单个核细胞中分离出CD133细胞,并分别对单个核细胞和CD133细胞在体外短期培养中的动态变化进行了初步观察和比较。结果:经免疫磁性纳米粒子分离的脐血中CD133细胞平均数为(5±1．4)×10~7/ml,占单个核细胞数的(3±0．3)%;单个核细胞(对照组)和CD133细胞(实验组)分别进行红、粒系集落扩增培养14天、21天,实验组中两种造血祖细胞集落扩增倍数都明显高于对照组(P＜0．01)。结论:使用自制的免疫磁性纳米粒子能较好的分离脐血中的CD133细胞,分离与纯化出来的CD133细胞不仅细胞活力不受影响,而且与单个核细胞相比具有更强的增殖能力。     

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.     

内源CD133~+细胞示踪小鼠模型的制备和鉴定

目的:为了制备可追踪CD133阳性神经干细胞分化谱系的小鼠模型。方法:将两种C57B16背景的转基因小鼠CD133-Cre-ERT2和Rosa26-CAG-LSL-ZsGreen杂交,获得CD133-Cre ER;CAG-ZsGreen小鼠模型。结果:免疫组化和激光扫描共焦成像分析表明,经Tamoxifen作用后,该杂交小鼠在侧脑室SVZ区、第三脑室和第四脑室的室管膜区域均表达绿色荧光蛋白ZsGreen,且这些区域的绿色荧光与CD133~+红色荧光重合。结论:在室管膜区CD133~+是静息态神经干细胞的标志,因此,通过分析CD133-Cre ER;CAG-ZsGreen小鼠中的ZsGreen阳性细胞可追踪神经干细胞的细胞分化谱系。成功制备了内源CD133~+细胞示踪小鼠模型,为探讨大脑中CD133~+神经干细胞的激活、增殖、迁移和分化提供了帮助。