人卵巢癌组织肿瘤干细胞的分离和鉴定

目的 从人卵巢癌组织中分离并鉴定卵巢癌干细胞.方法 取新鲜卵巢癌组织,以胶原酶和透明质酸酶充分消化,获得单细胞悬液,经红细胞裂解液处理,CD133磁珠孵育进行磁性细胞分选,流式细胞仪验证分选效率.将阳性细胞置于含人表皮生长因子、碱性成纤维细胞生长因子、牛血清白蛋白和人胰岛素的无血清培养基中,待细胞成球状生长后进行免疫荧光、分化实验和成瘤实验,鉴定其干细胞特性.结果 从人卵巢癌组织中成功分离出肿瘤干细胞,流式细胞仪检测其CD133的表达率为82.5％.该细胞在无血清培养基中呈悬浮球状生长,具有自我更新能力、分化潜能和更强的致瘤能力,具备干细胞特性.结论 人卵巢癌组织中存在卵巢癌干细胞,利用CD133磁珠分选得到的肿瘤干细胞具备干细胞的相关特性,可用于后续卵巢癌干细胞生物学特性的研究.     

纳米材料应用于卵巢癌细胞分离和检测的研究进展

近年来,纳米材料凭借其独特的光、声、电、磁、热和力学特性在生物医学领域中得到广泛应用,也为卵巢癌的早期诊断、精确分期和肿瘤病灶的定位带来福音。在卵巢癌细胞分离中,修饰靶向分子后的磁性纳米材料能特异性地捕获卵巢癌细胞,并在外加磁场作用下将其分离,为卵巢癌患者体内扩散的癌细胞的检测和清除提供了技术手段;在卵巢癌检测技术中,纳米材料制成的生物传感器能够有效提高卵巢癌患者样本分析的效率、选择性及特异性;纳米材料制成的造影剂不仅能同时适用于多种成像技术,还可以提高其对卵巢癌组织的成像灵敏度与分辨率,实现卵巢癌的分子成像;纳米材料与质谱分析结合后,可有效提高其检测的灵敏度,在卵巢癌的蛋白组学分析中发挥更好的作用。     

人恶性胶质瘤干细胞的分离与鉴定

目的:从人恶性胶质瘤中分离并鉴定胶质瘤干细胞。方法:采用神经干细胞无血清培养方法,分离胶质瘤干细胞,免疫细胞化学法、有限梯度稀释实验、二代球体形成分析、流式细胞分析等方法对胶质瘤干细胞进行鉴定。结果:3例患者原代培养胶质瘤细胞中CD133 细胞分别为7.4%、2.8%和4.2%。每100个原代胶质瘤细胞中可形成1个左右的胶质瘤细胞球;胶质瘤细胞球细胞均表达神经干细胞标记CD133和Nestin,不表达分化标志GFAP和MAP2,少数细胞表达分化标记MBP。每100个原代胶质瘤细胞球体细胞可形成6.07±2.15个悬浮生长的二代胶质瘤细胞球,二代细胞球细胞表达CD133和Nestin。胶质瘤细胞球细胞分化后细胞多数表达GFAP,少数表达MAP2和MBP。胶质瘤球体干细胞的增殖较原代胶质瘤细胞慢。胶质瘤球体细胞1×103个时可成瘤,原代培养的胶质瘤细胞需1×107。结论:采用神经干细胞培养条件可以很简便的分离出悬浮生长的胶质瘤球体干细胞。分离出的胶质瘤球体干细胞表达CD133抗原,具备肿瘤干细胞的基本特征。     

人卵巢癌细胞系干/祖细胞分离及其鉴定

目的:自人卵巢癌细胞系SK-OV-3中分离干/祖细胞并进行鉴定。方法:采用无血清球形体形成法从SKOV-3中分离培养卵巢癌干/祖细胞;采用实时定量PCR和蛋折质印迹法测定球形体细胞干/祖细胞相关标志ABCG2、Oct-4、Nanog基因和蛋白的表达;流式细胞仪检测其耐药性;双层软琼脂检测其克隆形成能力;NOD/SCID小鼠检测其体内致瘤性。结果:球形体细胞表达干/祖细胞相关标志Oct-4、ABCG2、Nanog;对顺铂高耐药;在双层软琼脂上克隆形成率达(13.67±1.48)%;1 000个球形体形成细胞就能在NOD/SCID鼠中成瘤。结论:采用无血清培养基中球形体形成法从SKOV-3细胞系中可以分离出具有干/祖特性的卵巢癌细胞,可为今后研究卵巢癌的发生、发展、复发及其化疗药物筛选提供简便实用的体外模型。     

悬浮培养在肿瘤干细胞研究中的应用及局限性

肿瘤干细胞被认为是肿瘤起源、生长和转移的关键因素,已成为当下肿瘤研究的热点之一.由于大多数肿瘤干细胞缺乏特异性的标志物,且组织定位和形态特征不明确,因此无法直接从肿瘤细胞中分离,当前常利用肿瘤干细胞与普通肿瘤细胞功能上的差异区分这2类细胞.悬浮培养法是常用的组织干细胞及肿瘤干细胞的体外研究方法,它能够直观地在单细胞层面上反映细胞的自我更新及增殖能力,因此被广泛用于肿瘤干细胞的鉴定、富集和纯化.然而,悬浮培养获得的细胞与体内真正的肿瘤干细胞存在本质的差别,获得的肿瘤球并不一定具克隆性,且并不是所有具备干细胞性质的细胞都能在悬浮条件下存活.因此,当使用悬浮培养研究肿瘤干细胞时,必须意识到它的局限性,本文旨在对这一问题的研究作一综述.     

宫颈癌细胞系中干细胞球样细胞的筛选与鉴定研究

目的探讨从人宫颈癌细胞系Ca Ski中分离培养宫颈癌干细胞球样细胞的可行性,并观察其体外生物学特性。方法采用肿瘤干细胞无血清悬浮培养法富集Ca Ski细胞系球样细胞,通过体外传代培养、添加血清诱导分化等技术鉴定其干性,利用流式细胞技术检测细胞周期,并应用蛋白印记法及细胞免疫荧光等方法从分子水平检测相关因子的表达。结果从Ca Ski细胞系中分离到的干细胞球样细胞可以在无血清培养基中悬浮生长成细胞球,并可在体外稳定传代,具有自我更新能力和分化潜能;肿瘤干细胞球样细胞多处于G0/G1期。与其他分子标志物相比,干细胞球样细胞的CD44明显高表达;与亲本细胞相比,干细胞球样细胞的干性相关因子SOX2、OCT4、β-catenin表达较高。结论利用肿瘤干细胞无血清悬浮培养法可以从Ca Ski细胞系中分离富集到具有稳定肿瘤干细胞性质的细胞群。     

人肺腺癌干细胞的分离及鉴定

目的：从人肺腺癌细胞株中分离及鉴定具有干细胞特征的高致瘤性癌干细胞。方法：采用磁性细胞分选（magnetic activated cell sorting，MACS）技术将肺腺癌细胞分成多个亚群，然后应用流式细胞及RT-PCR技术检测干细胞相关标志表达，通过NOD-SCID小鼠移植评估其致瘤性。结果：在A549和SPC-A肺腺癌细胞中发现一个新颖的癌细胞亚群，此类癌细胞的表型特征为CD24^＋IGF-1R^＋，具有高侵袭性和高致瘤性，在NOD-SCID小鼠中仅需移植100个细胞即可形成肿瘤，其致瘤能力是上述标志阴性细胞的1000倍。此外，这些细胞表达胚胎干细胞标志（OCT4和Bmi-1）及肺干细胞标志（CCSP，SP-C，TTF-1和Gremlin），类似小鼠肺脏细支气管肺泡干细胞（bronchioalveolar stem cells，BASC），并具有自主生长特性，能够在无血清条件下长期培养。结论：人体肺腺癌中CD24^＋IGF-1R^＋细胞属于BASC样癌干细胞。     

三氧化二砷诱导人卵巢癌细胞凋亡机制的研究

目的：探讨三氧化二砷（arsenic trioxide,As2O3)诱导人卵巢癌细胞株3AO细胞凋亡的机制。方法：以人卵巢癌细胞株3AO细胞为体外实验对象。通过台盼蓝染色法，测定不同浓度As2O3作用不同时间对3AO细胞的生长抑制率；采用流式细胞仪检测法，通过碘化丙（PI）／罗丹明123（Rhodammine 123,Rh123)双重染色测定3AO细胞线粒体跨膜电位（△ψm) ；通过吖啶橙染色，荧光显微镜观察As2O3作用后3AO细胞的形态变化。结果：As2O3对3AO细胞的生长具有明显的抑制作用，且具有时间与剂量依赖性（P＜0．05）；3.0μmol/L的As2O3作用48h后3AO细胞中PI－Rh123^-细胞与对照组相比，差异有显著性（P＜0．05）；As2O3作用后，3AO细胞呈现典型的凋亡细胞形态特征。结论：As2O3通过诱导凋亡来抑制人卵巢癌细胞株细胞的生长，其机制与线粒体跨膜电位△ψm下降有关。     

人卵巢癌细胞系A2780肿瘤干细胞分离培养与鉴定

目的:从人卵巢癌细胞系A2780中分离培养卵巢癌干细胞,并对其特性进行鉴定.方法:将A2780细胞消化,离心制成单细胞悬液,悬浮于含有生长因子的无血清培养基(SFM)中获得肿瘤细胞微球体,流式细胞仪检测细胞表面分子标志CD44和CD133的表达;Transwell小室侵袭实验检测肿瘤微球体的体外侵袭能力;CCK-8检测微球体细胞的增殖能力,比较两种细胞的倍增时间;将肿瘤微球体置于含有血清的培养基使其诱导分化,并观察其形态学变化.结果:A2780可在SFM中形成可悬浮生长、稳定传代的肿瘤细胞微球体.与贴壁的肿瘤细胞相比,微球体高表达干细胞标志CD44和CD133(P＜0.05),具有更强的体外侵袭力(t=9.354,P＜0.05)和增殖能力(t=13.682,P＜0.05),及更短的倍增时间(t=3.773,P＜0.05),在血清环境下可分化为普通的肿瘤细胞.结论:用含有生长因子的SFM悬浮卵巢癌细胞系A2780可获得卵巢癌肿瘤细胞微球体,此细胞球体中富集有肿瘤干细胞.     

人肺腺癌细胞系中肿瘤干细胞的分离培养及鉴定*

目的：从人肺腺癌细胞系A 549 及H 1299中分离富集含肿瘤干细胞的细胞球并鉴定其生物学特性。方法：用无血清悬浮培养的方法从人肺腺癌A 549 及H 1299细胞株中富集得到肿瘤细胞球。将肿瘤细胞球传代扩增,体外利用CCK-8 法、平皿克隆以及Transwell 小室实验,研究细胞球的增殖情况、自我更新和侵袭转移能力;通过RT-PCR 检测干细胞特异性转录因子Oct4、Nanog基因表达情况;体内利用裸鼠移植瘤形成实验研究肺癌细胞球的成瘤能力。鉴定细胞球的肿瘤干细胞特性。结果：在无血清悬浮培养下,A 549 及H 1299细胞株3~ 6 天后能形成稳定传代的肿瘤悬浮球,悬浮球的体外自我更新、克隆形成和侵袭转移等能力均高于其亲本细胞（P < 0.05）;干细胞核心基因Oct4 和Nanog的mRNA 表达水平明显升高（P < 0.05）;A 549 悬浮球可以明显提高裸鼠体内成瘤能力。结论：通过无血清悬浮培养法可有效富集A 549 及H 1299细胞系中的干细胞成分,该法可成为快速易行构建肺腺癌干细胞模型的方法。     

Generation of pluripotent cancer-initiating cells from transformed bone marrow-derived cells

Our previous studies indicated that pluripotent cancer-initiating cells existed in chemical carcinogen-transformed mouse bone marrow-derived cells (BMDCs). In this report, we named them as bone marrow-derived pluripotent cancer-initiating (bmPCI) cells. The bmPCI cells showed properties of embryonal carcinoma (EC) cells/embryonic stem (ES) cells in morphology and gene expression, differentiated into multiple somatic tumor cells, generated oocyte-like cells and later developed into blastocyst-like structures in vitro while causing teratocarcinomas in vivo. These findings demonstrate that pluripotent cancer-initiating cells can originate from BMDCs. It provides an accessible in vitro model for copying the "embryonal rest" phenomenon from adult cells.     

Induction of cytotoxic T lymphocytes against ovarian cancer-initiating cells

The majority of patients with stage III/IV ovarian carcinoma that respond initially to standard therapies ultimately undergo relapse due to the survival of small populations of cells with tumor-initiating potential. These ovarian cancer (OVCA)-initiating cells (OCIC) are sometimes called cancer stem cells (CSC) because they express stem cell markers, and can survive conventional therapies such as chemotherapy, which usually target rapidly replicating tumor cells, and give rise to recurrent tumors that are more chemo-resistant and more aggressive. Thus, it would be desirable to develop a therapy that could selectively target OCIC and be used to complement the conventional therapies. In this study, we isolated a subset of OVCA cells with a CD44(+) phenotype in samples from patients with OVCA that possess CSC properties including the formation of spheroids in culture, self-renewal and the ability to be engrafted in immune-compromised mice. We next explored the use of immunotherapy using fusions of dendritic cells and OCIC to specifically target the OCIC subpopulations. Fusion cells (FCs) prepared in this way activated T cells to express elevated levels of IFN-γ with enhanced killing of CD44(+) OVCA cells. We envision a combined approach where conventional therapies such as chemotherapy kill the bulk of tumor cells, whereas OCIC-reactive cytotoxic T lymphocytes target the resistant OCIC fraction. A combined therapy such as this may represent a promising approach for the treatment of OVCA.     

Compact spheroid formation by ovarian cancer cells is associated with contractile behavior and an invasive phenotype

Ovarian cancer cells are present in malignant ascites both as individual cells and as multicellular spheroid aggregates. Although spheroid formation affords protection of cancer cells against some chemotherapeutic agents, it has not been established whether a relationship exists between invasive behavior and predisposition to spheroid formation. Aspects of spheroid formation, including cell‐matrix adhesion, remodeling and contractility are characteristic myofibroblast‐like behaviors associated with fibrosis that contribute to tumor growth and dissemination. We explored the possibility that cell behaviors that promote spheroid formation also facilitate invasion. Our analysis of 6 human ovarian cancer cell lines indicated that ovarian cancer cells possessing myofibroblast‐like properties formed compact spheroids and invaded 3D matrices. These cells readily contracted collagen I gels, possessed a spindle‐like morphology, and had elevated expression of genes associated with the TGFβ‐mediated fibrotic response and/or β1 integrin function, including fibronectin (FN), connective tissue growth factor (CTGF/CCN2), lysyl oxidase (LOX1), tissue transglutaminase 2 (TGM2) and urinary plasminogen activator receptor (uPAR). Whereas cell aggregation was induced by TGFβ, and by β1‐integrin overexpression and activation, these treatments did not stimulate the contractile activity required for spheroid compaction. The positive relationship found between compact spheroid formation and invasive behavior implies a preferential survival of an invasive subpopulation of ovarian cancer cells, as cells in spheroids are more resistant to several chemotherapeutics. Preventing the formation of ovarian cancer spheroids may represent a novel strategy to improve the efficacy of existing therapeutics. © 2008 Wiley‐Liss, Inc.     

人结肠上皮细胞的原代培养

目的:探讨人结肠上皮细胞的分离、体外培养方法,为研究结肠功能及相关疾病提供细胞模型。方法:人正常结肠黏膜取自结肠癌病人手术切除的癌旁正常组织,运用胶原酶和嗜热菌蛋白酶消化分离,接种于适当培养液内,根据成纤维细胞贴壁时间的差异并运用胶原酶来纯化。结果:联合运用Ⅰ、Ⅳ型胶原酶和嗜热菌蛋白酶消化可分离出健全绒毛隐窝单位,在适当的培养条件下可长出单层不规则形细胞,经鉴定为人结肠上皮细胞。结论:使用合适的方法及培养液,可以分离培养出正常人结肠上皮细胞。     

Endoglin is necessary for angiogenesis in human ovarian carcinoma-derived primary endothelial cells

Endoglin (CD105, END) is upregulated in proliferating endothelial cells, suggesting potential therapeutic properties. However, it is not clear whether endoglin mediates an enhanced proliferative rate or may be upregulated as part of a negative feedback loop. To gain insights into context-dependent and cell type-dependent regulatory effects of endoglin, we studied its role properties in human ovarian carcinoma-derived endothelial cells (ODMECs). We isolated and cultured primary ODMECs from epithelial ovarian carcinoma tissue. ODMECs had higher expression of endoglin and VEGFR-2, and also exhibited enhanced spontaneous formation of vessel-like structures in vitro. Transfection of siRNA targeting endoglin in ODMECs cells resulted in the reduction of the proliferation and tube formation. These results indicate that a subset of ODMECs display abnormal angiogenic properties and this phenotype was blocked by decreasing endoglin levels, suggesting endoglin is essential for stimulating angiogenesis, and targeting it may be an attractive approach to anti-angiogenesis therapy for ovarian carcinoma.     

不同来源骨髓间质干细胞分离培养及成骨分化比较

目的 间质干细胞(MSCs)是多能成体干细胞,近年关于其在肿瘤相关领域的研究得到了国内外广泛关注.我们分离、鉴定并纯化不同来源的骨髓间质干细胞,比较其在成骨分化的差异,为下一步试验选择良好的细胞来源.方法 采用差速贴壁法分离SD大鼠及Balb/c小鼠骨髓间质干细胞,通过传代纯化,并观察MSCs在此过程中的纯化程度及增殖状况.流式细胞术鉴别两种MDSCs细胞表面抗原的表达.比较不同来源间质干细胞在成骨分化的差异.结果 在SD大鼠MDSCs分离和培养过程中,其增殖能力显著强于Balb/c小鼠MDSCs,在第三代时即可见典型的漩涡状贴壁生长特性.经流式细胞术检测两种来源的细胞均表达间质干细胞表面抗原.在成骨方面SD大鼠MDSCs表现出了较强的多能特性,且SD大鼠出现程度大于Balb/c小鼠.结论 SD大鼠来源MDSCs较易获得纯度较高且增殖旺盛的细胞源,在成骨方面的能力强于Balb/c小鼠,在一定程度上其可成为良好的细胞来源.     

人卵巢上皮癌微血管内皮细胞分离培养与鉴定

目的:建立人卵巢上皮癌微血管内皮细胞的原代分离纯化及培养方法。方法:收集2012-03-01-2012-10-31山东省肿瘤医院妇瘤科手术切除的卵巢上皮癌标本21例,采用胶原酶消化法分离人卵巢上皮癌微血管内皮细胞,免疫磁珠法提取纯化内皮细胞。采用形态学观察,免疫荧光技术及自发管腔形成实验对提取的内皮细胞进行鉴定;流式细胞仪检测细胞纯度;MTT法测定细胞增殖并绘制细胞生长曲线。结果:提取人卵巢微血管内皮细胞呈圆形或类圆形,周围有抗CD31免疫磁珠附着,活细胞计数测定>95%,流式细胞仪检测纯度>95%,细胞贴壁后呈长梭形、星形或多角形,胞内含有CD31磁珠,3～6d细胞间互相融合呈单层片状生长,铺满后呈典型铺路石样特征,荧光显微镜下观察表达内皮细胞特异性标志vWF,管腔形成实验证实体外有二维管腔形成。通过观察发现,内皮细胞24h内贴壁,7～11d可完全融合并出现接触抑制现象。通过MTT法测定内皮细胞生长分为增殖期、对数增长期和平台期3个阶段,生长曲线为"S"型。结论:应用免疫磁珠法成功建立了一种操作简单,且可获得纯度高和活性好的人卵巢上皮癌微血管内皮细胞分离及原代培养体系。