ALDH+乳腺癌干细胞的原代分离培养及生物学特性分析

目的 证实乳腺癌组织中存在ALDH+乳腺癌干细胞,并研究ALDH+乳腺癌干细胞体外增殖、侵袭能力等生物学特性.方法 应用流式细胞法从人乳腺癌组织细胞中分离并培养ALDH+乳腺癌干/祖细胞,通过克隆形成实验,生长曲线测定,以及transwell法等实验方法检测ALDH+乳腺癌干细胞的生物学特性.结果 在乳腺癌组织内,我们检测到ALDH+ CD24-/lowCD44+约占总细胞量的16％,无血清培养72 h后逐渐出现细胞球,2～3周时细胞球数量达到高峰.Transwell法检测ALDH+乳腺癌干细胞侵袭性较ALDH-乳腺癌干细胞侵袭性强.结论 乳腺癌组织中存在ALDH+ CD24-/low CD44+乳腺癌干细胞,ALDH可以作为鉴定乳腺癌干细胞的分子标志之一.     

雌激素对MCF-7中肿瘤干细胞相关亚群影响的实验研究

目的:探讨雌二醇(E2)及拮抗剂他莫西芬(TAM)对ER阳性乳腺癌细胞系MCF-7中肿瘤干细胞相关亚群(CD44+CD24-/low细胞)的影响。方法:E2组设10-7、10-8及10-9moL/L3个浓度,E2+TAM组再加入10-6moL/LTAM,对照组加等量药物溶剂(乙醇),培养10和20d时分别用流式细胞仪检测各组中CD44+CD24-/low细胞比例。结果:培养10d时,E2组中CD44+CD24-/low细胞比例分别为(2.28±0.38)%、(6.35±0.57)%和(2.86±0.29)%,与对照组中(0.83±0.03)%相比均增高,P<0.05。E2+TAM组中CD44+CD24-/low细胞比例分别为(1.85±0.19)%、(4.11±0.41)%和(1.73±0.33)%,与相同浓度E2组中相比比例均下降,P<0.05。培养20d时,E2组中CD44+CD24-/low细胞比例分别为(2.41±0.25)%、(6.18±0.41)%和(2.47±0.15)%,E2+TAM组中比例分别为(1.93±0.21)%、(4.26±0.39)%和(1.34±0.23)%,E2+TAM组中CD44+CD24-/low细胞比例与相同浓度E2组中相比均下降,P<0.05。结论:不同浓度E2作用下,MCF-7中肿瘤干细胞相关亚群含量增加,而在相同浓度的E2作用下,添加抗雌激素药物TAM能够使MCF-7中肿瘤干细胞相关亚群含量降低。     

不同乳腺癌干细胞亚群的自我更新和致瘤能力差异

目的 分析原代培养乳腺癌细胞中不同乳腺癌干细胞亚群的自我更新、致瘤能力差异。方法 采用流式细胞术和免疫荧光实验,从乳腺癌原代培养细胞中分选、鉴定CD44⁺CD24^-/low、ALDH1⁺和ALDH1⁺CD44⁺CD24^-/low细胞亚群,分析各分选细胞占总肿瘤细胞的比例;通过克隆形成实验观察各组细胞的自我更新能力;通过裸鼠致瘤实验观察各组细胞的致瘤能力。结果 CD44+CD24^-/low细胞占总细胞比例的7.2%,ALDH1⁺细胞所占比例为4.6%,ALDH1⁺CD44⁺CD24^-/low细胞所占比例为1.5%。三组细胞自我更新和致瘤能力由强至弱依次为ALDH1⁺CD44⁺CD24^-/low、ALDH1⁺、CD44⁺CD24^-/low细胞,各组细胞间比较差异均存在统计学意义（P<0.05）。 结论 乳腺癌组织中CD44+CD24-/low、ALDH1⁺和ALDH1⁺CD44⁺CD24^-/low三组乳腺癌干细胞亚群之间的自我更新和致瘤能力有明显差异,ALDH1⁺CD44⁺CD24^-/low亚群细胞具有最强的自我更新和致瘤能力,提示ALDH1⁺CD44⁺CD24^-/low可能是更具有特异性的乳腺癌干细胞标志物。     

乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC),寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系,倒置显微镜观察各细胞形态;流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞,其中CD44+CD24-为乳腺癌干细胞,其余三类为对照组;MTT法计数细胞,绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线;MCF-7细胞系进行无血清悬浮培养1个周期,流式细胞仪检测分子表面标记物CD44+CD24-含量,贴壁培养的CD44+CD24-乳腺癌干细胞为对照组;将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验,鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞;ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞;生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长,MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快;通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%;成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%,（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞;CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物;MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快;无血清悬浮培养法可简便、高效地富集乳腺癌干细胞;CD44+CD24-乳腺癌干细胞干性表达较强。     

CD44+CD24-/low乳腺癌干细胞分选鉴定及其多药耐药性研究

目的:观察MACS免疫磁珠法分选CD44+CD24-/low乳腺癌干细胞活性,并检测其与多药耐药的关系。方法:运用MACS免疫磁珠法从多药耐药乳腺癌细胞株MCF-7/ADR中分选CD44+CD24-/low乳腺癌干细胞,流式细胞术测定分选前后CD44+CD24-/low细胞比例,微球体培养法检测分选细胞自我更新能力,流式检测CD44+CD24-/low细胞表面P-糖蛋白(P-gp)表达水平,Real-time PCR检测多药耐药相关基因MDR1表达水平。结果:MACS免疫磁珠法分选后,CD44+CD24-/low细胞比例为93.85%,其成球能力明显强于non-CD44+CD24-/low细胞亚群。MCF-7/ADR细胞株和CD44+CD24-/low乳腺癌干细胞P-gp表达强度分别为101 177.10±2 171.86和114 906.70±2 560.19,P<0.05。CD44+CD24-/low乳腺癌干细胞MDR1基因表达水平为MCF-7/ADR细胞株的(1.07±0.02)倍,P<0.05。结论:经MACS免疫磁珠法分选所得CD44+CD24-/low细胞亚群有更强的自我更新能力,高表达P-gp蛋白和MDR1基因可能是引起乳腺癌多药耐药的重要原因。     

内分泌及化疗对ER+乳腺癌CD44+CD24-/low细胞亚群的实验与临床研究

目的:探讨内分泌治疗及化疗对雌激素受体(estrogen receptor,ER)阳性的乳腺癌CD44+CD24-/low细胞亚群的影响.方法:用3个浓度的雌二醇(estradiol,E2)和E2+他莫西芬(tamoxifen,TAM)处理乳腺癌MCF-7细胞,用工作浓度的化疗药物(多西他赛、紫杉醇、表柔比星和5-氟尿嘧啶)处理MCF-7细胞,FCM法检测各组细胞中CD44+CD24-/low的细胞比例,并检测ER+的转移性乳腺癌(metastatic breast cancer,MBC)患者化疗前、后外周静脉血中CD45-CD44+CD24-/low的细胞比例.结果:不同浓度E2培养MCF-7细胞10和20 d后,CD44+ CD24-/low细胞比例均高于对照组,E2+TAM组低于相应浓度E2组.各化疗药物作用MCF-7细胞24 h后,CD44+CD24-/low细胞比例均上升;而继续培养至20 d后,CD44+ CD24-/low细胞比例降低.部分缓解和疾病稳定的MBC患者,化疗后外周血CD45-CD44+CD24-/low细胞比例降低;而疾病进展者化疗后外周血CD45-CD44+CD24-/low细胞比例上升.结论:E2促进MCF-7细胞中CD44+CD24-/low细胞亚群的生长,TAM可抑制MCF-7细胞中CD44+CD24-/low细胞亚群的生长;MCF-7细胞中CD44+CD24-/low细胞亚群对多西他赛、紫杉醇、表柔比星和5-氟尿嘧啶均有抵抗能力;化疗有效患者外周血中CD45-CD44+CD24-/low细胞比例降低.     

MCF-7异质性及肿瘤干细胞相关亚群初步研究

目的:探讨人乳腺癌细胞系MCF-7的异质性及其肿瘤干细胞相关亚群(SP细胞或CD44+CD24-/low细胞)的性质。方法:采用Percoll不连续密度梯度法分离MCF-7,流式细胞仪检测各亚群中SP和CD44+CD24-/low细胞比例及各亚群细胞周期时相分布,各亚群细胞体外培养获得其生长曲线,平板克隆实验检测各亚群的增殖能力。结果:经Percoll分选MCF-7可形成A、B、C、D和E 5个亚群。各亚群中SP细胞比例分别为1.43%、2.15%、6.43%、25.23%和2.09%,其中D亚群中SP细胞富集。细胞周期时相分布显示,D亚群中G0/G1静止期细胞比例最高(P<0.05),为80.58%。生长曲线显示D亚群生长速度最快,以下依次为A亚群、B亚群、C亚群和E亚群。各亚群克隆形成率分别为(32.5±3.7)%、(41.3±4.8)%、(50.8±4.3)%和(68.2±3.5)%,其中D亚群具有最高的克隆形成率(P<0.05),而E亚群未形成明显的克隆性集落。但各亚群中均未检测到明显的CD44+CD24-/low细胞。结论:乳腺癌细胞系MCF-7具有异质性特征,经Percoll分选MCF-7可形成5个异质性的亚群,其中D亚群富含肿瘤干细胞相关亚群(SP细胞),但MCF-7在长期体外培养过程中,乳腺癌干细胞表面标志CD44+CD24-/low可能已经改变。     

乳腺癌细胞系中肿瘤干细胞相关亚群的初步研究

目的 通过乳腺癌细胞系及其干细胞的培养,化疗药干预和流式细胞仪筛选鉴定,探讨不同乳腺癌细胞系中CD44⁺CD24^-/low细胞比例,及富集乳腺癌干细胞相关亚群的方法。方法 通过细胞培养乳腺癌细胞系MCF-7、MDA-MB-231,观察生长曲线,比较化疗药物干预下生长情况;利用流式细胞仪检测两种乳腺癌细胞系中CD44⁺CD24^-/low细胞比例;无血清悬浮培养,化疗药(多西紫杉醇、表阿霉素)干预这两种乳腺癌细胞系,观察其是否形成细胞球。结果 (1)MDA-MB-231细胞系倍增时间短,生长速率高于MCF-7细胞系;(2)MCF-7细胞系中可能存在较大比例肿瘤干细胞,其对化疗抵制,能自我更新;(3)化疗敏感性用两独立样本t检验,MCF-7细胞,差异没有统计学意义;MDA-MB-231细胞,差异有统计学意义,提示MDA-MB-231细胞系对该方案化疗较敏感;(4)无血清悬浮培养,MDA-MB-231细胞系未发现明显细胞球;MCF-7细胞系初次无血清培养约6天出现细胞球。加入化疗药筛选后两种细胞,见大部分肿瘤细胞逐渐死亡,未发现明显细胞球;(5)流式细胞仪检测,MCF-7、MDA-MB-231两种细胞系中主要是CD44⁺CD24⁺亚群和CD44^-CD24⁺亚群,CD44⁺CD24^-/low细胞比例分别2.07%和0.20%。结论 (1)MDA-MB-231细胞系增值较快,恶性度相对较高,其对TA联合化疗药物较敏感;MCF-7细胞系中可能存在少量肿瘤干细胞,对化疗抵制,能自我更新;(2)无血清培养液培养MCF-7细胞系能形成悬浮细胞球;流式细胞仪检测两种细胞系中CD44⁺CD24^-/low细胞比例小;(3)CD44⁺CD24^-/low表型可能不是乳腺癌干细胞唯一特异性的表面标志。     

CD4+CD25+调节性T 细胞对乳腺癌细胞上皮间质转化和ALDH1+干样细胞的影响*

目的:研究CD4+CD25+调节性T 细胞（regulatory T cells ,Tregs）对乳腺癌细胞上皮间质转化（epithelial-mesenchymal transition ,EMT ）、细胞迁移侵袭能力,及ALDH1+干样细胞比例的影响。方法:采用免疫磁珠法分离乳腺癌患者外周血中CD4+CD25+Tregs,CD4+CD25+Tregs与乳腺癌BT474、MCF-7 细胞系共培养（共培养组）,BT474、MCF-7 单独培养（对照组）。 检测共培养组和对照组乳腺癌细胞EMT 相关标志物表达的变化,及细胞迁移和侵袭能力的变化。此外,检测BT474 细胞中ALDH1+干样细胞、微球形成能力和自我更新能力的变化。结果:CD4+CD25+Tregs诱导BT474 和MCF-7 细胞间质性标志物表达增高,诱导MCF-7 细胞上皮性标志物E-cadherin 表达降低。CD4+CD25+Tregs诱导BT474 和MCF-7 细胞迁移和侵袭能力上调。共培养组BT474 细胞中ALDH1+干样细胞比例、微球体形成能力、自我更新能力较对照组增强。结论:CD4+CD25+Tregs可诱导乳腺癌细胞发生EMT ,增强细胞体外迁移和侵袭能力,同时促进ALDH1+干样细胞增加。      

小白菊内酯体内杀伤小鼠乳腺癌肿瘤干细胞

目的： 探讨小白菊内酯（parthenolide,PTL）对小鼠乳腺癌肿瘤干细胞（cancer stem cell,CSC）的杀伤作用,为临床应用PTL治疗乳腺癌提供实验依据。 方法： 采用5-氟尿嘧啶（5-fluorouracil, 5-FU）化疗法制备富含CSC的小鼠4T1细胞乳腺癌模型,随机分为对照组、5-FU组、PTL组。4周后脱颈处死小鼠,检测各组小鼠肿瘤的体积和重量,流式细胞术检测小鼠肿瘤组织中CD44+CD24-/low细胞比例,Hoechst33342染色法检测侧群（side population,SP）细胞的比例,免疫组化法检测CD55和乙醛脱氢酶1（aldehyde dehydrogenase1,ALDH1）蛋白的表达,倒置显微镜观察乳腺癌细胞微球体的形成。 结果： 成功制备富含CSC的小鼠乳腺癌细胞移植瘤模型,PTL可下调小鼠肿瘤组织中CD44+CD24-/low细胞的比例\[（42.5±3.7）% vs （68.7±32）%,P<0.05\],有效降低荷瘤小鼠肿瘤组织中SP细胞的比例\[（39.2±1.8）% vs （61.3±2.6）%,P<0.05\],下调小鼠移植瘤组织中CD55和ALDH1蛋白的表达\[（18.9±1.5）% vs （30.1±1.3）%,（8.1±2.3）% vs （18.0±1.4）%;均P<0.05\],抑制小鼠肿瘤细胞在无血清培养条件下形成微球体,并可抑制小鼠移植瘤的体积和重量\[（0.625±0.159）cm3 vs （1.715±0184）cm3,（1.467±0.373）g vs （3.367±0.398）g;均P<0.05\]。 结论： PTL在荷瘤小鼠体内可以明显降低肿瘤组织CSC含量,提示PTL可用来靶向杀伤乳腺癌CSC。     

克隆柱法提取MCF-7乳腺癌细胞系中癌干细胞

目的通过克隆柱法提取MCF-7乳腺癌细胞中的乳腺癌干细胞,并进行鉴定。方法采用单细胞克隆加低密度干细胞培养基筛选获得呈“球样”生长的乳腺癌干细胞,行CD44、CD24免疫荧光细胞化学染色及诱导分化实验。结果CD44＋CD24^-/Low细胞的比例为12．1％,且在全克隆中富含CD44＋CD24^-/Low细胞。在形成的细胞球中富含CD44＋CD24^-/Low细胞,并且在诱导分化时可见明显类管腔样结构形成,并表达CK18及CK14。结论克隆柱法是提取细胞系中癌干细胞的简单有效方法。     

呼肠孤病毒诱导乳腺癌微球体细胞凋亡

目的:探讨呼肠孤病毒对乳腺癌微球体细胞凋亡的影响。方法:以干细胞培养液培养乳腺癌细胞MCF-7和BT474,形成微球体,分别用呼肠孤病毒和表柔比星处理微球体,FCM检测MCF-7、BT474微球体细胞中CD44+CD24-/low细胞的比例,TUNEL法检测微球体细胞的凋亡,Western blotting检测MCF-7细胞及其微球体细胞中Ras的表达。结果:呼肠孤病毒对乳腺癌MCF-7和BT474微球体细胞的感染率显著高于亲本细胞(均P<0.05)。表柔比星作用下MCF-7和BT474微球体细胞中CD44+CD24-/low细胞比例明显升高[(8.1±0.4)%vs(18.0±4.5)%,P<0.01;(0.6±0.2)%vs(0.9±0.13)%,P<0.05]。呼肠孤病毒感染后MCF-7和BT474微球体细胞中CD44+CD24-/low细胞比例明显下降[(8.1±0.4)%vs(0.8±0.2)%,(0.6±0.2)%vs(0.2±0.1)%;均P<0.05]。MCF-7和BT474微球体细胞在呼肠孤病毒感染后凋亡率明显增加[(1.90±0.21)%vs(5.0±1.4)%,P<0.05;(0.20±0.1)%vs(1.0±0.16)%,P<0.05]。MCF-7细胞及其微球体细胞均高表达Ras,且两者水平相似。结论:与表柔比星相比,呼肠弧病毒对乳腺癌微球体细胞的抑制作用明显,还可明显诱导乳腺癌微球体细胞凋亡。     

Repression of mammosphere formation of human breast cancer cells by soy isoflavone genistein and blueberry polyphenolic acids suggests diet-mediated targeting of cancer stem-like/progenitor cells

Mammary stem cells are undifferentiated epithelial cells, which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we addressed whether dietary factors selectively target mammary epithelial cells that display stem-like/progenitor subpopulations with previously recognized tumor-initiating potential. Using estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 human breast cancer cell lines and freshly isolated epithelial cells from MMTV-Wnt-1 transgenic mouse mammary tumors, we demonstrate that sera of adult mice consuming soy isoflavone genistein (GEN) or blueberry (BB) polyphenol-containing diets alter the population of stem-like/progenitor cells, as measured by their functional ability to self-renew and form anchorage-independent spheroid cultures in vitro at low frequency (1-2%). Serum effects on mammosphere formation were dose-dependently replicated by GEN (40 nM >2 μM) and targeted the basal stem-like CD44+/CD24-/ESA+ and the luminal progenitor CD24+ subpopulations in MDA-MB-231 and MCF-7 cells. GEN inhibition of mammosphere formation was mimicked by the Akt inhibitor perifosine and was associated with enhanced tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) expression. In contrast, a selected mixture of BB phenolic acids was only active in MDA-MD-231 cells and its CD44+/CD24-/ESA+ subpopulation, and this activity was independent of induction of PTEN expression. These findings delineate a novel and selective function of distinct dietary factors in targeting stem/progenitor cell populations in estrogen receptor-dependent and -independent breast cancers.     

The BMP2/7 heterodimer inhibits the human breast cancer stem cell subpopulation and bone metastases formation

Accumulating evidence suggests that a subpopulation of breast cancer cells, referred to as cancer stem cells (CSCs), have the ability to propagate a tumor and potentially seed new metastases. Furthermore, stimulation of an epithelial-to-mesenchymal transition by factors like transforming growth factor-β (TGFβ) is accompanied with the generation of breast CSCs. Previous observations indicated that bone morphogenetic protein-7 (BMP7) antagonizes the protumorigenic and prometastatic actions of TGFβ, but whether BMP7 action is mechanistically linked to breast CSCs has remained elusive. Here, we have studied the effects of BMP7, BMP2 and a BMP2/7 heterodimer on the formation of human breast CSCs (ALDH(hi)/CD44(hi)/CD24(-/low)) and bone metastases formation in a preclinical model of intra-cardiac injection of MDA-MB-231 cells in athymic nude (Balb/c nu/nu) mice. The BMP2/7 heterodimer was the most efficient stimulator of BMP signaling and very effectively reduced TGFβ-driven Smad signaling and cancer cell invasiveness. The tested BMPs-particularly the heterodimeric BMP2/7-strongly reduced the size of the ALDH(hi)/CD44(hi)/CD24(-/low) CSC subpopulation. In keeping with these in vitro observations, pretreatment of cancer cells with BMPs for 72 h prior to systemic inoculation of the cancer cells inhibited the formation of bone metastases. Collectively, our data support the notion that breast CSCs are involved in bone metastasis formation and describe heterodimeric BMP2/7 as a powerful TGFβ antagonist with anti-metastatic potency.     

长程多柔比星化疗富集乳腺肿瘤干细胞

目的:探讨长程多柔比星( adriamycin,ADR)作用乳腺癌细胞株MCF-7对富集肿瘤干细胞的可能性.方法:采用长程ADR诱导的方法建立ADR耐药细胞株MCF-7/ADR'.ALDEFLUOR法检测亲本MCF-7细胞及ADR耐药细胞MCF-7/ADR'中乙醛脱氢酶1(aldehyde dehydrogenase 1,ALDH1)阳性的肿瘤干细胞亚群的比例,然后采用无血清悬浮培养和裸鼠成瘤实验分别检测两者在体外形成干细胞微球体和体内成瘤能力的差异.结果:MCF-7和MCF-7/ADR'细胞中ALDH1+肿瘤干细胞亚群的比例分别为(0.82±0.77)％和(8.21±2.38)％,两者差异有统计学意义(P＜0.05);两者经无血清悬浮培养形成干细胞微球体的比例分别为(2.17±0.70)％和(7.87±1.39)％,差异有统计学意义(P＜0.05).MCF-7/ADR'细胞在裸鼠体内的成瘤能力明显强于MCF-7细胞.结论:长程ADR作用MCF-7后可富集肿瘤干细胞.     

Mesenchymal stem cells expressing GD2 and CD271 correlate with breast cancer-initiating cells in bone marrow

Purpose: The bone marrow microenvironment is considered a critical component in the dissemination and fate of cancer cells in the metastatic process. We explored the possible correlation between bone marrow mesenchymal stem cells (BM-MSC) and disseminated breast cancer-initiating cells (BCIC) in primary breast cancer patients. Experimental design: Bone marrow mononuclear cells (BM-MNC) were collected at the time of primary surgery in 12 breast cancer patients. BM-MNC was immunophenotyped and BCIC was defined as epithelial cells (CD326+CD45-) with a "stem-like" phenotype (CD44+CD24low/-, ALDH activity). BM-MSC was defined as CD34-CD45-cells that co-expressed GD2, CD271, and/or CD200 within CD326-depleted BM-MNC. Results: The percentages of BCIC (Aldefluor+CD326+CD44+CD24-) correlated with the percentages of BM-MSC, either CD45-GD2+CD200+CD271+ (Kedall's tau = 0.684, p = 0.004) or CD45-GD2+CD271+ in the bone marrow (Kedall's tau = 0.464, p = 0.042). Conclusions: There was a positive correlation between mesenchymal stem cells expressing GD2 and CD271 and breast cancer-initiating cells in BM of patients with primary breast cancer.