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。     

CD44和CD24标记的宫颈癌细胞亚群特性

目的 考察Siha细胞系中的CD44^+/CD24⁺能否富集宫颈癌干细胞。方法 用流式细胞仪分选出CD44⁺/CD24⁺Siha细胞,用无血清悬浮培养观察成球能力、裸鼠移植瘤实验观察成瘤能力、透射电子显微镜观察辐射前后两组细胞形态变化,并通过Transwell侵袭实验比较细胞侵袭能力的差异。结果 耐放疗细胞中CD44^+/CD24⁺Siha细胞比例明显高于其在亲代Siha细胞中的比例;无血清培养CD44⁺/CD24⁺Siha细胞组可以形成致密且体积较大的细胞球,CD44^+/CD24⁺Siha细胞组致瘤时间早,成瘤率高;辐射后CD44⁺/CD24⁺Siha细胞较亲代Siha细胞更抗凋亡;CD44⁺/CD24⁺Siha细胞组的迁移细胞数明显高于亲代Siha细胞组,所有数据均具有统计学意义。结论 CD44⁺/CD24⁺Siha具备部分干细胞特性,CD44⁺/CD24⁺可能成为宫颈癌干细胞特异性表面标志物。     

乳腺浸润性导管癌组织中ALDH1+/CD133+干细胞样细胞与血管生成的关系

目的 探讨乳腺浸润性导管癌组织中肿瘤干细胞标志物ALDH1、CD133的表达及其与肿瘤血管生成的关系。方法 应用免疫组织化学双染法检测120例乳腺浸润性导管癌组织中ALDH1⁺/CD133⁺ 干细胞样细胞,单染法检测血管性标记CD34、CD105及VEGF的表达情况。统计ALPH1⁺/CD133⁺干细胞样细胞与临床病理因素;CD34、CD105与VEGF的关系。结果 25.83％（31/120）的病例存在ALDH1⁺/CD133⁺干细胞样细胞,ALDH1⁺/CD133⁺干细胞样细胞与ER、VEGF的表达及MVD均相关（P＜0.05）,但与年龄、肿瘤直径、PR、Her-2、组织学分级及淋巴结转移均无关（P＞0.05）。结论 乳腺浸润性导管癌组织中ALDH1⁺/CD133⁺干细胞样细胞可能通过调节VEGF的表达促进肿瘤新生血管的生成。     

大肠癌细胞HCT-15肿瘤干细胞样细胞分离及成瘤性比较

目的:依据人大肠癌细胞HCT-15的表面标志物,分离其中的干细胞亚群。建立裸鼠体内原代大肠癌荷瘤模型,比较各亚群肿瘤体积和质量。方法:利用免疫磁珠分选技术,分离其中CD133/CD44干细胞亚群。分选得到CD133+CD44+、CD133+CD44-亚群分别接种于裸鼠体内,并观察肿瘤大小和质量。结果:CD133+CD44+和CD133+CD44-细胞亚群成功的从HCT-15细胞中分离出来。接种CD133+CD44+细胞的裸鼠形成的肿瘤体积［（2.76±0.22）cm3］和质量［（5.2±0.21）g］明显高于接种CD133+CD44-细胞裸鼠的肿瘤体积与质量［（1.56±0.34）cm3,（3.4±0.18）g］（P＜0.05）。结论:CD44阳性的大肠癌肿瘤干细胞成瘤能力明显高于CD44阴性的大肠癌肿瘤干细胞。     

高转移肺癌细胞株的建立及其肿瘤干细胞生物学特性

 目的 研究高转移肺腺癌细胞株的肿瘤干细胞生物学特征。方法 肺腺癌细胞系A549接种裸鼠皮下成瘤后,取肺的转移灶,通过机械分离法获取肺转移细胞,体外扩大培养后,再次接种裸鼠。如此反复接种裸鼠,获得稳定肺转移的细胞株A549-V13。采用无血清悬浮培养及PKH26染色确定A549-V13存在肿瘤干细胞。流式细胞术（FACS）检测和分选A549和A549-V13中肿瘤干细胞标志物CD133的表达并进行体内外生物学特征实验。结果 建立稳定高转移A549-V13细胞株。无血清悬浮培养A549-V13,7天后形成的球形细胞团中存在单个PKH26阳性细胞。FACS检测显示,与A549中CD133⁺细胞相比,高转移A549-V13细胞株中CD133⁺细胞的表达比例显著提高4.85倍。A549-V13中CD133⁺细胞具有更强的自我更新能力,侵袭能力提高1.42倍,耐药能力也显著增强,其IC₅₀提高1.26倍,A549-V13的CD133⁺细胞在裸鼠皮下接种2×102个细胞3月致瘤4/6,而接种2×10²个A549的CD133⁺细胞3月才致瘤2/6。结论 建立高转移肺癌细胞株A549-V13,伴随CD133⁺细胞表达比例的增加,其体内外生物学功能显著增强。     

喉癌细胞CD44+CD133+高致瘤亚群的生物学特性

目的 筛选喉癌细胞中的高致瘤亚群,探讨其作为肿瘤干细胞的生物学特性.方法 原代培养喉癌细胞,采用流式细胞仪分选CD44+、CD133+、CD44+CD133+、CD44+CD133-细胞,并分别以1×106、1×105、1×104、1×102个/只接种于裸小鼠左腋皮下,观察各亚群成瘤时间、成瘤率、平均瘤重及肿瘤体积,筛选出在极低细胞浓度下的高致瘤亚群.采用Boyden小室体外侵袭实验,检测各亚群细胞的侵袭能力.采用免疫细胞化学染色检测各亚群细胞中干细胞抗原-1(SCA-1)及整合素β1的表达.采用逆转录聚合酶链反应(RT-PCR)和Westem blot法,检测各亚群细胞中Bmi-1基因的表达.结果 CD44+CD133+细胞以1×103个/只接种于裸小鼠,可以成瘤.当各亚群细胞以1×106个/只接种于裸小鼠4周后,CD44+CD133+细胞接种组棵小鼠的肿瘤体积和重量分别为(7726.81±196.93)mm3和(5.51±0.12)g,均大于CD44+、CD44+CD133-及未分选细胞接种组(均P<0.05),而与CD133+细胞接种组裸小鼠的肿瘤体积和重量差异无统计学意义(P>0.05).CD44+CD133+细胞24 h的侵袭细胞数为83.62±1.61,显著高于其他各亚群喉癌细胞.CD44+CD133+细胞高表达整合素β1和SCA-1.Bmi-1 mRNA在CD44+CD133+细胞中的表达水平为0.951±0.112,显著高于CD44+CD133-细胞和未分选的喉癌细胞(0.532±0.214和0.268±0.193,均P<0.01);Bmi-1蛋白在CD133+和CD44+CD133+细胞中高表达,而在CD44+、CD44+CDi33-和未分选的喉癌细胞中仅有少量表达.结论 CD44+CD133+喉癌细胞具有高致瘤性及肿瘤干细胞的生物学特性,可能是喉癌恶性增殖的根源,并有望成为喉癌治疗的新靶点.     

人滑膜肉瘤肿瘤干样细胞分离与鉴定

目的:分离鉴定滑膜肉瘤（SW982）肿瘤干样细胞。方法:体外培养人滑膜肉瘤细胞系SW982,用CD133标记方法检测SW982中肿瘤干细胞的数量,用免疫磁珠分选法进行分选,分选后所得CD133+和CD133-细胞群分别进行以下实验。通过无血清悬浮培养基培养获得CD133+悬浮细胞球,并将其进行重新贴壁、重新成球以检测其自我更新能力。顺铂（cisplatin,CDDP）和阿霉素（doxorubicin,DXR）分别处理CD133+与CD133-贴壁细胞,CD133+贴壁细胞与CD133+悬浮细胞球,MTS检测每两种细胞药物敏感性。Real-time PCR及Western blotting 检测CD133+和CD133-贴壁细胞干细胞相关基因ABCG2、Bmi1、c-Myc、Nanog、Oct3/4、Sox2表达情况。将CD133+和CD133-贴壁细胞分别接种于6~8周雌性BALB/c裸鼠,观察成瘤情况,并将接种后所得肿瘤进行免疫组化,观察接种瘤含CD133情况。结果:SW982细胞系中CD133含量为8.59%,CDDP、DXR对CD133+和CD133-贴壁细胞抑制具有浓度依赖性。CD133+和CD133-细胞均表达干细胞相关基因ABCG2、Bmi1、c-Myc、Nanog、Oct3/4、Sox2。相对于CD133-细胞,CD133+细胞ABCG2、Nanog、Oct3/4、Sox2表达明显升高。CD133+细胞裸鼠体内成瘤率高于CD133-细胞,CD133+接种瘤含CD133较CD133-接种瘤多。结论:SW982细胞系CD133+细胞具有高自我更新能力、高耐药性、高表达干细胞相关基因、高成瘤性,是肿瘤干细胞。     

CD44+/CD24+表型的宫颈癌Siha细胞对顺铂的耐药及机制研究

背景与目的：肿瘤干细胞的存在是肿瘤细胞抗拒化疗的原因之一。该研究探讨CD44⁺/CD24⁺宫颈癌Siha细胞对顺铂的耐药性及相关机制。方法：体外培养Siha细胞,流式荧光激活细胞分选仪分选CD44⁺/CD24⁺Siha细胞,噻唑蓝(thiazolyl blue,MTT)法检测不同浓度顺铂对细胞的体外抑制情况,采用流式细胞仪检测10 μg/mL顺铂作用于CD44⁺/CD24⁺ Siha细胞24、48和72 h时的细胞凋亡率,实时定量聚合酶链式反应(quantitutive real-time polymerase chain reaction,qRT-PCR)和蛋白［质］印迹法(Western blot)检测CD44⁺/CD24⁺ Siha细胞中Oct-4、ABCG2、Bcl-2的表达,同时设立亲代Siha细胞作为对照。结果：不同浓度顺铂(0.1、1、5、10、15和20 μg/mL)对CD44⁺/CD24⁺ Siha细胞的增殖抑制作用较亲代Siha细胞小［(88.4±1.5)% vs (92.9±1.5)%,(79.9±1.0)% vs (84.7±1.1)%,(69. 8±0.8)% vs (75.1±2.9)%,(59.0±0.7)% vs (65.8±2.7)%,(49.6±0.9)% vs(52.1±0.5)%,(45.1±0.7)% vs (48.8±1.0)%,P<0.05］;与亲代细胞相比,当10 μg/mL顺铂作用于CD44⁺/CD24⁺Siha细胞时,发现在24、48和72 h时的细胞凋亡率均较小［(3.05±0.16)% vs (5.17±0.27)%,(17.94±2.02)%vs (32.60±4.28)%和(40.14±3.01)% vs (56.62±5.32)%,P<0.05］。qRT-PCR和Western blot实验均显示CD44⁺/CD24⁺ Siha细胞高表达Oct-4、ABCG2和Bcl-2,与亲代Siha细胞比较差异有统计学意义(P=0.015)。结论：CD44⁺/CD24⁺ Siha细胞可以抵抗顺铂诱导的细胞凋亡,具有化疗抵抗性,并且高表达肿瘤干细胞表面标志物如Oct-4和ABCG2,该结果在宫颈癌肿瘤干细胞的有效分选以及肿瘤靶向治疗方面有深远意义。     

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。     

外阴鳞状细胞癌肿瘤干细胞的分选和鉴定

目的:分离外阴鳞状细胞癌A-431细胞系中CD44+的细胞亚群,鉴定其具有肿瘤干细胞特性。方法:采用流式细胞术分选出CD44免疫荧光抗体标记的CD44+细胞亚群,检测CD133、CD34蛋白在CD44+和CD44-中表达的差异,检测CD44+及CD44-细胞亚群的细胞周期。结果:A-431细胞系中CD44+细胞亚群约占4%,胚胎干细胞因子CD133、CD34在CD44+和CD44-的VSCC中表达存在显著性差异,主要在细胞膜上表达,CD44+的肿瘤细胞亚群97.69%处于G1期,CD44-的肿瘤细胞亚群61.12%处于S期。结论:在 A-431细胞系中 CD44 可以作为分选肿瘤干细胞的特异性表面标志物,CD133、CD34 在 CD44+的肿瘤细胞高表达,可能成为分选外阴鳞癌肿瘤干细胞的特异性表面标志物,CD44+肿瘤细胞具有典型的干细胞增殖特点。     

CD15及CD133在高、低级别胶质瘤中的表达及意义

目的探讨CD15在胶质瘤的表达及分布特征,与目前公认的脑肿瘤干细胞标志物CD133作对照,并探讨两者相关性。方法采用免疫组织化学法检测80例胶质瘤组织（高级别39例,低级别41例）CD15和CD133的表达。用Image-Pro Plus 6.0图像分析处理软件计算CD15和CD133阳性细胞积分光密度值（IOD）。结果CD15与CD133在高、低级别的胶质瘤均有表达。CD15及CD133阳性细胞数以及壁龛随胶质瘤病理级别增高而增多。在高、低级别胶质瘤中CD15⁺细胞与CD133⁺细胞IOD值差异均有统计学意义（z=－3.432,P=0.001;z=－4.192,P=0.000）;CD15⁺细胞与CD133⁺细胞IOD值呈显著正相关（r=0.535,P=0.000）;CD133还表达于含有肥胖细胞成分的胶质瘤肥胖细胞中,并可见CD133+血管。结论CD15及CD133阳性细胞数以及壁龛随胶质瘤病理级别增高而增多。CD15及CD133表达成正相关关系。含有肥胖细胞成分的胶质瘤中大量肥胖细胞CD133阳性,与其不良临床预后相符。关键词：     

Homing-Associated Cell Adhesion Molecule (H-CAM/CD44) on Human CD34+ Hematopoietic Progenitor Cells

Human CD34⁺ hematopoietic progenitor cells (HPCs) express CD44 and can directly adhere to hyaluronate (HA) via CD44. Furthermore, CD44 may also be involved in the regulation of CD34⁺ HPC proliferation and development. The expression of CD44 molecules on CD34⁺ hematopoietic progenitor cells is significantly lower on bone marrow (BM) CD34⁺ cells compared with circulating CD34⁺ cells in cord blood and peripheral blood. Myeloid and erythroid progenitor cells are found predominantly in CD34⁺CD44^- cell fractions. More interestingly, CD34⁺CD44⁺ cells expressing B-lymphocyte-associated CD10 and CD19 would represent unique B-lymphocyte committed precursors in the BM, which might undergo apoptotic cell death in the early steps of B-cell differentiation.     

In vitro proliferative and differentiating characteristics of CD133(+) and CD34(+) cord blood cells in the presence of thrombopoietin (TPO) or erythropoietin (EPO). Potential implications for hematopoietic cell transplantation

We investigated the characteristics of cord blood (CD) CD133⁺ and CD34⁺ cells, by flow cytometry, clonogenic assays and assessment of the replating ability (area under the curve (AUC)) following 7-day liquid culture in the presence of early acting growth factors and either thrombopoietin (TPO) or erythropoietin (EPO). The CD34⁺ population showed a more effective proliferation in all parameters tested and TPO proved to be more effective than EPO. On the contrary, the CD133⁺ cell fraction retained and expanded more immature elements in a modest but consistent manner with either TPO or EPO. We conclude that CD133⁺ and CD34⁺ expanded cord blood cells could potentially be used in combination to overcome the shortcomings of cord blood transplantation in older children and adults.     

Proportion of CD4+CD25+ regulatory T cell is increased in the patients with ovarian carcinoma

Objective: To study the frequency of the CD4⁺CD25⁺ regulatory T cells (Tregs) in the patients with ovarian carcinoma and its possible mechanism. Methods: The percentages of CD4⁺CD25⁺ Tregs in the peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs) and tumor associated lymphocytes (TALs) from 13 patients with ovarian carcinoma and in the PBLs from 14 healthy women were determined by flow cytometry. The expression of CD69 on CD4⁺PBLs from the patients was detected. PBLs from healthy women were cultured in complete RPMI 1640 containing the supernatant from SKOV3 cell line with or without PHA (phytohemagglutinin) stimulation for 72 hours, then the percentage of CD4⁺CD25⁺ T cells was detected. Results: CD4⁺CD25⁺ Tregs in the PBLs from patients with ovarian carcinoma were significantly increased compared with those from the control. The percentage of CD4⁺CD25⁺ Tregs in TILs was higher than that in PBLs and TALs from the patients, but not significantly. The expression of CD69 on CD4⁺PBLs from the patients was negative. The percentages of CD4⁺CD25⁺ T cells in PBLs cultured with SKOV3 supernatant elevated significantly compared with those without supernatant whether PHA was added or not (P = 0.001 and 0.001, respectively). Conclusion: There is an increasing of the proportion of CD4⁺CD25⁺ Tregs in PBLs, TILs and TALs of the patients with ovarian carcinoma, which probably results from up-regulation of soluble factor secreted by ovarian carcinoma cells.     

中晚期宫颈鳞癌患者治疗前外周血Treg计数分析

背景与目的：中晚期宫颈鳞癌同期放化疗(concurrent chemoradiotherapy,CCRT)治疗前性价比高的疗效判断方法较有限,该研究拟通过检测治疗前外周血CD4⁺CD25⁺CD127^Low/-调节性T细胞(regulatory T cells,Tregs)亚群计数及血清鳞癌抗原(squamous cell carcinoma antigen,SCC-Ag)水平,评价两者预测临床疗效的可行性。方法：采集44例Ⅱ_B~Ⅳ_A期宫颈鳞癌患者行CCRT治疗前的外周血标本,分别利用流式细胞免疫表型分析和酶联免疫法检测外周血CD4⁺CD25⁺CD127^Low/- Treg计数及血清SCC-Ag水平。收集临床和病理资料,并统计检验2个指标对疗效的预测作用。结果：治疗前外周血CD4⁺CD25⁺CD127^Low/- Treg计数在临床有效组低于无效组［(8.78±2.80)% vs (10.95±2.56)%,P<0.05］,血清SCC-Ag在不同临床疗效组间差异无统计学意义,且这2个、指标之间未发现相关性(Spearman’rho=-0.093,P=0.540)。经受试者工作特征(receiver operating characteristic,ROC)曲线确定治疗前外周血CD4⁺CD25⁺CD127^Low/- Treg及血清SCC-Ag最佳界值分别为9.76%与9.50 ng/mL。单因素分析显示,治疗前外周血CD4⁺CD25⁺CD127^Low/- Treg计数(OR=1.901,95%CI：1.112~3.219,P=0.017)对CCRT疗效有预测作用,而血清SCC-Ag水平无预测作用(OR=0.998,95%CI：0.001~4.253,P=0.897)。多因素Logistic回归分析显示,治疗前外周血CD4⁺CD25⁺CD127^Low/- Treg为独立的临床疗效预测因子(OR=3.115,95%CI：1.253~7.742,P=0.014)。结论：治疗前外周血CD4⁺CD25⁺CD127^Low/- Treg计数用于中晚期宫颈鳞癌患者CCRT临床疗效预测具有可行性。     

Both CD133+ and CD133− subpopulations of A549 and H446 cells contain cancer-initiating cells

Tumors have been known to contain a small population of cancer stem cells that initiate tumor growth and promote tumor spreading. CD133 alone or in combination with other markers is currently being used for identification and isolation of the putative cancer stem cell population from malignant tumors. To determine whether the CD133⁺ cells constitute the stem cell populations of lung cancer cells A549 and H446, CD133⁺ and CD133⁻ subpopulations were sorted from A549 and H446 cells by magnetic cell separation and characterized for their in vitro stem cell-like properties. Interestingly, both the CD133⁺ and CD133⁻ cells displayed similar abilities of colony formation, self-renewal, proliferation, differentiation, and invasion, as well as resistance to chemotherapy drugs. Furthermore, colony formation assays showed that more than 40% of cells in both the CD133⁺ cells and CD133⁻ subpopulations could form large colonies capable of regenerating the unsorted populations and forming tumors in nude mice. These results suggest that CD133 alone cannot be used as a stem cell marker for the lung cancer cells A549 and H446, and both the CD133⁺ and CD133⁻ subpopulations contain similar numbers of cancer stem cells. ( Cancer Sci 2009; 100: 1040–1046)     

Alterations in lymphocyte subsets in blood may predict resectability in carcinoma of cardia or oesophagus

Impaired immune responses in patients with carcinoma of cardia or oesophagus have previously been reported. However, we do not know whether resectability correlates with specific immunological variables. Immunological assessment was performed in 35 such cancer patients including measurement of total T cells (CD3⁺) and T cell subsets (CD4⁺ and CD8⁺), NK cells (CD16⁺) and B cells (CD19⁺) in blood. In vitro lymphocyte responses to phytohemagglutinin (PHA) separated from peripheral blood were quantitated. The numbers in peripheral blood of both total T cells (CD3³⁺) and B lymphocytes (CD19⁺) were significantly lower in the inoperable patients compared to resected patients (P < 0.01). The number of NK cells (CD16⁺) was, however, not significantly lower in the inoperable patients compared to the patients operated for cure. Lymphocyte responses to PHA in vitro were similar in resectable and non-resectable patients, but significantly lower in inoperable patients compared to the controls (P < 0.01). In conclusion, resectability in carcinoma of cardia or oesophagus is associated with changes in both T (CD3⁺) and B (CD19⁺) cell subsets.