常见恶性肿瘤侧群细胞的研究进展

侧群细胞(SP)是指利用荧光染料Hoechst33342外排特性而筛选出来的细胞群体.SP具有肿瘤干细胞特性,在肿瘤的发生、发展中发挥极其重要的作用.目前国内外对于各类常见恶性肿瘤SP都进行了较为广泛的研究.对SP的研究可为肿瘤的诊断和治疗提供新的思路和方法.     

肺癌侧群细胞microRNA表达谱检测及初步分析

背景与目的 目前研究认为侧群细胞(side population cell,SP cell)富集了肿瘤干细胞,是肿瘤生长和发展的根源;并且SP细胞对放化疗高度耐受,成为肿瘤复发转移的重要因素.为此,我们探讨了肺癌SP细胞与非SP细胞(non-SP)miRNA分子表达谱差异,旨在为进一步研究miRNA在肺癌干细胞中的作用机制打下基础.方法 利用Hoechst 33342染料法,通过流式细胞仪紫外激光分选功能分选出A549细胞中的SP细胞和non-SP细胞,分别采用Trizol一步法提取两者的总RNA,利用miRNA芯片检测系统进行miRNA表达谱差异分析.结果 肺癌SP细胞和non-SP细胞miRNA表达谱存在明显差异.与non-SP细胞相比,在SP细胞中上调2倍以上的miRNA有9条,下调2倍以上的miRNA有25条.结论 差异表达的miRNA能参与肺癌干细胞的发生发展过程,为进一步揭示肺癌干细胞发生的分子机制提供了依据.     

侧群细胞研究进展与肿瘤治疗策略

肿瘤组织具有无限增殖的能力,这和干细胞具有众多的类似[1]。新近研究表明肿瘤细胞自我更新和无限增殖的根源是肿瘤干细胞的存在。肿瘤干细胞在肿瘤组织中只是极小的一部分,却有着和干细胞类似的自我更新能力,它们本身分裂次数很少,却能产生类似前体细胞的快速增长的子代细胞群体。它们还和干细胞一样,能经受得起在血管中的长途游走,在适合的部位繁衍起来。总之,肿瘤干细胞是肿瘤细胞的祖细胞,是肿瘤的真正种子,它们虽然仅占肿瘤细胞中极少的一部分,但具有自我更新能力和不定分化潜能,是形成不同分化程度肿瘤和肿瘤不断生长的根源,是肿瘤发生扩散、复发等过程中的起始细胞或动力细胞。传统的治疗方法不能有效的作用于肿瘤干细胞,开发针对肿瘤干细胞的靶向治疗能给肿瘤治疗模式带来全新的改变,有望彻底改善肿瘤患者的预后。1侧群细胞定义1996年,Goodell[2]等首次在小鼠骨髓中用流式细胞仪分离出一小群SP(S ide Popu lation)细胞,因有很强的外排染料的功能而呈现Hoechst33342低染(Hoechst33342是一种核酸结合染料,在紫外光激发下可发出蓝色和红色两种荧光),这群细胞不仅表达造血干细胞的表面标志,而且仅正常量的千分...     

人乳腺癌MCF-7细胞中SP亚群细胞的分离及其生物学行为研究

目的:了解肿瘤干细胞的生物学行为及相关的调控机制.方法:采用干细胞对Hoechst33342染料外排的特性进行MCF-7 细胞系肿瘤干细胞的流式分选.并对其增殖、自我更新、分化能力等生物学行为进行研究.还利用流式细胞仪、免疫荧光技术、荧光定量PCR等技术进行了与干细胞生物学行为密切相关的Wnt信号转导通路中相关分子的表达特征研究.结果:MCF-7细胞系中包含SP亚群(side population);且SP细胞具有分化潜能,高增殖能力,及自我更新特性;而且Wnt通路的各种关键分子在该细胞系SP亚群细胞中呈活化状态.结论:MCF-7细胞系的SP亚群可以代表该细胞系的肿瘤干细胞,Wnt通路在该肿瘤细胞系肿瘤干细胞的自我更新等其它生物学行为的调控中起重要作用.     

SGC-7901和MGC-803肿瘤干细胞中不同分子标志表达的观察

目的：探讨ABCG2、CD133、CK20、CyclinB1和DNA-PKcs在SGC-7901和MGC-803细胞SP（side population）亚群中的表达,探讨其为胃癌干细胞（cancer stemcell）标志的可行性。方法：用Hoe-chest33342染色检测胃癌细胞SGC-7901和MGC-803中SP细胞的比例;通过荧光免疫细胞化学染色比较ABCG2、CD133、CK20、Cyclin B1和DNA-PKcs在SP细胞与非SP细胞中表达的差异。结果：经Hoechst33342和荧光免疫细胞化学染色显示,SGC-7901细胞中SP细胞占3.8%;SP细胞中ABCG2和CK20表达阳性的细胞比率显著低于非SP细胞,P〈0.05;CD133、Cyclin B1和DNA-PKcs在SP细胞和非SP细胞中的表达差异无统计学意义,P〉0.05。经Hoechst33342和荧光免疫细胞化学染色显示,MGC-803细胞中SP细胞占2.9%;SP细胞中Cyclin B1的阳性表达率显著高于非SP细胞中的（P〈0.05）,但ABCG2、CD133、CK20及DNA-PKcs的阳性表达率显著低于非SP细胞中的,P〈0.05。结论：CK20^-/Cyclin B1^＋或CD133^＋可能是胃癌干细胞的特异性分子标志,而单项ABCG2、CD133、Cyclin B1及DNA-PKcs在鉴定胃癌干细胞时无特异性。     

人结肠癌ccl227细胞系肿瘤干细胞生长特性研究

目的：研究体外培养条件下人结肠癌ccl227细胞系肿瘤干细胞的生长特性。方法：用荧光染料Hoechst33342染色培养在SSM和SFM中的ccl227细胞,荧光显微镜观察SP细胞。用6孔板接种的方法绘制ccl227细胞的生长曲线。显微镜观察肿瘤球的生长。结果：在两种培养条件下均观察到ccl227细胞系中存在浅染和拒染的SP细胞。生长曲线显示在两种培养条件下,ccl227细胞的繁殖能力不同。在玻璃培养瓶和低吸附6孔板中均可以培育出肿瘤球,但形态有些不同。结论：SSM和SFM培养条件下,ccl227细胞中均存在SP细胞;在SFM中,ccl227细胞增殖缓慢;SFM可培育出富含肿瘤干细胞的肿瘤球。在低吸附条件下,更易形成悬浮的肿瘤球。     

SGO-7901和MGC-803肿瘤干细胞中不同分子标志表达的观察

目的:探讨ABCG2、CD133、CK20、CyclinB1和DNA-PKcs在SGC-7901和MGC-803细胞SP(side population)亚群中的表达,探讨其为胃癌干细胞(cancer stem cell)标志的可行性.方法:用Hoe-chest33342染色检测胃癌细胞SGC-7901和MGC-803中SP细胞的比例;通过荧光免疫细胞化学染色比较ABCG2、CD133、CK20、Cyclin B1和DNA-PKcs在SP细胞与非SP细胞中表达的差异.结果:经Hoechst 33342和荧光免疫细胞化学染色显示,SGC-7901细胞中SP细胞占3.8%;SP细胞中ABCG2和CK20表达阳性的细胞比率显著低于非SP细胞,P<0.05;CD133、Cyclin B1和DNA-PKcs在SP细胞和非SP细胞中的表达差异无统计学意义,P0.05.经Hoechst33342和荧光免疫细胞化学染色显示,MGC-803细胞中SP细胞占2.9%;SP细胞中Cyclin B1的阳性表达率显著高于非SP细胞中的(P<0.05),但ABCG2、CD133、CK20及DNA-Pkes的阳性表达率显著低于非SP细胞中的,P<0.05.结论:CK20-/Cyclin B1+或CD133+可能是胃癌干细胞的特异性分子标志,而单项ABCG2、CD133、Cyclin B1及DNA-PKcs在鉴定胃癌千细胞时无特异性.     

人肺癌A549细胞系肿瘤干细胞的筛选和鉴定

背景与目的肺癌干细胞是肺癌恶性表型的根源和潜在的治疗靶点,从人肺癌A549细胞株中分离肺癌干细胞,观察特异性干细胞标志物分子的表达,为进一步的干细胞研究提供试验基础。方法接种肺癌A549细胞株,经流式细胞术,特异性筛选分离肺癌干细胞,观察克隆形成能力、细胞增殖能力和体外致瘤能力的差别,并分别用RT-PCR和Westernblot的方法分析干细胞标志物分子CD133和ABCG2的表达。结果经过流式细胞仪成功分选了人肺腺癌A549细胞系SP细胞亚群,结果表明此SP细胞亚群约占A549细胞总数的5.93%,经维拉帕米处理后Hoechest33342阴性/弱阳性细胞含量下降为0.32%,SP细胞克隆形成能力,细胞增殖能力和体外致瘤能力均明显高于非SP细胞。RT-PCR和Westernblot结果发现,筛选分离的肺癌SP细胞群高表达干细胞标志物分子CD133和ABCG2。结论通过流式细胞术可以筛选分离高表达CD133和ABCG2分子的肺癌干细胞,可用于进一步的研究中。     

人小细胞肺癌细胞株H446侧群细胞的生物学特征

背景与目的:肿瘤干细胞学说的提出为肿瘤治疗提供了新的靶点和方向,但肿瘤干细胞的分离纯化一直是个难题。本研究拟从人小细胞肺癌细胞株H446中分离并鉴定出具有干细胞特性的侧群(SP)细胞,研究其生物学特征,为肿瘤干细胞的分离纯化奠定基础。方法:采用荧光激活细胞分选(FACS)技术分选得到H446细胞中SP细胞和非侧群(NSP)细胞,并检测纯度。观察形成悬浮肿瘤细胞球的能力,采用逆转录-聚合酶链反应(RT-PCR)及荧光定量PCR检测这两种细胞亚群中CD133、ABCG2、NucleosteminmRNA水平。MTT法比较SP细胞、NSP细胞及未分选细胞体外增殖能力及耐药性差异,流式细胞仪检测体外分化能力,裸鼠成瘤实验检测体内成瘤能力。结果:荧光显微镜下H446细胞中Hoechst33342阴性细胞约为(5.1±0.2)%。流式细胞分选结果显示,H446中SP细胞比例为(6.3±0.1)%。SP细胞在无血清培养基中形成悬浮肿瘤细胞球的能力强于NSP细胞。CD133、ABCG2在SP细胞中的表达是NSP细胞的(21.60±0.26)倍、(7.10±0.14)倍,差异有统计学意义(P<0.01);Nucleostemi...     

人脑胶质瘤干细胞SHG-44s的克隆及初步鉴定

目的:探讨人脑胶质瘤体外细胞系中存在肿瘤干细胞的可能性.方法:将SHG44细胞系和SHG44-9细胞株,分别应用含血清培养基(DMEM 10?S)和无血清培养基(DMEM/F12,添加bFGF、LIF和EGF)培养.用CD133免疫磁珠分离干细胞、Hoechst33342和NESTIN流式细胞仪、Nestin、NSE、GFAP免疫细胞化学染色检测肿瘤干细胞及其分化细胞.结果:CD133磁珠分离得到的干细胞的比例:在血清组SHG44和SHG44-9分别为0.021%和0.035%:无血清组分别为1.2%和2.3%.而Hoechst33342和CD133标记后流式细胞仪检测干细胞比例:血清组中SHG44、SHG44-9分别为1.5%、0.37%;无血清组分别为16.4%和29.1%.并且这些细胞能够增殖和分化成神经元和胶质细胞.而Nestin标记后SHG44、SHG44-9中分别有51.05%、77.53%阳性细胞.结论:体外长期传代培养的人脑胶质瘤细胞系中存在肿瘤干细胞SHG44s,CD133磁珠和Hoechst33342流式细胞仪分离和检测胶质瘤干细胞是行之有效的方法,而Nestin 细胞是干细胞分化后的祖细胞或前体细胞,不能作为分离和检测干细胞特异性标记物使用.     

Fluorouracil selectively enriches stem-like cells in the lung adenocarcinoma cell line SPC

Most adult stem cells are in the G0 or quiescent phase of the cell cycle and account for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. This study sought to enrich cancer stem cells and explore cancer stem-like cell clones using 5-fluorouracil (5-FU) in the lung adenocarcinoma cell line, SPC. Proliferation inhibition was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, according to which half maximal inhibitory concentration values were calculated. Expression levels of stem cell markers after treatment with 5-FU were examined using immunofluorescence and Western blotting. Additionally, side population (SP) cells were sorted using FACS. Properties of SP cells were evaluated by using Transwell, colony-forming assays, and tumor formation experiments. 5-FU greatly inhibits proliferation, especially of cells in S phase. SP cells possess greater invasive potential, higher clone-forming potential, and greater tumor-forming ability than non-SP cells. Treatment with 5-FU enriches the SP cells with stem cell properties in human lung adenocarcinoma cell lines.     

Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer

Background:

Side population (SP) fraction cells, identified by efflux of Hoechst dye, are present in virtually all normal and malignant tissues. The relationship between SP cells, drug resistance and cancer stem cells is poorly understood. Small-cell lung cancer (SCLC) is a highly aggressive human tumour with a 5-year survival rate of <10%. These features suggest enrichment in cancer stem cells.

Methods and results:

We examined several SCLC cell lines and found that they contain a consistent SP fraction that comprises <1% of the bulk population. Side population cells have higher proliferative capacity in vitro, efficient self-renewal and reduced cell surface expression of neuronal differentiation markers, CD56 and CD90, as compared with non-SP cells. Previous reports indicated that several thousand SP cells from non-small-cell lung cancer are required to form tumours in mice. In contrast, as few as 50 SP cells from H146 and H526 SCLC cell lines rapidly reconstituted tumours. Whereas non-SP cells formed fewer and slower-growing tumours, SP cells over-expressed many genes associated with cancer stem cell and drug resistance: ABCG2, FGF1, IGF1, MYC, SOX1/2, WNT1, as well as genes involved in angiogenesis, Notch and Hedgehog pathways.

Conclusions:

Side population cells from SCLC are highly enriched in tumourigenic cells and are characterised by a specific stem cell-associated gene expression signature. This gene signature may be used for development of targeted therapies for this rapidly fatal tumour.     

肺腺癌细胞株SPC-A1中侧群细胞的分离与肿瘤干细胞样特征的鉴定

背景与目的近来已有充分的研究表明在实体肿瘤中存在肿瘤干细胞,肿瘤干细胞的发现改变了我们对肿瘤发生机制及化疗耐药的观点。本文拟从人肺腺癌细胞株中分离并鉴定具有干细胞特征的肿瘤干细胞样细胞。方法采用无血清培养结合流式细胞术分离人肺腺癌细胞株中的边群细胞(SP,side population),通过增殖能力、克隆形成、裸鼠成瘤及表型分析鉴定其干细胞特性。结果肺腺癌细胞株中存在SP细胞,经无血清培养后,SP细胞的比例显著提高,SP细胞具有高增殖活性、高克隆形成率及强致瘤性,具有肿瘤干细胞的特性。结论人肺腺癌细胞株中存在具有肿瘤干细胞性质的SP细胞,无血清培养可以富集肺腺癌细胞株中的SP细胞。     

The polycomb gene product BMI1 contributes to the maintenance of tumor-initiating side population cells in hepatocellular carcinoma

Side population (SP) cell analysis and sorting have been successfully applied to hepatocellular carcinoma (HCC) cell lines to identify a minor cell population with cancer stem cell properties. However, the molecular mechanisms operating in SP cells remain unclear. The polycomb gene product BMI1 plays a central role in the self-renewal of somatic stem cells in a variety of tissues and organs and seems to be implicated in tumor development. In this study, we determined the critical role of BMI1 in the maintenance of cancer stem cells with the SP phenotype in HCC cell lines. BMI1 was preferentially expressed in SP cells in Huh7 and PLC/PRF/5 HCC cells compared with the corresponding non-SP cells. Lentiviral knockdown of BMI1 considerably decreased the number of SP cells in both Huh7 and PLC/PRF/5 cells. Long-term culture of purified SP cells resulted in a drastic reduction in the SP subpopulation upon the BMI1 knockdown, indicating that BMI1 is required for the self-renewal of SP cells in culture. More importantly, the BMI1 knockdown abolished the tumor-initiating ability of SP cells in nonobese diabetic/severe combined immunodeficiency mice. Derepression of the INK4A and ARF genes that are major targets for BMI1 was not necessarily associated with impaired self-renewal of SP cells caused by BMI1 knockdown. In conclusion, our findings define an important role for BMI1 in the maintenance of tumor-initiating SP cells in HCC. BMI1 might be a novel therapeutic target for the eradication of cancer stem cells in HCC.     

Isolation and characterization of cancer stem-like side population cells in human oral cancer cells

Recent studies suggest that cancer stem cells may be responsible for tumorigenesis and contribute to some individuals’ resistance to cancer therapy. Some studies demonstrate that side population (SP) cells isolated from diverse cancer cell lines harbor stem cell-like properties; however, there are few reports examining the role of SP cells in human oral cancer. To determine whether human oral cancer cell lines contain a SP cell fraction, we first isolated SP cells by fluorescence activated cell sorting, followed by culturing in serum-free medium (SFM) using the SCC25 tongue cancer cell line, so that SP cells were able to be propagated to maintain the CSC property. Differential expression profile of stem cell markers (ABCG2, Oct-4 and EpCAM) was examined by RT-PCR in either SP cells or non-SP cells. Growth inhibition by 5-FU was determined by the MTT assay. Clonogenic ability was evaluated by colony formation assay. SCC25 cells contained 0.23% SP cells. The fraction of SP cells was available to grow in SFM cultures. SP cells showed higher mRNA expression of stem cell markers (ABCG2, Oct-4 and EpCAM) as compared with non-SP cells. Moreover, SP cells demonstrated more drug resistance to 5-FU, as compared with non-SP cells. The clone formation efficiency of SP cells was significantly higher than non-SP cells at an equal cell number (P < 0.01). We isolated cancer stem-like SP cells from an oral cancer cell line. SP cells possessed the characteristics of cancer stem cells, chemoresistance, and high proliferation ability. Further characterization of cancer stem-like SP cells may provide new insights for novel therapeutic targets.     

Identification of cancer stem cells in the "side population"

Both normal somatic stem cells and cancer cells are thought to be capable of unlimited proliferation. Paradoxically, however, some cancers seem to contain stem-like cells (cancer stem cells). There is increasing evidence that cancers might contain their own stem cells. Many cancers, like normal organs, seem to be maintained by a hierarchical organization that includes slowly dividing stem cells,rapidly dividing transit amplifying cells (precursor cells), and differentiated cells. Malignant gliomas, for example,often contain both undifferentiated and differentiated cells and sometimes contain cells that express neuronal markers as well as cells that express glial markers, suggesting that they may contain multipotent neural stem cell-like cells. We have shown that some cancer cell lines contain a small side population (SP), which, in many normal tissues, is thought to contain the stem cells of the tissue. We provide evidence that SP cells in the C6 glioma cell line can produce both neurons and glial cells and thus have cancer stem cell property. Taken together with studies on normal neural stem cells, studies on cancer stem cells will help us to understand a link between normal stem cells and cancer stem cells.     

Novel antigens in non-small cell lung cancer: SP17, AKAP4, and PTTG1 are potential immunotherapeutic targets

Lung cancer is the leading cause of cancer deaths in both genders worldwide, with an incidence only second to prostate cancer in men and breast cancer in women. The lethality of the disease highlights the urgent need for innovative therapeutic options. Immunotherapy can afford efficient and specific targeting of tumor cells, improving efficacy and reducing the side effects of current therapies. We have previously reported the aberrant expression of cancer/testis antigens (CTAs) in tumors of unrelated histological origin. In this study we investigated the expression and immunogenicity of the CTAs, Sperm Protein 17 (SP17), A-kinase anchor protein 4 (AKAP4) and Pituitary Tumor Transforming Gene 1 (PTTG1) in human non-small cell lung cancer (NSCLC) cell lines and primary tumors. We found that SP17, AKAP4 and PTTG1 are aberrantly expressed in cancer samples, compared to normal lung cell lines and tissues. We established the immunogenicity of these CTAs by measuring CTA-specific autoantibodies in patients'' sera and generating CTA-specific autologous cytotoxic lymphocytes from patients'' peripheral blood mononuclear cells. Our results provide proof of principle that the CTAs SP17/AKAP4/PTTG1 are expressed in both human NSCLC cell lines and primary tumors and can elicit an immunogenic response in lung cancer patients.     

Endosialin: a novel malignant cell therapeutic target for neuroblastoma

Endosialin emerged recently as a potential therapeutic target for sarcoma. Since some sarcoma subtypes, such as Ewing's sarcoma, show characteristics of neuroendocrine differentiation, we wondered whether cancers with neuro-endocrine properties and/or neuroectodermal origin, such as neuroblastoma, small cell lung cancer and melanoma, may express endosialin. Endosialin protein expression was surveyed in neuroblastoma, small cell lung cancer and melanoma in human clinical specimens by immunohistochemistry (IHC) and in human cell lines by flow cytometry. Side population cells were examined to determine whether cancer stem cells can express endosialin. Endosialin-expressing neuroblastoma cell lines were implanted in immunodeficient mice and allowed to grow. The xenograft tumors were resected and tested for endosialin expression by IHC. In human clinical specimens, vascular endosialin staining was observed in neuroblastoma, small cell lung cancer and melanoma. Malignant cell staining was strongest in neuroblastoma, weak in melanoma and rare in small cell lung cancer. In human cell lines, endosialin was detected in neuroblastoma cell lines, including cancer stem cell-like side population (SP) cells, but was absent in melanoma and was both rare and weak in small cell lung cancer. Human neuroblastoma xenograft tumors were found to be positive for endosialin. Our work suggests that endosialin may be a suitable therapeutic target for neuroblastoma.     

卵巢癌SKOV3细胞中侧群细胞的生物学特性

目的 分选人卵巢癌细胞系SKOV3中的侧群（side population, SP）细胞,并探讨其是否具有肿瘤干细胞的生物学特性。方法 流式细胞仪检测、分选经DNA染料Hoechst 33342染色后SKOV3中的SP细胞,并对侧群细胞（SP）与非侧群细胞（non-SP）作细胞生物学鉴定,包括增殖能力、克隆形成能力、侵袭及迁移能力、自我更新能力及细胞周期。结果 SKOV3细胞系中SP细胞比例为（1.12±0.104）%,SP细胞增殖速度快于non-SP细胞,差异有统计学意义（P<0.05）。接种相同数量的细胞,SP细胞克隆形成率高于non-SP细胞（P<0.05）。Transwell实验显示,SP细胞侵袭与迁移的细胞数与non-SP相比均明显增多（P<0.05）。SP细胞体外能分化为Non-SP细胞,具有自我更新能力。SP细胞大多处于细胞周期的G0/G1期。结论 卵巢癌细胞系SKOV3中的SP细胞具有肿瘤干细胞的生物学特性,SP细胞表型可考虑作为富集卵巢癌干细胞样细胞的有效方法。