喉癌CD133+肿瘤干细胞与化疗抵抗

目的 筛选喉癌细胞中的CD133+细胞亚群,探讨其肿瘤干细胞特性及化疗抵抗性,并分析其原因。 方法 流式细胞仪检测Hep-2细胞经顺铂,氟尿嘧啶,紫杉醇作用后,CD133表达率的变化。采用流式细胞分选技术分离CD133+,CD133-细胞亚群,观察各亚群对上述药物的反应。采用软琼脂克隆形成实验检测各亚群的克隆形成能力,采用transwell小室体外侵袭实验,检测各亚群细胞的侵袭能力。采用逆转录聚合酶链反应(RT-PCR)和western blot法,检测各亚群细胞中耐药基因ABCG2基因的表达。 结果：Hep-2细胞中CD133表达率为1-2%. 化疗药物作用后CD133+亚群被富集。CD133+细胞克隆形成率为(41.9±3.9%)显著高于CD133-细胞(11.7±1.6%), P<0.01。CDl33+细胞侵袭细胞数为(88±9.7)显著高于CD133-细胞（53±7.2）, P<0.01。CD133+细胞ABCG2的表达水平显著高于CD133-细胞。 结论： CDl33+喉癌细胞具有肿瘤干细胞的生物学特性, CD133+喉癌肿瘤干细胞存在化疗抵抗,ABCG2的高表达是其中的主要原因。针对CD133+细胞的治疗势必会促进对喉癌治疗的进展。     

桑辛素对喉癌干细胞干性表型调控的作用研究

  目的  探索桑辛素对喉癌干细胞干性表型调控的影响。  方法  流式细胞仪分选并检测CD133+喉癌干细胞比例;肿瘤球形成实验检测CD133+喉癌干细胞的自我更新能力;Transwell实验检测CD133+喉癌干细胞的迁移能力;改良MTT实验检测化疗药物对CD133+喉癌干细胞的细胞毒性作用;免疫荧光染色、实时荧光定量PCR（RT-qPCR）及Western blot检测CD133+喉癌干细胞的干细胞标志物表达;在不同浓度的桑辛素处理CD133+喉癌干细胞后（以桑辛素0 μmol/L为对照）,通过肿瘤球形成实验、Transwell实验、改良MTT实验及Western blot,分别检测CD133+喉癌干细胞的自我更新能力、迁移能力、化疗药物的细胞毒性作用及干细胞标志物表达变化。  结果  流式细胞仪分选结果显示,CD133+喉癌干细胞占喉癌细胞的比例为（3.50±0.34）%;经过培养富集后,其比例可达（93.20±5.23）%。肿瘤球形成实验结果显示,与喉癌细胞相比,CD133+喉癌干细胞具有增强的自我更新能力 (P<0.001);Transwell实验显示,与喉癌细胞相比,CD133+喉癌干细胞的迁移能力增强（P<0.05）;改良MTT实验结果显示,与喉癌细胞相比,CD133+喉癌干细胞抵抗化疗药物（5-氟尿嘧啶及顺铂）的细胞毒性作用（P<0.05）;免疫荧光染色、RT-qPCR及Western blot结果显示,干细胞标志物（CD133、ALDH1、Sox2、ABCG2及N-cadherin）在CD133+喉癌干细胞中呈高表达水平。通过不同浓度的桑辛素处理CD133+喉癌干细胞,其自我更新能力降低（P<0.05）;迁移能力亦下降（P<0.05）;此外,桑辛素处理的CD133+喉癌干细胞对化疗药物的细胞毒性作用更加敏感（P<0.05）;Western blot结果显示,不同浓度的桑辛素处理的CD133+喉癌干细胞,其上述的干细胞标志物表达水平下调（P<0.05）。  结论  CD133+喉癌干细胞具有干性表型特征;桑辛素可减弱喉癌干细胞的干性表型,可能与下调其干细胞标志物表达相关。     

喉癌干细胞的培养及其放射抗拒相关miRNA的筛选研究

目的通过培养Hep-2喉癌细胞株,分离并扩增出肿瘤干细胞,放疗后筛选出调控放射抗拒的miRNA。方法含生长因子的无血清培养基培养Hep-2喉癌细胞株,流式细胞仪检测细胞中侧群细胞的比例。当侧群细胞比例达25%时,收集全部细胞球,筛选出CD133+、CD44+细胞作为喉癌干细胞,X线照射喉癌干细胞。miRNA生物芯片检测照射与未照射的喉癌干细胞,筛选出喉癌干细胞中2倍差异表达的miRNA,确定用以调控放射抗拒的miRNA。结果成功培养出侧群细胞,流式细胞仪检测其比例达到26.2%,筛选出的CD133+、CD44+细胞的比例均高于91%。通过miRNA芯片成功检测出喉癌干细胞中2倍差异表达的miRNA。结论本研究成功培养出喉癌干细胞,成功筛选出喉癌干细胞中2倍差异表达的miRNA,发现了可能用以调控放射抗拒的miRNA。     

小细胞肺癌细胞系H446中肿瘤干细胞的分选与鉴定

 目的 明确能否以表面黏附分子CD133为干细胞标志物,使用免疫磁珠分选（magnetic cell separation,MACS）法分离小细胞肺癌细胞系H446细胞中的肿瘤干细胞。方法 以CD133为特异性分子标志物,使用MACS方法分选H446细胞,比较CD133阳性（CD133+）细胞与CD133阴性（CD133-）细胞在集落形成、自我更新、增殖分化、侵袭性、耐药性和成瘤性方面的不同。结果 CD133+细胞和CD133-细胞在集落形成能力、自我更新能力、增殖能力、分化能力、侵袭能力和耐药性方面基本相同。集落形成和成瘤性实验结果显示：CD133+或CD133-细胞中40%以上的细胞都能够形成含有100个细胞以上的大集落,增殖成为与未分选细胞相同的细胞群,并能在裸鼠身上成瘤。结论 表面黏附分子CD133不能作为分选小细胞肺癌H446细胞系中肿瘤干细胞的分子标志物,分选后的CD133+与CD133-细胞中含有相同的肿瘤干细胞。     

miRNA-29c在喉癌组织中的表达及对喉癌Hep-2细胞增殖、侵袭的影响

目的 观察miRNA-29c 在喉癌组织和喉癌Hep-2 细胞株中的表达情况,研究其与肿瘤临床分期、淋巴转移及预后等临床病理参数的关系,并探讨miRNA-29c 对喉癌Hep-2 细胞增殖、侵袭的影响。 方法 选取2006 年1 月～2016 年12 月在我院确诊的86 例喉癌患者的蜡块标本,利用实时荧光定量聚合酶链式反应检测喉癌组织和癌旁正常喉黏膜上皮组织中miRNA-29c 的表达,并分析其与临床病理学特征的关系;通过Q-RT-PCR 检测转染后每组细胞中miRNA-29c 的表达;CCK-8 检测细胞增殖能力变化情况;Transwell 实验检测跨膜细胞数量的变化。 结果miRNA-29c 在喉癌组织中相对表达量明显低于癌旁组织（P<0.01）,其表达量与不同临床分型、TNM分期、淋巴转移密切相关（P<0.05）;转染miRNA-29c 模拟物组细胞中的miRNA-29c 表达水平显著高于无关序列组和空白对照组（P<0.01）,并可抑制喉癌Hep-2 细胞的增殖和侵袭力（P<0.01）。 结论 在喉癌中,miRNA-29c 是一个抑制性miRNA,miRNA-29c 低表达可能是影响喉癌预后的重要因素;过表达miRNA-29c 可显著降低喉癌Hep-2 细胞的增殖和侵袭能力,miRNA 表达水平的下调可能参与了喉癌的发生发展及侵袭转移过程。     

分割剂量射线照射富集宫颈癌肿瘤干细胞的研究

【目的】 研究分割剂量射线照射的宫颈癌Hela细胞表达肿瘤干细胞相关蛋白情况及裸鼠体内成瘤能力,探讨经此法富集宫颈癌肿瘤干细胞的可行性。【方法】 Hela细胞分组接受分割剂量分别为2、4、6、8、10及12 Gy的射线照射,总照射剂量24 ~ 42 Gy。流式细胞术检测各照射组及对照组细胞的ABCG2、CD44及CD133表达率;各组细胞接种裸鼠皮下,检测其体内成瘤能力。【结果】 各照射组细胞的ABCG2表达率均明显上调,以Hela-R2组(4 Gy×10次)细胞的表达率最高[(46.89±1.20)%],且随着照射次数的增加而升高(P < 0.05);各组细胞的CD44及CD133表达率变化均无明显趋势;Hela-R2组细胞的裸鼠体内成瘤能力最高,其次为Hela-R5组(10 Gy×3次)。【结论】 分割剂量射线照射可在体外富集宫颈癌肿瘤干细胞,以分割剂量为4 Gy组富集效果最好,诱导Hela细胞株表达ABCG2显著上调,及提高其裸鼠体内成瘤能力。     

人卵巢肿瘤细胞系3AO中肿瘤干细胞的分离鉴定

目的利用CD133抗体偶联的免疫磁珠分选法从人卵巢肿瘤3AO细胞系中分离CD133+细胞,并且通过检测其抗凋亡能力和耐药性进一步鉴定其特征。方法通过免疫磁珠分选方法分离人卵巢肿瘤细胞系中CD133+肿瘤干细胞,流式细胞仪检测肿瘤干细胞自我更新能力和抗凋亡能力,裸鼠移植实验反映成瘤性,MTT法检测其耐药性。结果通过免疫磁珠手段可成功分离得到CD133+卵巢肿瘤肿瘤干细胞,细胞增殖活力好,自我更新能力强,随着培养时间的延长可产生大量的CD133-细胞。裸鼠移植成瘤实验表明CD133+细胞成瘤率是10/10,成瘤平均时间是(58±6)d;而CD133-成瘤率是4/10,平均成瘤时间是(145±8)d,CD133+细胞成瘤能力明显强于CD133-细胞。MTT检测结果表明CD133+细胞的IC50值为CD133-细胞的2.5倍左右,提示对顺铂药物不敏感。对使用顺铂的细胞进行吖啶橙染色后发现,大量CD133-细胞呈现细胞凋亡状态,而CD133+细胞形态基本正常。进一步的AnnexinⅤ-FITC和PI双染结果表明,药物处理后的CD133+细胞比CD133-细胞具有更强的抗凋亡能力。结论免疫磁珠分选得到的CD133+细胞具有自我更新、增殖和分化为CD133-细胞等肿瘤干细胞的特征。CD133+细胞比CD133-细胞具有较强的成瘤能力、耐药性及抗凋亡能力。     

HepG2、Hep3B细胞中肿瘤干细胞相关标志分子的表达

目的富集培养肝癌干细胞,研究肝癌干细胞相关标志物CD90、CD133、八聚体4(Oct4)和ATP结合盒转运蛋白ABCG2在肝癌细胞HepG2、Hep3B中的表达,并初步分析其意义。方法使用流式细胞仪从肝癌细胞(检测HepG2、Hep3B两种细胞系)中分选肝癌干细胞并行无血清成球培养。设肝癌细胞组为对照组。单细胞克隆形成实验检测细胞增殖能力。分别给予肝癌细胞及肝癌干细胞阿霉素处理,MTT法测阿霉素处理后各组细胞的存活率,Real-time PCR及Western blot分别检测各组细胞CD90、CD133、Oct4和ABCG2 mRNA及蛋白水平的表达。结果肝癌干细胞单个细胞增殖能力强于肝癌细胞,克隆形成分析显示培养第14天克隆形成率Hep3B细胞组低于Hep3B CSCs组[8/27(30%)vs 12/23(52%),P<0.05];HepG2细胞组克隆形成率也低于HepG2 CSCs组[7/38(18%)vs 9/26(35%),P<0.05]。用阿霉素处理肝癌细胞及肝癌干细胞48 h后,MTT法测定结果显示肝癌干细胞组细胞活性显著高于对照组(P<0.05):HepG2细胞组细胞活性为(38.17±6.92)%、HepG2 CSCs组为(69.88±5.43)%;Hep3B细胞组(50.16±4.89)%,Hep3B CSCs组为(78.53±7.86)%。Real-time PCR结果显示肝癌干细胞组中CD90、CD133、Oct4及ABCG2基因mRNA表达较亲代细胞组显著上调(P<0.05)。Western blot结果显示肝癌干细胞组Oct4及ABCG2蛋白水平显著上调,与亲代细胞组间表达差异有显著性(P<0.05)。结论肝癌干细胞相关标志CD90、CD133、Oct4及ABCG2均高表达于肝癌干细胞,并且Oct4及ABCG2基因的高表达有可能与肝癌干细胞耐药性相关。     

Influence of CD133~+ expression on patients' survival and resistance of CD133~+ cells to anti-tumor reagents in gastric cancer

Objective:To investigate the influence of CD133+expression on patients'survival and resistance of CD133+cells to anti-tumor agents in gastric cancer(GC).Methods:Influence of CD133 expression on prognosis was analyzed employing samples from patients with GC.GC cell lines were utilized to separate CD133+and CD133-subpopulations by immunomagnetic separation and to analyze the biological features of two subpopulations in vitro and in vivo,especially in resistant to anti-tumor reagents and its apoptotic mechanism.Results:The lower CD133+group showed a significantly better survival compared with the higher CD133+group.The highest content of CD133+subpopulations for KATO-III cells had stronger proliferative ability than CD133-subpopulations.A single CD133+cell was capable of generating new cell colony and the tumorigenicity rate in nude mice was100%for CD133+clonal spheres or for CD133+cells,but 0%for CD133-cells.Furthermore,the higher expression levels of Oct-4,Sox-2,Musashi-1 and ABCG2 in CD133+clonal spheres were identified compared with CD133+cells or CD133-cells.Under the treatment of anti-tumor reagents,CD133+cells had lower suppression rates compared with CD133-cells while lower level of Bcl-2 and higher level of Bax were found in CD133+cells compared with CD133-cells.Conclusions:The patients with lower CD133+expression had a better survival.Enriched CD133+cells in clonal sphere shared the ability to be self-renewable,proliferative,tumorigenic and resistant to anti-tumor agents as probably regulated by Bcl-2 and Bax.     

Rac1+ cells distributed in accordance with CD 133+ cells in glioblastomas and the elevated invasiveness of CD 133+ glioma cells with higher Rac1 activity

Background  Recent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma (GBM) cells is also implicated in the failure of current therapies, it is not clear how glioma stem cells (GSCs) are involved in invasiveness. Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement. In this study, we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells, and analyze the migration and invasion potential of these cells.

Methods  A series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011. Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance. Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens. Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression. Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line. Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133- U87 cells. The migration and invasive ability of CD133+ and CD133- U87 cells were determined by cell migration and invasion assays in vitro. Student’s t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.

Results  In the central parts of GBMs, CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining. In the peripheral parts of the tumors, CD133+ cells were lined up along the basement membrane of the vessels and myelinated nerve fibers. Rac1 expression was high and diffused in the central parts of the GBMs, and the Rac1+ cells were distributed basically in accordance with CD133+ cells both in the central and peripheral parts of GBMs. In double-labeling immunofluorescence, Rac1 was expressed in (83.14±4.23)% of CD133+ cells, and CD133 and Rac1 co-expressed cells were located around the vessels in GBMs. Significantly higher amounts of Rac1-GTP were expressed in the CD133+ cells (0.378±0.007), compared to CD133- cells (0.195±0.004) (t=27.81; P <0.05). CD133+ cells had stronger ability to migrate (74.34±2.40 vs. 38.72±2.60, t=42.71, P <0.005) and invade (52.00±2.28 vs. 31.26±1.82, t=30.76, P <0.005), compared to their counterpart CD133- cells in transwell cell migration/invasion assay.

Conclusions  These data suggest that CD133+ GBM cells highly express Rac1 and have greater potential to migrate and invade through activated Rac1-GTP. The accordance of distribution between Rac1+ cells and CD133+ cells in GBMs implies that Rac1 might be an inhibited target to prevent invasion and migration and to avoid malignant glioma recurrence.

     

丹酚酸B对人喉癌Hep-2细胞增殖、凋亡及侵袭转移能力的影响及机制研究

目的研究丹酚酸B对人喉癌Hep-2细胞增殖、凋亡及侵袭转移能力的影响,并探讨其作用机制。方法运用CCK-8检测丹酚酸B对人喉癌Hep-2细胞增殖活性的影响。运用流式细胞仪检测丹酚酸B对人喉癌Hep-2细胞凋亡率的影响。划痕实验检测丹酚酸B对人喉癌Hep-2细胞迁移能力的影响。Transwell侵袭实验比较丹酚酸B对人喉癌Hep-2细胞侵袭转移能力的影响。Western blot法检测丹酚酸B对人喉癌Hep-2细胞PI₃K、Akt、Bcl-2蛋白表达水平的影响。结果CCK-8法发现丹酚酸B能抑制人喉癌Hep-2细胞增殖,其作用呈浓度依赖性。流式细胞术分析发现丹酚酸B能诱导人喉癌Hep-2细胞发生凋亡。划痕实验发现丹酚酸B能抑制人喉癌Hep-2细胞的迁移能力。Transwell实验发现丹酚酸B能抑制人喉癌Hep-2细胞的体外侵袭能力。Western blot法检测发现测丹酚酸B能有效降低人喉癌Hep-2细胞PI₃K、Akt、Bcl-2蛋白表达水平。结论丹酚酸B在体外能够有效抑制人喉癌Hep-2细胞增殖,诱导细胞凋亡,同时抑制喉癌细胞的体外侵袭转移能力。其作用可能与下调PI₃K、Akt、Bcl-2蛋白表达水平相关。     

转录因子Slug和Sox9对鼻咽癌干细胞侵袭迁移能力的实验研究

［摘要］目的　研究Slug及Sox9在鼻咽癌干细胞侵袭、转移中的作用．方法　通过CD133+分选CNE-2鼻咽癌干细胞, 利用siRNA技术共沉默鼻咽癌CD133+干细胞中Slug和Sox9,通过划痕实验观察其对细胞侵袭、迁移能力等生物学行为的影响．结果　CNE-2/CD133+ 组在24 h、48 h迁移能力明显高于各干扰组及CNE-2组,各干扰组在24 h与CNE-2组差异无统计学意义（P＞0.05）,48 h较CNE-2组减弱,在72 h及96 h CNE-2/CD133+ 组与CNE-2明显强于各干扰组（P＜0.05）,各干扰组在各时间无统计学意义（P＞0.05）．结论　 Slug和Sox9的沉默对鼻咽癌进展及转移有影响,其低表达与肿瘤的复发、患者生存期等的关系仍需进一步研究．     

X线辐射联合替莫唑胺化疗对胶质瘤CD133、ABCG2表达的影响

［摘要］目的　分析X线辐射联合替莫唑胺化疗对胶质瘤CD1333、ABCG2表达的影响,为临床治疗提供参考．方法　以武警陕西总队医院在2013年1月至2015年2月人脑胶质瘤64例为观察对象,依照分级不同分为低级别组25例和高级别组39例,均给予X射线符合和替莫唑胺化疗,分析不同级别胶质瘤干预前后胶质瘤干细胞标志物CD133和ADCGG表达变化．结果 干预前,高级别组CD133表达（89.7％）和ABCG2表达（92.3％）均显著高于低级别组CD133表达（68.0％）和ABCG2表达（81.8％）,P＜0.05,干预后高级别组和低级别组CD133表达和ABCG2表达均显著降低,P＜0.05,高级别组CD133表达（56.4％）和ABCG2表达（56.4％）均显著高于低级别组CD133表达（20.0％）和ABCG2表达（24.0％）,P＜0.05,干预前后,CD133（r＝-0.782）和ABCG2（r＝-0.652）表达与细胞恶化程度呈现负相关,P＜0.05．结论　不同恶化程度胶质瘤细胞CD133和ABCG2表达存在一定差异,X线辐射联合替莫唑胺化疗治疗能够有效改善CD133和ABCG2表达．     

Glioblastoma stem cells resistant to temozolomide-induced autophagy

Background Recent studies have demonstrated the existence of a small fraction of cells with features of primitive neural progenitor cells and tumor-initiating function in brain tumors. These cells might represent primary therapeutic target for complete eradication of the tumors. This study aimed to determine the resistant phenotype of glioblastoma stem cells （GSCs） to temozolomide （TMZ） and to explore the possible molecular mechanisms underlying TMZ resistance. Methods Freshly resected glioblastoma specimen was collected and magnetic isolation of GSCs was carried out using the Miltenyi Biotec CD133 Cell Isolation kit. The cytotoxic effect of TMZ on CD133^＋ and CD133^- glioblastoma cells was determined by using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Autophagy-related proteins （Beclin-1, LC3 and Atg5） and cleaved caspase-3 （p17） were analyzed by Western blotting. Immunofluorescent staining was used to detect Atg5, glial fibrillary acidic protein （GFAP） and CD133 expression in glioblastoma cells. Statistical analysis was carried out using SPSS 10.0 software. For all tests, the level of statistical significance was set at P 〈0.05. Results CD133^＋ glioblastoma ceils exhibited neurosphere-like growth in vitro and high expression of CD133 stem cell marker. The growth-inhibiting rate in CD133- glioblastoma cells treated with 5 or 50 pmol/L TMZ was significantly higher than that in CD133^＋ glioblastoma cells （（14.36±3.75）% vs （2.54±1.36）% or （25.95±5.25）% vs （2.72±1.84）%, respectively, P 〈0.05）. Atg5, LC3-11 and Beclin-1 levels were significantly lower in CD133^＋ glioblastoma cells than those in autologous CD133^- cells after TMZ treatment （P 〈0.05）. Caspase-3 was mildly activated only in CD133^- glioblastoma cells after exposure to TMZ （P 〈0.05）. Immunofluorescent staining revealed elevated expression of Atg5 in GFAP^＋ cells following TMZ treatment. Conclusions The GSCs display strong capability of tumor＇s resistance to TMZ. This resistance is probably contributed by the CD133^＋ cells with down-regulation of autophagy-related proteins. Future treatment should target this small population of cancer stem cells in tumors to improve survival of patients.     

肺癌肿瘤干细胞研究新进展

自从小鼠非小细胞肺癌模型最初阶段分离到支气管肺泡干细胞(BASCs)后;陆续发现A549肺癌细胞株Holoclone型克隆集落存在不断自我更新的肿瘤干细胞,人肺腺癌中CD24+IDF-1R-细胞为BASCs样癌干细胞。研究表明,CD133分子、ABCG2可作为分选肺癌肿瘤干细胞细胞表面标志物,BMI-1基因过表达可能与肺癌肿瘤干细胞增殖相关。进一步发现侧群细胞细胞中存在肺癌干细胞。本文对肺癌肿瘤干细胞及相关研究进展予以综述。     

肿瘤干细胞标志物CD133、ABCG2、p75~（NTR）在非小细胞肺癌组织的表达及其生物学意义

目的探讨肿瘤干细胞标志物CD133、ABCG2及p75NTR在非小细胞肺癌（NSCLC）组织中的表达及其生物学意义。方法采用免疫组织化学方法,检测44例NSCLC组织、17例癌旁组织、17例非肿瘤组织中的CD133、ABCG2及p75NTR蛋白的表达并对结果进行分析。结果 （1）CD133、ABCG2及p75NTR蛋白在44例NSCLC组织中的阳性表达率分别为68.2%（30/44）、31.8%（14/44）、47.7%（21/44）,在17例癌旁组织中分别为5.9%（1/17）、0（0/17）、11.8%（2/17）,在17例非肿瘤组织中分别为47.1%（8/17）、11.8%（2/17）、17.6%（3/17）,差异均有统计学意义（P〈0.05）;其中NSCLC组织中p75NTR蛋白的阳性表达与肿瘤的远处转移密切相关（P〈0.05）;（2）随肿瘤恶性度的增加,CD133蛋白的阳性表达率也明显增加,差异接近统计学意义（P=0.07）。结论 NSCLC组织中CD133、ABCG2及p75NTR的表达明显高于癌旁及非肿瘤组织,或许这三者与NSCLC恶性演进有关;肿瘤干细胞标记物CD133的表达与NSCLC的分化密切相关,CD133蛋白或许能成为NSCLC的肿瘤干细胞标记物。     

免疫磁珠法分离纯化人肾癌CD133~+细胞实验研究

目的:本研究通过分析CD133+细胞在肾细胞癌患者肿瘤组织的表达情况,寻找肾癌中CD133+细胞分离纯化方法,探寻CD133+细胞与肾癌发生中的作用。方法:①应用胎盼蓝拒染试验检测细胞活性。②应用免疫磁珠法分离纯化肿瘤组织中CD133+细胞。③应用流式细胞仪检测肾透明细胞癌标本中CD133+细胞表达情况。结果:30例肾透明细胞癌中CD133+比为(13.08±1.39)%,与病理分级、临床分期无关。免疫磁珠法分离纯化CD133+纯度为(75±2.4)%,细胞活性不受影响。结论:免疫磁珠技术纯化肾癌CD133+细胞成熟有效,CD133+细胞可能是肾癌干细胞。     

靛玉红对喉癌Hep-2细胞侵袭能力的影响及机制

目的探讨靛玉红甲肟（Indirubin-3＇-monoxime,IRO）对人喉癌Hep-2细胞迁移和侵袭的影响及其机制。方法MTT法检测不同浓度、不同作用时间IRO对Hep-2细胞增殖活性的影响。Boyden chamber实验计算各组穿膜的细胞数以比较靛玉红甲肟对细胞侵袭能力的变化。明胶酶谱检测不同浓度靛玉红甲肟处理喉癌Hep-2细胞后细胞MMP-9和MMP-2活性的变化。结果 MTT结果发现IRO对Hep-2细胞具有明显的增殖抑制作用,且表现为剂量依赖性（P〈0.01）。Boydenchamber体外迁移和侵袭实验结果显示：靛玉红甲肟可以显著抑制人喉癌Hep-2细胞的侵袭能力（P〈0.05）。明胶酶谱检测发现以靛玉红甲肟处理24h后人喉癌Hep-2细胞MMP-9和MMP-2活性明显降低,不同浓度组间存在显著性差异。结论靛玉红甲肟具有抑制人喉癌Hep-2细胞株增殖和侵袭的作用,其作用机制与显著抑制MMP-9和MMP-2活性有关。     

RNAi沉默PCDGF基因对喉鳞癌Hep-2细胞生物学特性的影响

目的 研究RNAi沉默畸胎瘤细胞源性生长因子(PCDGF)表达对喉鳞癌Hep-2细胞增殖、凋亡及侵袭能力的影响.方法 RT-PCR检测喉鳞癌组织中PCDGF的mRNA表达水平;Hep-2细胞分为空白对照组、阴性对照组和PCDGF-siRNA组,Western blot检测转染效果及Bcl-2、Bax、E-cadherin和MUC1蛋白表达;CCK8实验检测细胞增殖;流式细胞仪检测细胞凋亡;Transwell 小室检测细胞侵袭能力.结果 喉鳞癌组织中的PCDGF mRNA表达水平显著高于声带息肉组织(t=6.88,P=0.005).PCDGF在喉鳞癌组织中高表达.PCDGF-siRNA组喉鳞癌Hep-2细胞中PCDGF、Bcl-2、MUCI蛋白表达显著低于空白对照组(P<0.05),Bax、E-cadherin蛋白表达显著高于空白对照组(P<0.05),细胞增殖率显著低于空白对照组(P<0.05),细胞侵袭数显著低与空白对照组(P<0.05),细胞凋亡率显著高于空白对照组(P<0.05).结论 抑制PCDGF的表达能够抑制喉鳞癌Hep-2细胞的增殖和侵袭能力,诱导细胞发生凋亡.     

Enhanced invasion in vitro and the distribution patterns in vivo of CD133+ glioma stem cells

Background  Recent studies have suggested that cancer stem cells cause tumor recurrence based on their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma cells is also implicated in the failure of current therapies, it is not clear whether cancer stem cells are involved in invasiveness. This study aimed to assess invasive ability of glioma stem cells (GSCs) derived from C6 glioma cell line and the distribution patterns of GSCs in Sprague-Dawley (SD) rat brain tumor. 

Methods  Serum-free medium culture and magnetic isolation were used to gain purely CD133⁺ GSCs. The invasive ability of CD133⁺ and CD133^– C6 cells were determined using matrigel invasion assay. Immunohistochemical staining for stem cell markers and luxol fast blue staining for white matter tracts were performed to show the distribution patterns of GSCs in brain tumor of rats and the relationship among GSCs, vessels, and white matter tracts. The results of matrigel invasion assay were estimated using the Student’s t test and the analysis of Western blotting was performed using the one-way analysis of variance (ANOVA) test.

Results  CD133⁺ GSCs (number: 85.3±4.0) were significantly more invasive in vitro than matched CD133^– cells (number: 25.9±3.1) (t=14.5, P <0.005). GSCs invaded into the brain diffusely and located in perivascular niche of tumor-brain interface or resided within perivascular niche next to white fiber tracts. The polarity of glioma cells containing GSCs was parallel to the white matter tracts.

Conclusions  Our data suggest that CD133⁺ GSCs exhibit more aggressive invasion in vitro and GSCs in vivo probably disseminate along the long axis of blood vessels and transit through the white matter tracts. The therapies targeting GSCs invasion combined with traditional glioblastoma multiforme therapeutic paradigms might be a new approach for avoiding malignant glioma recurrence.