乙醛脱氢酶阳性乳腺癌干细胞的分离培养及其生物学特性分析

 目的　证实人类乳腺癌MCF-7细胞系中存在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　应用流式细胞术从MCF-7细胞中分离并培养ALDH+ 乳腺癌干／祖细胞,通过划痕试验、MTT法生长曲线测定、以及Transwell法等检测ALDH+乳腺癌干细胞的生物学特性。结果　MCF-7细胞系中,CD-／low24 CD+44 细胞比例约为1.4 %,ALDH+ CD-／low24 CD+44 细胞比例约为1.2 %; ALDH+乳腺癌干细胞与CD-／low24 CD+44 细胞两者的生长曲线基本一致;CD-／low24 CD+44 ,ALDH+ CD-／low24 CD+44 组细胞划痕区细胞间距离明显缩短,其迁移能力明显强于对照组,且两群干细胞之间存在差异;Transwell实验结果,与对照组相比,CD-／low24 CD+44 、ALDH+ CD-／low24 CD+44 两组细胞有大量细胞过膜,三组MTT检测吸光度值分别为1.05±0.098、1.56±0.075、1.67±0.032。结论　MCF-7细胞系中存在CD-／low24 CD+44 和ALDH+ CD-／low24 CD+44 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。     

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。     

长程无血清培养人乳腺癌细胞球的生物学特性

目的:从人乳腺癌原代细胞中分离培养乳腺癌干/祖细胞,并通过体外连续传代培养,研究其体外增殖、分化等生物学特性.方法:应用非黏附性乳腺球(mammosphere,MS)悬浮培养法富集乳腺癌干/祖细胞后,通过连续的单克隆实验检测乳腺球细胞(mammosphere-derived cells,MSDC)的克隆形成和自我更新能力,并经血清诱导后观察其分化能力.FCM法检测CD44~+/CD24~(-/low)细胞的含量.结果: 接种于含生长因子无血清培养液的原发乳腺癌标本细胞均有MS生成,内含具有干细胞特征的肿瘤细胞.随着培养中传代次数的增加,贴壁分化的细胞增多,形成的MS减少,球体变小,且更易贴壁分化.CD44~+/CD24~(-/low)细胞仅存在于基底样乳腺癌标本.来自不同标本的MS的生物学特性表现出一定差异.结论:具有自我更新、无限增殖和多向分化等干细胞样特性的肿瘤细胞在人乳腺癌组织中广泛存在,其生物学行为可能因标本来源不同有所差异,也会因环境因素的作用而改变.     

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。     

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。     

CD24+CD44+胰腺癌细胞的获取及其干细胞特性的初步鉴定

背景与目的:近年研究发现,肿瘤内存在极小一部分细胞,决定肿瘤的恶性表型及生物学特性,被称为"肿瘤干细胞".本研究拟从人原发胰腺癌组织中找到具有干细胞特性的胰腺癌细胞,为今后靶向干预胰腺癌干细胞,解决胰腺癌治疗的临床难题提供研究基础.方法:将人原发胰腺癌组织种植于NOD/SCID小鼠成瘤,对移植瘤细胞进行流式细胞分选;将流式细胞仪分选获得的细胞用无血清、含有生长因子的DMEM/F12培养基培养,观察细胞的生长、传代及体外干细胞球形成能力;将细胞接种于非肥胖型糖尿病/重症联合免疫缺陷型(Nonobese diabetic/severe combined immunodeficiency,NOD/SCID)小鼠,观察、比较成瘤率,免疫组化法检测干细胞移植瘤肿瘤分化相关抗原Ki67、CK7及甲基化调控因子MBD1的表达;免疫荧光检测细胞Hedgehog、BMI-1的表达.结果:人原发胰腺癌组织种植于NOD/SCID小鼠可部分成瘤,对移植瘤细胞进行流式细胞分选,获得CD24+CD44+细胞(获得率0.6%～1.8%);将CD24+CD44+细胞用无血清、含有生长因子的DMEM/F12培养基培养,细胞呈悬浮球状生长,可连续传代并能再次形成干细胞球;将102个CD24+CD44+细胞接种于NOD/SCID小鼠,成瘤率12.5%(1/8小鼠成瘤),将103个CD24+CD44+细胞接种于NOD/SCID小鼠,4周后100%成瘤,而同样数量的CD24 CD44胰腺癌细胞则无法成瘤(P＜0.05),免疫组化法检测干细胞移植瘤中部分细胞表达Ki67、CK7及MBD1阳性;免疫荧光检测证实CD24+CD44+细胞阳性表达Hedgehog、BMI-1,表达量分别高于CD24-CD44-细胞9.95倍和2.74倍(P＜0.05).结论:所获得的CD24+CD44+细胞具有胰腺癌干细胞特性,可作为今后胰腺癌干细胞研究的实验基础.     

小鼠乳腺癌4T1细胞中肿瘤干细胞样细胞的富集和鉴定

目的：无血清培养基(serum-free medium, SFM)悬浮培养小鼠乳腺癌细胞株4T1,富集并鉴定4T1细胞株中肿瘤干细胞样细胞。方法：通过含EGF、bFGF和B27等细胞因子的SFM培养富集4T1细胞中肿瘤干细胞样细胞,将其接种于含血清培养基（serum-supplemented medium, SSM),观察4T1肿瘤干细胞样细胞分化情况。应用细胞表面标志CD44+CD24-/low和Hoechst 33342染色法检测4T1细胞中肿瘤干细胞样细胞的比例,小鼠致瘤实验验证不同培养条件下4T1肿瘤干细胞样细胞的致瘤能力。结果：4T1细胞在SFM中能够存活、增殖,并形成细胞球,细胞球可连续传代,若重新接种于SSM中可贴壁分化。4T1细胞球中CD44+CD24-/low细胞比例为6.4%~68.9%,侧群 (side population,SP) 细胞比例为7.3%~61.2%,均显著高于SSM中培养的4T1细胞（P<0.05）;随着SFM中细胞球传代次数增加,CD44+CD24-/low细胞和SP细胞的比例逐渐升高。小鼠致瘤实验结果显示,富集了肿瘤干细胞的细胞球比常规培养4T1细胞的致瘤性更强。结论：乳腺癌4T1细胞可在含多种生长因子的SFM中悬浮生长并形成细胞球,4T1细胞中含有的乳腺癌干细胞样细胞可通过SFM培养法富集。     

卵巢癌组织及腹腔积液中肿瘤干细胞的分离与培养

目的:从人卵巢癌组织和腹腔积液中分离培养肿瘤干细胞,分析细胞表型和化疗药物敏感性。方法:取卵巢浆腺癌患者的原发肿瘤组织及腹腔积液,通过无血清培养形成悬浮生长的球体。采用FCM及免疫荧光法检测干细胞标志物的表达。体外药敏实验检测获得的球体细胞和分化细胞对紫杉醇和卡铂的药物敏感性。结果:卵巢癌组织及腹腔积液中的卵巢癌细胞经无血清悬浮培养可以形成球体,后者具有CD44+CD24-表型并表达Oct4、Nanog及Sox2等干细胞标志物;腹腔积液及组织来源的球体细胞经血清培养液培养3周后发生分化,CD44+CD24-细胞比例分别下降至47%和3%;分化细胞对紫杉醇和卡铂的敏感性均显著高于球体细胞(P<0.01)。结论:卵巢癌的肿瘤干细胞属于CD44+CD24-细胞,后者表达Oct4等干细胞标志并具有显著耐药性。     

乳腺癌干细胞上皮-间质转化标志物表达变化及意义

目的 探讨乳腺癌干细胞上皮．间质转化标志物表达变化及其临床意义。方法采用无血清悬浮培养法,从MCF-7细胞培养乳腺癌微球体细胞,应用流式细胞仪检测微球体细胞中CD44和CD24的表达,采用Westernblot方法检测微球体细胞中E-钙黏素、N-钙黏素、纤维连接蛋白、波形蛋白的表达水平,体外穿膜实验检测肿瘤细胞的迁移、侵袭能力。结果乳腺微球体富集了CD44＋CD24-的乳腺癌干细胞,并能在含血清培养基中增殖分化。该微球体细胞的上皮标志物E-钙黏素表达水平下调,而间质标志物N-钙黏素、纤维连接蛋白、波形蛋白的表达水平上调,同时乳腺癌干细胞的迁移、侵袭能力显著增强。结论乳腺癌干细胞具有上皮-间质转化的特征,具有显著增强的迁移、侵袭能力。     

小细胞肺癌干细胞样细胞的分离鉴定

背景与目的:小细胞肺癌是高度侵袭的恶性肿瘤,患者的5年生存率不足10%,提示小细胞肺癌组织中可能富含较多的肿瘤干细胞.本研究设计从小细胞肺癌组织中原代培养肿瘤细胞并建立细胞系,从早期传代的细胞系中分离鉴定肺癌干细胞.方法:用无血清培养基原代培养肺癌细胞,传代后建立细胞系.应用流式细胞分析及分选技术,从细胞系第3代或第4代的细胞中寻找肺癌干细胞.应用单细胞克隆形成实验、平板集落形成试验及细胞球形成实验,研究肺癌干细胞的生物学特性.结果:应用原代细胞培养技术成功地从1例小细胞肺癌标本中培养出高纯度的原代肺癌细胞,其传代次数可达25代以上.流式细胞学分析显示该细胞系中有5.1%的细胞CD44呈强阳性表达(CD44++),相比于占主群的CD44弱阳性细胞(CD44+),CD44++细胞具有较强的平板集落形成能力,只有它们可以形成完全克隆(holoclone),在超低黏附的96孔培养板中可形成细胞球,而CD44+细胞不能形成完全克隆和细胞球.这些结果提示小细胞肺癌干细胞富集在CD44++细胞群中,而CD44+细胞中没有干细胞.应用CD44和CD90共同标记,可将小细胞肺癌细胞系中的细胞分为4群,分别是CD44+CD90-、CD44+CD90+、CD44++CD90-和CD44++CD90+细胞,其中CD44++CD90+细胞所占比例为1.9%.相比于其他细胞群,CD44++CD90+细胞形成细胞球的能力最强,提示CD90可能也是肺癌的干细胞标记.结论:小细胞肺癌细胞系中存在肿瘤干细胞样细胞,CD44和CD90可能是小细胞肺癌的干细胞标记.     

乳腺肿瘤干细胞分选的相关研究

“肿瘤干细胞”学说的提出为肿瘤包括乳腺肿瘤在内的治疗提供了新的思路。乳腺肿瘤干细胞正确的分选纯化是研究的关键,其分选主要历经侧群细胞分选、无血清悬浮培养、细胞表面分子标记分选、ALDEFLUOR试剂盒检测乙醛脱氢酶1高活性分选法4个阶段;由于化疗会富集肿瘤干细胞,部分学者把化疗耐药也作为一种分选的方法。目前有关乳腺肿瘤干细胞分选结果的争论主要集中在标志物CD44＋CD24-、ALDH1＋之间。哪种分选方法或标志物的选择更适宜分选乳腺肿瘤干细胞成为亟待解决的问题。     

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。     

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.     

Metformin-induced preferential killing of breast cancer initiating CD44+CD24-/low cells is sufficient to overcome primary resistance to trastuzumab in HER2+ human breast cancer xenografts

Trastuzumab-refractory breast cancer stem cells (CSCs) could also explain the high rate of primary resistance to single-agent trastuzumab in HER2 gene-amplified breast cancer patients. The identification of agents with strong selective toxicity for trastuzumab-resistant breast CSCs may have tremendous relevance for how HER2+ breast cancer patients should be treated. Using the human breast cancer cell line JIMT-1, which was established from the pleural metastasis of a patient who was clinically resistant to trastuzumab ab initio, we examined whether preferential killing of the putative CD44+CD24-/low breast CSC population might be sufficient to overcome primary resistance to trastuzumab in vivo. Because recent studies have shown that the anti-diabetic biguanide metformin can exert antitumor effects by targeted killing of CSC-like cells, we explored whether metformin's ability to preferentially kill breast cancer initiating CD44+CD24-/low cells may have the potential to sensitize JIMT-1 xenograft mouse models to trastuzumab. Upon isolation for breast cancer initiating CD44+CD24-/low cells by employing magnetic activated cell sorting, we observed the kinetics of metformin-induced killing drastically varied among CSC and non-CSC subpopulations. Metformin's cell killing effect increased dramatically by more than 10-fold in CD44+CD24-/low breast CSC cells compared to non-CD44+CD24-/low immunophenotypes. While seven-weeks treatment length with trastuzumab likewise failed to reduce tumor growth of JIMT-1 xenografts, systemic treatment with metformin as single agent resulted in a significant two-fold reduction in tumor volume. When trastuzumab was combined with concurrent metformin, tumor volume decreased sharply by more than four-fold. Given that metformin-induced preferential killing of breast cancer initiating CD44+CD24-/low subpopulations is sufficient to overcome in vivo primary resistance to trastuzumab, the incorporation of metformin into trastuzumab-based regimens may provide a valuable strategy for treatment of HER2+ breast cancer patients.     

食管癌耐阿霉素EC9706中干细胞富集及上皮间质化

目的:建立耐阿霉素(ADM)食管癌细胞株,命名为EC9706/ADM,探讨该细胞株的上皮间质化(EMT)及肿瘤干细胞富集现象.方法:用中等浓度间歇法建立食管癌耐阿霉素细胞株,MTT法测定细胞株对ADM、5-FU、顺铂、环磷酰胺的耐药指数.流式细胞仪检测EC9706/ADM及EC9706中CD44+CD24-细胞比例,MTT法、Transwell、平板克隆形成和球囊培养实验分别检测耐药对细胞增殖、迁移与转移、体外成瘤和克隆形成能力的影响;荧光定量PCR检测EC9706/ADM干细胞标志ALDH1、CD133及Oct4及EMT相关分子标志物E-钙黏蛋白及间质细胞分子标记N-钙黏蛋白、波形蛋白、纤维连接蛋白的表达.结果:EC9706/ADM的耐药指数为17.8,对5-FU、顺铂、环磷酰胺产生了交叉耐药性.与EC9706细胞相比,EC9706/ADM耐药细胞转移能力明显增强,表现出更高的微球体形成能力,E-cadherin表达降低而Fibronectin表达显著增高.结论:食管癌耐阿霉素细胞EC9706/ADM发生了EMT现象,转移迁移能力增强,细胞表现出更高的干细胞特性,化疗药物有助于肿瘤干细胞的富集.     

Hypoxia promotes the phenotypic change of aldehyde dehydrogenase activity of breast cancer stem cells

Stable breast cancer cell (BCC) lines are valuable tools for the identification of breast cancer stem cell (BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC‐derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase (ALDH) activity is just such a recognized biomarker of BCSCs with a CD44⁺/CD24^? phenotype. We isolated BCSCs with high ALDH activity (CD44⁺/CD24^?/Aldefluor^pos) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity (CD44⁺/CD24^?/Aldefluor^neg). Moreover, we found Aldefluor^pos BCSCs had a greater hypoxic response and subsequent induction of HIF‐1α expression compared to the Aldefluor^neg BCSCs. We also found that knocking down HIF‐1α, but not HIF‐2α, in Aldefluor^pos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial‐mesenchymal transition. Indeed, HIF‐1α overexpression in Aldefluor^neg BCSCs led to Slug and Snail mRNA increase and the associated repression of E‐cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluor^neg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes.