条件性重编程技术分离培养人原代乳腺癌干细胞

目的 应用条件性重编程细胞(Conditionally Reprogrammed Cells,CRCs)技术建立高效、稳定的人乳腺癌千细胞体外培养,建立潜在的乳腺癌干细胞靶向药研究模型.方法 收集人乳腺癌组织样本,采用CRCs技术分离培养原代乳腺癌细胞,用CD24免疫磁珠标记原代乳腺癌细胞后使用磁性细胞分选仪分选出乳腺...     

ErbB-2转基因小鼠乳腺癌干细胞的分离培养

目的:分离、培养ErbB-2转基因小鼠乳腺癌干细胞(BRCSCS),探讨其生物学特性及培养传代条件,为药敏试验及动物实验进行基础性研究。方法:从ErbB-2转基因小鼠乳腺癌组织中获取乳腺癌细胞(BRCCS),然后以FITC标记的CD24、CD44抗体标记细胞,通过流式细胞仪检测并分选CD44+CD24-/low细胞。结果:Erb-B2转基因小鼠乳腺癌组织中CD44+CD24-/low细胞约占细胞总数的2.6%(2.3%～2.9%)。结论:小鼠乳腺癌组织中存在CD44+CD24-/low且具有干细胞特性的BRCCS,利用流式细胞仪可成功地将其从小鼠乳腺癌组织中分离出来。     

乳腺癌干细胞VEGF表达的实验研究

目的探讨乳腺癌干细胞血管内皮生长因子（Vascular endothelial growth factor,VEGF）的表达及意义。方法应用流式细胞分选技术从乳腺癌细胞株MCF-7细胞分离乳腺癌干细胞和非干细胞,采用Northern blot技术测定干细胞与非干细胞VEGF表达情况。结果与乳腺癌非干细胞相比,乳腺癌干细胞VEGF表达水平明显增强。结论乳腺癌干细胞可通过高表达VEGF增强转移能力,是乳腺癌转移的关键因素之一。     

自身免疫病患者外周血CD34+细胞动员、采集和纯化研究

目的：探讨自身免疫病患外周血造血干细胞动员、采集和纯化的效果及安全性。方法：对17例确诊自身免疫病患予环磷酰胺＋粒细胞集落刺激因子（G－CSF）动员外周血干细胞，CliniMACS系统对其中11例患外周血单个核细胞进行CD34^ 细胞阳性分选，流式细胞仪检测分选前后CD34^ 细胞纯度及淋巴细胞各亚群。结果：1例动员中并发尿崩症，2例动员失败。11例分选后CD34^ 细胞纯度平均96．1％，回收率70．14％；CD3^ 、CD19^ 、CD14^ 细胞分别去除10^3/kg,10^2/kg,10^3/kg。结论：对自身免疫病患采用环磷酰胺＋G－CSF方案动员外周血干细胞方法可靠，安全性好；外周血干细胞的纯化方法可获得高纯度和高回收率的CD34^ 细胞，并极大限度的去除T、B等细胞。     

乳腺癌干细胞RNA-DCs疫苗诱导特异性细胞毒性T淋巴细胞抗肿瘤免疫应答

目的制备负载乳腺癌干细胞RNA的树突状细胞(DCs)疫苗,研究其诱导的特异性细胞毒性T淋巴细胞(CTLs)的抗肿瘤免疫反应。方法采用细胞毒性试剂盒检测2种乳腺癌干细胞疫苗[(A组,CD44+CD24-+MCF-7-CTLs)、(B组,MCF-7-CTLs)]和DC-CTLs(C组)的体外细胞杀伤能力。取24只小鼠均分为四组:A1组皮下接种活化的A组疫苗+MCF-7乳腺癌细胞;B1组接种活化的B组疫苗+MCF-7乳腺癌细胞;C1组皮下注射活化的DCs-CTLs+MCF-7乳腺癌细胞;D1组单用MCF-7乳腺癌细胞作为对照。分析裸鼠接种后肿瘤在体内的生长情况。结果体外对CD44+CD24-+MCF-7乳腺癌细胞杀伤能力强度:A组>B组>C组(P<0.05)。体外对MCF-7乳腺癌细胞杀伤能力强度:B组>A组>C组(P<0.05)。在体成瘤实验显示,B1组和A1组的成瘤时间分别为第7周和第6周,明显长于D1组(第1周)和C1组(第2周)(P<0.05)。结论 CD44+CD24-乳腺癌干细胞RNA-DCs疫苗诱导的CTLs能够产生特异性免疫应答,从而抑制乳腺癌细胞成瘤。     

阿霉素对乳腺癌干细胞的体外作用研究

目的通过实验观察研究阿霉素对于乳腺癌干细胞的病理药理的作用,从而来研究乳腺癌细胞系MCF-7干细胞通过使用了药物阿霉素在其体外所产生的变化,来选择乳腺癌干细胞更有针对性的治疗方案和研究比较更加行之有效的药物。方法使用流式细胞仪器,在乳腺癌细胞系(MCF-7),剥离乳腺癌干细胞CD24(-/low)、CD44(+)、ESA(+)。在浓度不同的阿霉素培养液中放置乳腺癌干细胞与没有进行分拣的细胞,测得阿霉素对不同干细胞的抑制生长生,进行细胞凋亡及克隆形成能力的分析。结果 MTT表明乳腺癌干细胞具有耐药性。药物阿霉素呈浓度依赖型抑制了乳腺癌未分类细胞及干细胞:未分类细胞IC50为5.5μmol/L;而分选出的干细胞IC50为29.1μmol/L(P<0.001)。乳腺癌干细胞在使用药物阿霉素可加速病变的恢复,随着用药时间的增加其效果愈加明显。研究中还发现,乳腺癌干细胞在阿霉素的作用下可减少其形成和扩散。结论乳腺癌干细胞在使用了药物阿霉素成瘤性有所降低,对于其克隆能力具有抑制作用,其变化呈时间依赖型与浓度依赖型。     

系统性红斑狼疮患者血清IL-6对造血干细胞分化发育为树突状细胞的影响

目的 观察系统性红斑狼疮(SLE)患者血清中的白细胞介素6(IL-6)对脐血CD34 造血干细胞(HPC)体外诱导分化为树突状细胞(DCs)的影响.方法 淋巴细胞分离液分离获得脐血单个核细胞,免疫磁珠阳性分选CD34 HPC,采用粒-巨噬细胞集落刺激因子(GM-CSF) IL- 4 肿瘤坏死因子α(TNF-α)方案体外诱导分化形成DCs,加入高IL-6水平的SLE患者血清观察对细胞分化的影响.在培养的7、10和14d收集细胞,流式细胞术检测表型,细胞增殖/毒性(CCK-8)试剂盒分析DCs刺激同种异体淋巴细胞增殖的能力.结果 在高IL-6水平SLE血清的作用下,由HPC分化而来的DCs表达CD80、CD86上升,刺激同种异体淋巴细胞增殖的能力增强;IL-6中和抗体能够降低这种DCs的CD80、CD86表达水平和对同种异体淋巴细胞增殖的刺激能力.结论 SLE血清中的IL-6对造血干细胞向DCs分化、发育及DCs功能的活化可能起促进作用.     

乳腺癌干细胞富集方法的初步研究

目的通过培养和富集乳腺癌细胞系及其干细胞,探讨不同乳腺癌细胞系中CD44+CD24-/low细胞比例,并分离鉴定乳腺癌干细胞。方法培养两种乳腺癌细胞系MCF-7、MDA-MB-231,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;通过无血清悬浮培养,观察其是否形成细胞球,再联合化疗筛选,富集乳腺癌干细胞。结果无血清悬浮培养,MDA-MB-231细胞未发现明显细胞球,MCF-7培养约6d出现细胞球;加入化疗药后,MCF-7细胞成球周期短至4d。流式细胞仪检测,MCF-7、MDA-MB-231两种细胞系中CD44+CD24-/low细胞比例分别2.07%和0.20%。结论利用CD44+CD24-/low标记流式细胞仪检测乳腺癌干细胞,简单有效;无血清悬浮培养,能富集乳腺癌干细胞,联合化疗双重筛选,能提高富集效率。     

miR-486对CD133+胶质瘤干细胞的影响

目的 研究miR-486对CD133 胶质瘤干细胞的影响。方法 CD133抗体标记U87胶质瘤细胞系,流式细胞分选纯化CD133 胶质瘤干细胞,实时定量PCR检测CD133 胶质瘤干细胞中miR-486的表达量。脂质体转染方法构建过表达miR-486的CD133 胶质瘤干细胞,MTT法检测其凋亡率,流式分析仪检测细胞增殖率与生长周期。结果 流式分选后CD133 胶质瘤干细胞纯度达到实验要求,CD133 胶质瘤细胞中miR-486表达量较CD133-胶质瘤细胞中下降（P<0.01）,转染miR-486模拟物的CD133 胶质瘤干细胞较转染miRNA对照模拟物的CD133 胶质瘤干细胞相对增殖率降低,细胞周期检测对照组细胞位于G0/G1期的比例低于转染miR-486后的细胞（P<0.05）,转染miR-486后细胞凋亡率显著提高（P<0.001）。结论CD133 胶质瘤干细胞中miR-486表达量减少,过表达miR-486的CD133 胶质瘤干细胞增殖受到抑制,生长周期被阻滞于G1/S期,凋亡率显著提高,可能是胶质瘤靶向治疗中队胶质瘤干细胞的有效靶点。     

人脐带血造血干细胞体外分化为NK细胞的效率及功能检测

目的检测人脐带血造血干细胞(HSCs)体外分化为自然杀伤(NK)细胞的效率及功能。方法从人脐带血中分离CD34+HSCs,接种于含20μg/L FMS样酪氨酸激酶3配体(Flt3L)、干细胞生长因子(SCF)、白细胞介素(IL)-7、IL-15及IL-21的SCGM培养基中,定向诱导CD34+HSCs分化为NK细胞。观察细胞生长状态,在分化的第7、14、21及28天,采用流式细胞术检测各阶段CD56、NKG2D、NKp46、CD3、CD19及CD34等细胞免疫表型的表达变化;分化的第21、28天,以分化得到细胞为效应细胞,K562细胞作为靶细胞,设置8∶1、4∶1、2∶1和1∶1 4组效靶细胞比,分别采用乳酸脱氢酶(LDH)细胞毒性检测法和7AAD/CFSE标记法,检测分化得到的细胞杀伤功能。结果脐带血来源的CD34+HSCs在体外适宜的条件下可大量增殖;整个分化进程的不同阶段中,CD3和CD19表达量差异无统计学意义,CD56、NKG2D及NKp46的表达量逐渐增加,最高达(72.57±1.60)%、(32.83±1.29)%和(29.53±2.40)%,CD34的表达量逐渐降低,最低为(12.13±2.01)%。LDH毒性检测法和7AAD/CFSE标记法测得最大杀伤效率可达(49.91±2.76)%和(40.87±1.12)%,8∶1、4∶1、2∶1和1∶1组NK细胞杀伤能力均依次降低(P<0.05),诱导分化28 d的NK细胞杀伤能力与21 d差异无统计学意义。结论人脐带血HSCs在体外适宜的培养条件下,可定向分化为NK细胞,诱导分化得到的NK细胞具有杀伤功能。     

纳米雄黄对MCF-7乳腺癌干细胞的体外抑制作用

目的 研究纳米雄黄对乳腺癌干细胞的体外抑制作用.方法 以人乳腺癌MCF-7亲本细胞为对象,采用无血清培养法培养获得乳腺癌干细胞.以阿霉素(1 mg/L)为阳性对照,以相同质量浓度的水飞雄黄为参照,采用CCK-8法检测纳米雄黄对MCF-7亲本细胞及干细胞增殖的影响;借助悬浮球形成及分化实验、划痕实验、Transwell侵...     

Reversing breast cancer stem cell into breast somatic stem cell

Stem cells have an important role in cell biology, allowing tissues to be renewed by freshly created cells throughout their lifetime. The specific micro-environment of stem cells is called stem cell niche; this environment influences the development of stem cells from quiescence through stages of differentiation. Recent advance researches have improved the understanding of the cellular and molecular components of the micro-environment--or niche--that regulates stem cells. We point out an important trend to the study of niche activity in breast cancers. Breast cancer has long been known to conserve a heterogeneous population of cells. While the majority of cells that make up tumors are destined to differentiate and eventually stop dividing, only minority populations of cells, termed cancer stem cell, possess extensive self renewal capability. These cancer stem cells possess characteristics of both stem cells and cancer cells. Breast cancer stem cells reversal to breast somatic stem cells offer a new therapy, that not only can stop the spread of breast cancer cells, but also can differentiate breast cancer stem cells into normal breast somatic stem cells. These can replace damaged breast tissue. Nevertheless, the complexity of realizing this therapy approach needs further research.     

槐耳颗粒联合DC-CIK细胞对乳腺癌MDA-MB-231干细胞体内外杀伤作用研究

目的 探究白屈菜红碱（CHE）对腺样囊性癌细胞（ACC2）生长的抑制作用及机制。方法 利用CCK8法、EdU法、Hoechst33342/PI双染色法、试剂盒法检测CHE对ACC2细胞活力、细胞增殖、细胞凋亡和活性氧（ROS）水平的影响;通过Western blotting技术检测CHE对Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达的影响;利用斑马鱼移植瘤模型检测CHE对斑马鱼体内ACC2细胞生长的抑制作用。结果 CCK-8结果显示：与对照组比较,2、3、4、5、6、7、8、9、10 μmol/L的CHE显著降低ACC2细胞的存活率（P<0.05、0.01）,且呈浓度相关性; ROS检测结果显示：与对照组比较,5、8 μmol/L的CHE导致ACC2细胞内的ROS水平显著上升（P<0.05、0.01）; EdU增殖检测结果表明：与对照组比较,5、8 μmol/L的CHE致使ACC2细胞的增殖能力显著下降（P<0.01）;Hoechst/PI染色结果显示：与对照组比较,CHE 5、8 μmol/L组ACC2细胞凋亡率显著上升（P<0.01）。抗氧化剂N-乙酰半胱氨酸（NAC）显著抑制CHE诱导的ROS水平升高、细胞凋亡增加（P<0.01）;Western blotting结果显示：2、5、8 μmol/L的CHE能够显著上调Cleaved-Caspase 3、PARP、NF-κB蛋白的表达（P<0.01）,且呈现浓度相关性,5、8 μmol/L的CHE能够显著上调p-JNK的蛋白表达（P<0.01）,8 μmol/L的CHE能够显著上调p-p38的蛋白表达（P<0.01）;NAC显著降低由CHE导致的Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达增加（P<0.01）,5、8 μmol/L CHE能够有效抑制斑马鱼体内肿瘤的生长（P<0.01）。结论 体外及斑马鱼移植瘤模型证明,CHE可以有效抑制ACC2细胞生长,其机制与提高细胞ROS水平,上调NF-κB、p-JNK、p-p38表达,从而抑制细胞增殖、诱导细胞凋亡相关。     

Curcumin: a calixarene derivative micelle potentiates anti-breast cancer stem cells effects in xenografted,triple-negative breast cancer mouse models

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease with no approved targeted therapy. Curcumin has shown therapeutic potential against TNBC, but it shows low bioavailability and low efficacy when administered as a free drug. Here we describe a novel vehicle for in vivo delivery of curcumin based on the phosphorylated calixarene POCA4C6. Curcumin-loaded POCA4C6 micelles (CPM) were prepared using the thin-film method and they showed a unilamellar structure with an average particle size of 3.86?nm. The micelles showed high curcumin encapsulation efficiency and loading was based on liquid chromatography-tandem mass spectrometry. Studies with cell cultures suggest that CPM can sustainably release curcumin in a pH-dependent manner. The micelles efficiently inhibited proliferation, invasion, migration and tumor spheroid formation by BT-549 human breast cancer cells. These effects involved increased apoptosis and reduced levels of nuclear β-catenin and androgen receptor. After injection into tumor xenografts, CPM persisted in the tumor tissue and efficiently inhibited tumor growth without causing obvious systemic toxicity. CPM also significantly reduced levels of CD44⁺/CD133⁺ breast cancer stem cells. Our results highlight the potential of CPM as an effective therapy against TNBC.     

负载抗原DC与CIK共培养对富集培养乳腺癌CTCs杀伤作用研究

[Abstract]ObjectiveTo study the kill activity of the whole tumor cell antigen (Ag) pulsed dendritic cell (DC) coculture with cytokine induced killer cell (CIK) for breast cancer circulating tumor cells (CTCs).Methods Peripheral blood mononuclear cells (PBMC) were isolated from breast cancer patients with CTCs using a blood cell separator instrument. The epidermal cell adhesion molecule (EpCAM ) (+) breast cancer CTCs enriched by MACS were cultured in vitro. The nested RT-PCR, cell immunofluorescence imaging (CK8/18), and immunohistochemistry (CK8/18and CK19) methods, were used for the detection and identification of the cells. EpCAM (-) cells were routinely induced to DC and CIK, which were grouped into Ag-DC-CIK group, DC-CIK group, DC group and CIK group. The cytotoxic activity of co-cultured DC-CIK against breast cancer CTCs was detected by flow cytometry and MTT assay. The cell morphology was observed by light microscopy and transmission electron microscopy.ResultsThe target band of CK19mRNA can be detected by nested RT-PCR. The expressions of CK8/18and CK19of EpCAM (+) cells in vitro were positive by immunofluorescence staining and immunohis?tochemical staining. The proliferative activity of co-cultured DC-CIK was higher than that of CIK (P＜0.001). The positive rates of CD1α+, CD83+CD86+, CD83+CD11C+, CD86+CD11C+DCs and CD3+CD8+, CD3+CD56+CIKs were significantly higher in Ag- DC- CIK group than those of DC- CIK group, DC group and CIK group (P＜0.05). The apoptosis of breast cancer CTCs was induced in Ag-DC-CIK, DC-CIK and CIK3groups, and apoptotic rates were (56.83±3.07)%,(31.43±1.77)% and (24.70±1.51)%, showing significant differences between them (P＜0.05). Transmission electron microscopy showed the typical micro-structure of breast cancer CTCs induced apoptosis.ConclusionMACS in combination with cell immunological methods can improve significantly the detective sensitivity of breast cancer CTCs. The co-cultured Ag-DC-CIK is highly effective immune cells,which shows a high er proliferation and cytoxicty against breast cancer CTCs. This may be used as a clinically immunotherapy means of anti-breast cancer recurrence and metastasis.