肾癌干细胞的培养及鉴定

目的从肾癌细胞中获取肾癌干细胞。方法将肾癌细胞悬浮于含有表皮生长因子（EGF）和成纤维生长因子（bFGF）的无血清培养基中培养。用流式细胞仪检测其CD133及CD34。收集无血清培养7d后的细胞重新常规培养并观察其分化情况。结果悬浮培养2d后出现肾癌干细胞球,培养7d后表达CD133^＋,CD34^-的细胞占8．33％±1．26％,高于肾癌细胞中表达CD133^＋CD34^-的细胞（P〈0．05）,后者只占1．24％±0．36％。结论用无血清培养基和悬浮培养的方法可以从肾癌细胞中获取肾癌干细胞。     

MiR-590-3p在胰腺癌干细胞中的表达

目的 应用无血清培养基分离胰腺癌干细胞,检测其miR-590-3p的表达。方法 运用无血清培养基克隆培养ASPC-1、PANC1细胞,检测其单克隆形成、分化及细胞周期、半数抑制浓度(IC50)和表面标记物CD24+、CD44+表达。实时定量PCR法检测细胞miR-590-3p的表达。结果 经无血清培养基培养,(0.94±0.53)％的ASPC-1细胞和(0.57±0.12)％的PANC1细胞能存活,呈克隆球样悬浮生长,并可以在体外连续传代。加入血清后细胞球又重新贴壁生长。ASPC-1细胞球G0/G1期比例和CD24+、C44+、CD24+ CD44+的细胞比例及IC50分别为(75.3±5.4)％、0.96％～2.01％、27.52％～34.47％、0.35％ ～0.44％和(224.37±5.71) μg/ml,均显著高于亲本细胞的(43.7±3.8)％、0.38％～0.42％、17.65％ ～ 18.25％、0.05％～ 0.08％、(11.43±2.10) μg/ml(P值均＜0.05)。PANC1细胞球G0/G1期比例和CD24+、CD44+、CD24 +CD44+的细胞比例及IC50分别为(80.1±4.7)％、5.31％ ～9.84％、72.05％ ～93.06％、4.91％ ～5.21％、(296.58±4.27) μg/ml,均显著高于亲本细胞的(46.1±5.3)％、4.09％～4.97％、47.71％～55.66％、1.48％ ～2.63％、(26.17±3.81) μg/ml(P值均＜0.05)。ASPC-1、PANC1细胞球miR-590-3p表达分别是亲本细胞的4.67和4.52倍。结论 应用无血清培养基可以从ASPC-1、PANC1细胞系中分离出具有干细胞特性的胰腺癌细胞球,其miR-590-3p表达上调,该基因可能是胰腺癌干细胞特性维持的关键基因。     

Wee1抑制剂MK-1775对GBC-SD细胞系胆囊癌干细胞样细胞自我更新的影响

目的 研究Wee1抑制剂MK-1775对GBC-SD细胞系胆囊癌干细胞样细胞自我更新的抑制作用.方法 体外培养GBC-SD细胞系,在无血清干细胞培养基中加入MK-1775后培养悬浮肿瘤干细胞球; Westernblot检测Wee1的表达,并分析比较肿瘤干细胞球体积大小和形成率的变化;建立裸鼠皮下移植瘤模型后MK-17751灌胃2 wk, 2 wk后检测分析移植瘤的重量.结果 加入MK-1775培养8 d后, GBC-SD细胞中Wee1表达下调,同时肿瘤干细胞球大小及形成率均受到抑制;经MK-17751处理后,裸鼠皮下移植瘤生长受到抑制.结论 MK-1775具有抑制GBC-SD细胞系胆囊癌干细胞样细胞自我更新的作用.     

无血清培养胆囊癌GBC-SD细胞形成肿瘤细胞球的鉴定

目的:分离扩增肿瘤干细胞,并鉴定其生物学性质.方法:用无血清培齐基培养人胆囊癌GBC-SD细胞得到肿瘤细胞球.将肿瘤细胞球传代扩增,并用含血清培养基培养促使其分化;将肿瘤球和普通GBC-SD细胞分别种入96孔板,MTT检测增殖能力,并将肿瘤球和普通GBC-SD细胞分别植入裸鼠皮下,观察移植瘤的形成:流式细胞术检测CD15s和CD24在肿瘤球细胞和普通GBC-SD细胞中的表达,筛选细胞表面标志物.结果:在无血清培养基中,胆囊癌细胞可以形成少量的肿瘤细胞球,并显示很强的自我更新和增殖能力,含血清环境能够诱导其分化而贴壁生长;在动物实验中,肿瘤球细胞较普通GBC-SD细胞显示更强的致瘤能力(80.00%vs 10.00%,P<0.05);标志物CD15s在肿瘤球的表达较普通GBC-SD细胞明显增高(2.56%±0.38%vs 10.77%±0.93%,t=18.25,P<0.05).结论:人胆囊癌细胞GBC-SD的肿瘤干细胞可以通过无血清培养环境来分离和扩增,CD15s可能为其细胞表面标志物.     

ALDH1作为卵巢癌干细胞标志物的实验研究

目的探讨含生长因子无血清培养基(SFM)培养的卵巢癌细胞株(SKOV3)细胞悬浮球对CD133与乙醛脱氢酶1(ALDH1)表达的影响以及ALDH1是否可作为卵巢癌干细胞(CSC)的标志物。方法用含生长因子的无血清培养基对卵巢癌SKOV3细胞进行培养作为实验组,以含血清培养基(SSM)组作为对照,四甲基偶氮唑蓝比色法(MTT)及平板克隆形成实验、Transwell侵袭实验检测两组细胞的自我增殖、克隆形成及侵袭能力,通过流式细胞技术检测卵巢癌SKOV3细胞和悬浮球中ALDH1、CD133表达情况;体外化疗实验富集ALDH1high细胞初步探索ALDH1在卵巢癌细胞中的作用。结果实验组自我增殖、克隆形成及侵袭能力明显高于对照组(P0.05);实验组ALDH1high、CD133+细胞比例明显高于对照组(P0.05);体外化疗实验表明顺铂连续作用于卵巢癌SKOV3细胞72 h后,实验组ALDH1high、CD133+细胞比例明显增加,ALDH1high细胞比例增加了3.23倍,CD133+细胞比例增加了2.34倍,与对照组相比较差异显著(P0.05)。结论含生长因子的无血清培养基培养卵巢癌SKOV3细胞能够形成肿瘤干细胞球及高表达ALDH1,ALDH1可能是卵巢癌干细胞的标志物之一。     

EGFR及ADAM9在胰腺癌干细胞与分化细胞中的表达及意义

目的 通过微球体培养富集胰腺癌干细胞,检测其表皮生长因子受体(EGFR)和去整合素-金属蛋白酶9(ADAM9)的表达,并探讨其意义.方法 运用无血清条件培养基悬浮培养胰腺癌PANC1细胞,并连续传代培养.将部分微球体接种于含血清及胶原底物的培养基中进行分化诱导.收集微球体细胞及分化细胞,流式细胞仪检测侧群(SP)细胞比例;实时PCR及蛋白质印迹法检测细胞EGFR、ADAM9 mRNA和蛋白的表达.结果 成功培养出胰腺癌PANC1细胞微球体,并能在体外连续传代.微球体细胞分化诱导后能重新贴壁生长.微球体细胞和分化细胞中SP细胞比例分别为(5.40±0.38)%和(2.80±0.42)%,两者差异具有统计学意义(P＜0.05).微球体细胞与分化细胞相比,EGFR及ADAM9 mRNA表达分别上调约2.5和3.0倍(P＜0.05);微球体细胞EGFR及ADAM9蛋白相对表达量分别为0.90±0.09和0.64±0.07,显著高于分化细胞的0.62±0.11和0.48±0.09(P＜0.05).结论 微球体培养能富集胰腺癌干细胞,ADAM9可能通过EGFR信号通路在胰腺癌的发生、发展中起重要作用.     

靶向乳腺癌干细胞DC瘤苗的核酸抗原制备

摘要 目的：应用无血清培养基细胞球培养法富集MCF-7乳腺癌干细胞,制备乳腺癌干细胞mRNA核酸抗原,为制备靶向乳腺癌干细胞树突细胞瘤苗奠定基础。方法：应用无血清培养基悬浮培养法富集MCF-7乳腺癌细胞,经单克隆形成、表面标志检测、NOD-SCID小鼠成瘤等实验对其肿瘤干细胞特性进行鉴定后,利用T7 mMESSAGE mMACHINE试剂盒进行mRNA体外扩增。结果：乳腺癌细胞MCF-7在无血清培养基中可形成悬浮状细胞球,其中具有CD44+CD24-表面标志的细胞约占90.16%。该细胞亚群具有较贴壁细胞更强的克隆形成能力和低剂量体内成瘤能力。细胞总RNA经体外扩增获得的mRNA,可作为核酸抗原。结论：应用无血清悬浮细胞球培养法可以获得高含量乳腺癌干细胞。通过体外扩增方法获得该细胞亚群的mRNA,可作为肿瘤核酸抗原,为下一步制备靶向乳腺癌干细胞的树突细胞瘤苗奠定了基础。     

单克隆培养法分离、培养胶质瘤干细胞

目的 观察单克隆培养法从人脑胶质瘤细胞系SHG44中分离、培养肿瘤干细胞的结果.方法 取对数生长期SHG44细胞,用无血清培养基进行肿瘤干细胞的分离培养及单克隆培养,免疫荧光染色法对分化前后的肿瘤球行CD133、神经巢蛋白(Nestin)、胶质纤维酸性蛋白质(GFAP)、小管蛋白3-Tubulin染色,MTT法检测肿瘤于细胞的增殖情况,流式细胞术检测CD133阳性细胞比例.结果 单克隆分离培养的细胞球表达CD133及Nestin干细胞标记物,分化后表达星形胶质细胞GFAP及神经元β-Tubulin标记物,细胞球生长约第5天增殖率达到峰值.结论 单克隆培养法可成功分离培养出胶质瘤干细胞,分化前后的细胞球可表达干细胞标记物、神经胶质细胞及神经元标记物,肿瘤球具有较强的增殖及分化能力,并含有一定数量的CD133阳性细胞.     

CD117阳性细胞亚群在肝癌细胞系HepG2中的分离及其特性

目的:观察从人肝癌细胞系HepG2中分离的CD117+细胞生物学行为,探讨肝癌中CD117+细胞亚群的干细胞特性.方法:采用无血清悬浮培养法培养HepG2细胞,流式细胞术检测HepG2及球体细胞中CD117的表达比例.用流式细胞分选技术从HepG2成球细胞中分离CD117+的肿瘤细胞,进行无血清悬浮培养,观察其成球能力.CCK-8法观察CD117+细胞的增殖能力和顺铂对CD117+细胞的抑制率,计算IC50和耐药指数(RI).结果:HepG2细胞能在无血清培养基中存活、增殖并形成细胞球,成球率为6.21%±2.03%;流式细胞检测发现球体细胞中CD117+细胞的比例比HepG2细胞提升了9倍;CD117+细胞在无血清培养基中成球率和增殖能力均显著高于未分选细胞和CD117细胞;CD117+细胞在各浓度的顺铂作用下抑制率均较未分选细胞和CD117细胞明显降低,三者IC50分别为12.229μmol/L、7.970μmol/L和7.345μmol/L,CD117+细胞和未分选细胞耐药系数RI为1.165和1.076.结论:人肝癌细胞系HepG2中的CD117+细胞是具有肿瘤干细胞特性的细胞亚群,CD117可能是肝...     

HT29和HCT116结肠癌细胞无血清培养形成肿瘤干细胞球特性的差异性研究

目的比较HT29和HCT116两种结肠癌细胞系中肿瘤干细胞的差异，初步探讨结肠癌干细胞研究模型。方法以无血清培养法培养HT29和HCT116细胞，观察其在不同时间形成肿瘤干细胞球的差异，用限制性稀释法计算两者的成球率；流式细胞术分析HT29和HCT116细胞系中CD44／CD24的表达情况；裸鼠体内成瘤实验鉴定HT29细胞球与HCT116细胞球成瘤能力。结果无血清培养法发现HCT116较HT29更易形成肿瘤干细胞球且所需时间更短，即HT29在无血清培养的第7天开始形成规则的球体，而HCT116则在第5天就已形成规则的球体，HCT116成球率（11．4±1．15）％高于HT29（3．31±0．27）％，且差异有统计学意义（P〈0．05）；HT29和HCT116中CD44±／CD24±各细胞含量有显著差异，结果显示具有干细胞特性的CD44＋／CD24＋结肠癌细胞在HCT116中所占比例（60．33±5．75）％明显高于HT29（9．23±2．15）％，差异有统计学意义（t=13．939，P〈0．05）；体内成瘤实验发现HCT116细胞球在裸鼠体内的成瘤能力明显强于HT29细胞球，HCT116细胞球的成瘤速度及瘤体生长速度都较HT29细胞球快。结论与HT29相比，HCT116结肠癌细胞系更适合作为肿瘤干细胞研究的模型。     

人胃癌细胞株MGC-803侧群细胞的分选与生物学特征

目的 从人胃癌细胞株MGC-803中分离侧群(SP)细胞,研究其生物学特征.方法 利用流式细胞分选术(FACS)从MGC-803细胞株中分选出SP细胞和非SP细胞亚群进行培养,采用克隆形成实验比较两组亚群细胞的体外增殖能力,NOD/SCID鼠成瘤实验检测两组亚群细胞体内成瘤能力.结果 FACS结果显示,细胞株MGC-803中SP细胞亚群占总细胞的0.3％ ～ 1.2％;SP细胞体外克隆形成率为(0.862±0.050)％,非SP细胞体外克隆形成率为(0.325 ±0.207)％,两者比较P＜0.05;SP细胞最低成瘤数量是1×103/只,非SP细胞为5×103/只.结论 人胃癌细胞株MGC-803中存在SP细胞,其生物学特性与干细胞基本符合.     

Facile isolation and the characterization of human retinal stem cells

This study identifies and characterizes retinal stem cells (RSCs) in early postnatal to seventh-decade human eyes. Different subregions of human eyes were dissociated and cultured by using a clonal sphere-forming assay. The stem cells were derived only from the pars plicata and pars plana of the retinal ciliary margin, at a frequency of approximately 1:500. To test for long-term self-renewal, both the sphere assay and monolayer passaging were used. By using the single sphere passaging assay, primary spheres were dissociated and replated, and individual spheres demonstrated 100% self-renewal, with single spheres giving rise to one or more new spheres in each subsequent passage. The clonal retinal spheres were plated under differentiation conditions to assay the differentiation potential of their progeny. The spheres were produced all of the different retinal cell types, demonstrating multipotentiality. Therefore, the human eye contains a small population of cells (approximately equal to 10,000 cells per eye) that have retinal stem-cell characteristics (proliferation, self-renewal, and multipotentiality). To test the in vivo potential of the stem cells and their progeny, we transplanted dissociated human retinal sphere cells, containing both stem cells and progenitors, into the eyes of postnatal day 1 NOD/SCID mice and embryonic chick eyes. The progeny of the RSCs were able to survive, migrate, integrate, and differentiate into the neural retina, especially as photoreceptors. Their facile isolation, integration, and differentiation suggest that human RSCs eventually may be valuable in treating human retinal diseases.     

Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34 cells after ex vivo expansion

OBJECTIVE: The relationship between phenotype and function in ex vivo-cultured human hematopoietic stem cells (HSC) remains poorly understood. We investigated the effects of a short-term serum-free culture on the relationship between stem cell phenotype, cell division history, and function in human CD34(+) cells. METHODS: G-CSF-mobilized peripheral CD34(+) cells were cultured for 4 days with stem cell factor, flt-3 ligand, and thrombopoietin. The phenotype (CD34, CD38, HLA-DR, c-kit), cell division history, colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and NOD/SCID repopulating activities were evaluated at Day 0 and 4. RESULTS: We observed a loss of CD38, HLA-DR, and c-kit surface expression resulting in a drastic increase in CD34(+)CD38(-), CD34(+)HLA-DR(-), and CD34(+)c-kit(-/low) cells at Day 4. In contrast, the frequency of Thy-1(+) cells was maintained. We observed a 1.3-fold expansion of CFC, a 4.8-fold increase in LTC-IC, and an overall maintenance of the NOD/SCID repopulating cell activity. CD34(+)CD38(-) and CD34(+)HLA-DR(-) cells detected at Day 4 displayed the most active pattern of division (4 to 5 divisions) whereas 60% of CD34(+)Thy-1(+) cells divided 0 to 2 times during the same period. At Day 4, the NOD/SCID repopulating activity was associated with Thy-1(+) cells with no more than 2 divisions. CONCLUSIONS: Our results show that the relationship between stem cell phenotype and function is dramatically altered in cultured CD34(+) cells. Thy-1 expression and cell division history appear to be superior to CD38, HLA-DR, and c-kit, or to homing molecules (CXCR4, VLA-4) as predictors of the repopulating activity of cultured peripheral CD34(+) cells.     

恶性脑胶质瘤U87细胞系肿瘤干细胞放疗敏感性的体外实验研究

目的探讨脑胶质瘤细胞系U87细胞及其肿瘤干细胞（CSCs）对体外放疗的敏感性。方法应用神经干细胞（NSCs）培养基分离培养形成细胞球克隆,检测细胞球细胞的NSCs特性。采用不同剂量放射线照射U87细胞及其CSCs,应用集落形成实验和生存曲线检测其生存情况。结果 U87细胞在NSCs培养基中形成悬浮的细胞球克隆,具有自我更新和多向分化能力,表达CD133、Nestin;两种细胞的存活率均随照射剂量增加而下降,且CSCs的存活率明显高于U87细胞（P〈0.01）。结论在体外条件下,CSCs的放射敏感性明显低于U87细胞,其可能是恶性脑胶质瘤放疗抵抗的主要原因。     

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

Background

Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.

Methods

Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.

Results

The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.

Conclusions

Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.     

干细胞培养基成球培养法筛选结肠癌干细胞的生物学特性

目的探讨干细胞培养基成球培养法筛选结肠癌干细胞的相关蛋白表达及生物学特性。方法用干细胞培养基成球培养法筛选结肠癌干细胞,Western blot检测干细胞相关蛋白的表达,流式细胞仪检测细胞周期;同时检测干细胞增殖、黏附、耐药及侵袭能力,并摸索结肠癌干细胞最佳冻存条件。结果用干细胞培养基成球培养法成功培养出了结肠癌细胞球,检测发现干细胞相关蛋白表达增强,静止期细胞比例明显升高,细胞增殖速度慢,黏附性、耐药性、侵袭性均强于亲本细胞。结论干细胞培养基成球培养法筛选的细胞球中富集了肿瘤干细胞,为结肠癌干细胞的研究提供了良好的细胞模型。     

Large-scale isolation of CD133+ progenitor cells from G-CSF mobilized peripheral blood stem cells

We have evaluated the feasibility of large-scale isolation of CD133+ progenitors from healthy mobilized adult donors for potential clinical use in autologous and allogeneic transplantation. A total of 11 healthy volunteer adult donors were mobilized with G-CSF. CD133+ stem cells were isolated from a single leukapheresis using the Clinimacs method. The median percentage of CD133 before positive selection was 0.75% (range 0.39-2.03%). After selection, the median purity and recovery was 94% (range 85.2-98.0%) and 69% (range 44-100%), respectively. The median log10 T-cell depletion obtained by CD133+ positive selection was 4.2 (range 3.8-4.7). The CD133+ progenitors were highly enriched in colony-forming units (CFU) and transplantation into NOD/SCID mice resulted in a high engraftment rate. Transplantation of sorted CD133+/CD34+ cells into NOD/SCID mice showed a higher engraftment compared to CD133-/CD34+ cells. Mobilized peripheral CD133+ stem cells can be purified in large scale for potential clinical use. The biological function of the cells is not impaired. The majority of the NOD/SCID repopulating cells are within the CD133+/CD34+ subpopulation. Therefore, clinical studies using purified CD133+ stem cells can be envisoned to further clarify the role of CD133+ stem cells in hematopoietic reconstitution after transplantation.     

阻断Hedgehog信号通路对人胰腺癌干细胞自我更新的影响

目的 观察Hedgehog信号通路特异阻断剂环巴明对胰腺癌干细胞自我更新的影响.方法 应用0.5、1、2、5、10 μmol/L环巴明处理胰腺癌PANC1干细胞、PANC1贴壁细胞和永生化胰腺导管上皮H6C7细胞24、48、72 h.采用实时RT-PCR法检测细胞Smo及Gli1 mRNA表达;CCK-8法检测细胞增殖抑制;流式细胞仪检测细胞周期及凋亡.结果 10 μmol/L环巴明处理72 h后,PANC1干细胞、PANC1细胞和H6C7细胞的Smo mRNA表达量分别为1、0.83、2.61;Glil mRNA为57.27、26.35、1;生长抑制率分别为(37.85±13.69)%、(8.53±4.43)%、(43.55±28.98)%.与PANC1细胞比较,PANC1干细胞的Smo、Gli1 mRNA表达显著增加,生长抑制率亦显著增强(P值＜0.05或＜0.01).经10 μmol/L环巴明处理72 h,PANC1干细胞G1期比例从(67.41±6.35)%显著降至(36.53±6.03)%(P＜0.05),细胞凋亡率从(10.95±5.68)%降至(5.73±1.42)%(P＞0.05);PANC1细胞G1期比例从(67.64±6.88)%显著降至(53.13±1.10)%(P＜0.05),细胞凋亡率从(12.08±4.12)%降至(5.66±1.33)%(P＞0.05);而H6C7细胞的G1期比例及凋亡无明显变化.结论 环巴明阻断Hedgehog信号通路可抑制PANC1干细胞增殖,其机制可能与细胞凋亡无关.