肠癌SW-620肿瘤细胞SP亚群研究

目的 观察肠癌细胞株SW-620中肿瘤干细胞相关的SP(side population)亚群比例,分选出相关的SP和NSP细胞,并鉴别相关的SP是否具有干细胞某些生物学特性.方法 制备SW-620细胞悬液,经Hoechst33342和PI染色(其中对照组同时加入拮抗剂维拉帕米),流式细胞仪分析SP亚群.通过流式细胞仪分选出肠癌细胞株SW-620中SP细胞.克隆形成实验和噻唑蓝(MTT)实验检测SP和NSP细胞增殖能力的差异.裸鼠体内成瘤对比实验验证SP细胞和NSP细胞致瘤性.结果 SP亚群占SW-620细胞的1.0%左右,维拉帕米阻断后减少为0.01%左右.流式细胞仪分选出SP细胞7.5×105个,大部分为NSP细胞.克隆形成实验和MTT证实SP细胞和NSP细胞增殖能力有差异.裸鼠体内成瘤实验发现:1×105个SP细胞可以致裸鼠成瘤,1×105个NSP细胞及以下数量级别细胞不能致裸鼠成瘤.结论 人肠癌细胞株SW-620中存在SP亚群细胞,维拉帕米可抑制染料外排而明显减少SP细胞的比例.可以通过流式细胞仪分选出SP细胞,通过克隆形成实验和MTT实验检测SP和NSP细胞增殖能力有差异.裸鼠成瘤对比实验证实SP比NSP细胞更具致瘤性.     

胰腺癌细胞株PANC-1中肿瘤干细胞的体外耐药性研究

目的:根据肿瘤干细胞学说理论,探讨胰腺癌耐药的新机制。方法:通过胰腺癌肿瘤干细胞表面标记途径和侧群(side population,SP)细胞途径,分离出人胰腺癌细胞株PANC-1中的SP/NSP(非SP)细胞及CD44+CD24+/CD44-CD24-细胞亚群,用MTT检测上述各亚群细胞在体外对化疗药物耐受的差异,用AnnexinV-PI双染法检测2种肿瘤细胞的抗凋亡能力,并采用实时荧光定量PCR检测两者耐药基因ABCG2、ABCB1和PLK-1表达的差异。结果:人胰腺癌细胞株PANC-1中的SP细胞比例为(7.64±0.96)%,CD44+CD24+细胞比例为(2.60±0.96)%。相对于NSP和CD44-CD24-细胞而言,SP和CD44+CD24+细胞具有更强的化疗耐受能力(P0.01)和抗凋亡能力(P0.01);荧光定量RT-PCR结果均提示,SP和CD44+CD24+细胞高表达耐药基因ABCG2、ABCB1和PLK1。结论:人胰腺癌细胞株PANC-1中SP及CD44+CD24+细胞具有更强的化疗耐受能力,研究胰腺癌肿瘤干细胞可为克服胰腺癌化疗敏感性差的现状提供新的实验基础和理论依据。     

肝癌SMMC-7721细胞中侧群细胞干细胞标记的表达

目的 分选肝癌细胞株SMMC-7721中的侧群(SP)细胞,并分析其干细胞标记的表达.方法 采用流式细胞荧光激活分选(FACS)技术将SMMC-7721细胞分为SP细胞和非侧群(NSP)细胞两个亚群,以实时荧光定量聚合酶链反应(real-time PCR)技术和流式细胞术对两个亚群细胞干细胞标记mRNA和蛋白表达进行分析.结果 SMMC-7721细胞株中分选出的SP细胞比例为(9.2±0.2)%.SP细胞ABCG2、CD133、Oct4、Sox2和NANOG等干细胞标记mRNA的表达水平分别是NSP细胞的7.132倍、4.985倍、8.642倍、5.095倍和5.164倍,差异均有统计学意义(P＜0.01);ABCG2、CD133、Oct4、Sox2和NANOG蛋白在肝癌SP细胞中的含量分别为(92.65±3.92)%、(12.75±1.62)%、(17.35±2.31)%、(9.57±1.71)%和(28.39±5.28)%,在NSP细胞中的含量分别为(0.26±0.06)%、(2.51±0.17)%、(1.74±0.38)%、(1.52±0.41)%和(3.37±1.02)%,差异有统计学意义(P＜0.01).结论 肝癌SMMC-7721细胞中的SP细胞可能富集了肝癌干细胞,联合应用多种干细胞标记筛选肝癌SP细胞可能会获得纯化的肝癌干细胞.

Abstract：

Objective To study the expression of stem cell markers in side population cells sorted from SMMC-7721 cell line. Methods Fluorescence-activated cell sorting (FACS) was used to sort side population (SP) cells and non-SP (NSP) cells from SMMC-7721 cell line. Real-time polymerase chain reaction (PCR) and flow cytometry (FCM) were used to evaluate the expression of several stem cell markers such as ABCG2, CD133, Oct4, Sox2 and NANOG in SP cells and NSP cells. Results FACS analysis indicated that (9.2 ±0. 2)% of the SMMC-7721 cells were SP cells. Real-time PCR analysis suggested that ABCG2, CD133, Oct4, Sox2 and NANOG were expressed in the SP cells at higher levels than the NSP cells by about 7. 132, 4. 985, 8. 642, 5.095 and 5. 164 folds, respectively ( P ＜0. 01 ). FCM analysis revealed that the expression of ABCG2, CD133, Oct4, Sox2 and NANOG proteins in SP cells was (92. 65 ±3.92)%, (12.75 ±1.62)%, (17.35 ±2.31)%, (9.57 ± 1.71)% and (28.39 ±5.28)% respectively,while in NSP cells that was (0. 26 ±0. 06)%, (2. 51 ±0. 17)%, ( 1.74 ±0. 38)%, ( 1.52 ±0. 41 )% and ( 3.37 ± 1.02) % respectively ( P ＜ 0. 01 ). Conclusion The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells. Purified liver cancer stem cells may be obtained by screening SP cells using a variety of stem cell markers.     

MHCC97中肝癌干细胞的分离及其特性

目的 分离肝癌细胞系MHCC97中肝癌干细胞并观察其干细胞特性.方法 利用流式分选法从MHCC97中分离出边缘群(SP)和主群(MP).分别采用软琼脂克隆形成和裸鼠成瘤实验观察SP和MP细胞体内、外成瘤能力;羟基喜树碱(HCPT)体外诱导,逆转录-聚合酶链反应(RT-PCR)分析其分化潜能.结果 SP细胞克隆形成率为57%,MP细胞仅为13%;1×103SP细胞可成瘤(2/8),MP细胞则需1×105才能成瘤(2/8).诱导3 d后,约15%SP细胞出现双核;诱导后SP细胞甲胎蛋白和r-谷氨酰转肽酶的表达降低,白蛋白表达增强,葡萄糖磷酸酶仅诱导后表达.结论 MHCC97中SP亚群富集具有干细胞特性的癌细胞,即肝癌干细胞.     

膀胱癌细胞株T24中侧群细胞的分离和功能鉴定

目的 观察膀胱癌细胞株T24中是否存在侧群(SP)细胞及其比例,并鉴定其功能.方法 利用双波长流氏细胞仪(FACS)检测T24中SP细胞的比例,并证实这些SP细胞是否具有癌干细胞的特点.结果 T24中SP细胞占34.7%;与非侧群(NSP)细胞比较,SP细胞有更强的生长增殖能力和克隆形成能力(P＜0.05),表达更高的ATP结合转运蛋白G超家族成员2(ABCG2)和干性基因,对放化疗有更强的抵抗能力,有更多的细胞处于G_0/G_1期(87.4%比63.3%,P＜0.05);分选后的sP和NSP细胞经过约10 d的常规培养,SP细胞中NSP的比例占76.2%,而NSP细胞中SP的比例只占2.6%.结论 膀胱癌细胞株T24中存在很高比例的SP细胞,而且这些SP细胞有癌干细胞的特点.     

人肝癌细胞系MHCC97H中侧群细胞初探

目的 从人肝癌细胞系MHCC97H中分选侧群细胞,探索其形态、表型和功能特征.方法 利用流式细胞仪从MHCC97H中分选得到侧群细胞,用相差显微镜和透射电镜观察其形态学特点;Western blotting观察其ABCG2表达;用流式细胞仪评估侧群细胞与干细胞基因CD133、CD117的关系;用克隆形成实验和NOD/SCID接种实验分别观察侧群细胞体外增殖和体内成瘤情况.结果 侧群细胞体积较小,呈圆形或短梭形;侧群细胞的细胞器如线粒体、内质网等明显少于非侧群细胞;流式检测显示侧群细胞中含有CD133、CD117阳性细胞;Western blotting显示侧群细胞和非侧群细胞中均有ABCG2表达,两者无明显差异;平板克隆形成实验显示SP具有较强的克隆形成能力;体内移植实验显示2000个SP细胞即能在体内形成肿瘤.结论 肝癌细胞系MHCC97H中侧群细胞具有肿瘤干细胞样细胞的表型和特性;ABCG2可能不是决定肝癌侧群细胞表型的主要因素.     

胰腺癌肿瘤干细胞对抗肿瘤药物敏感性的实验研究

目的 探讨胰腺癌肿瘤干细胞对抗肿瘤药物的敏感性.方法 FACS技术分选人胰腺癌PANC-1细胞;RT-PCR技术检测分选PANC-1细胞中CD133、ABCG2、Notch1的表达情况;MTT法检测分选细胞对抗肿瘤药物5-氟尿嘧啶和吉西他滨的耐药性.建立分选细胞的移植瘤模型,随机分为吉西他滨治疗组(n=3)和对照组(n=3),观察肿瘤生长情况,作CD133免疫组化染色.结果PANC-1细胞中含有SP亚群.SP细胞CD133、ABCG2、Notch1的mRNA的表达明显上调,non-SP细胞的表达量显著低于前者.在吉西他滨的干预下,SP细胞和non-SP细胞的OD值差异有统计学意义.而5-氟尿嘧啶的干预(10 μg/ml和100 μg/ml)则没有显著差异.移植肿瘤治疗组中CD133阳性细胞明显多于对照组(P=0.001).结论胰腺癌中存在SP亚群细胞.胰腺癌PANC-1的成瘤能力是由其中的SP亚群细胞决定的,而并非所有胰腺癌PANC-1细胞.胰腺癌肿瘤干细胞对抗肿瘤药吉西他滨具有较高耐药性.     

人胆囊癌细胞系GBC-SD侧群细胞致瘤特性的实验研究

目的 研究人胆囊癌细胞系GBC-SD侧群细胞(side population cells,SP)的致瘤特性。方法 利用流式细胞术从GBC-SD中分选SP、非SP细胞(non-SP),分别进行软琼脂克隆形成实验和非肥胖性糖尿病联合免疫缺陷(NOD/SCID)小鼠的移植瘤形成实验。通过流式细胞术检测5例人胆囊癌组织中SP细胞的比例。结果人胆囊癌细胞系GBC-SD中存在SP细胞,其比例约为0.87％;SP细胞的克隆形成率高于non-SP细胞（14.74％±3.53％比5.17％±1.05％,t=2.75,P＜0.05）。NOD/SCID小鼠移植瘤实验表明,5×103个SP细胞可成瘤(4/7),而non-SP细胞成瘤则需要1×105个细胞(1/7),且来源于SP细胞亚群的NOD/SCID小鼠移植瘤中存在SP、non-SP细胞。SP细胞存在于人胆囊癌组织中,其范围为0.27％～2.3％。结论来源于GBC-SD的SP细胞亚群具有类似肿瘤十细胞的高致瘤性。     

多种转移潜能不同的人前列腺癌细胞“上皮细胞间质转化态”特性鉴定及其意义

目的:对多种转移潜能不同的人前列腺癌细胞“上皮细胞间质转化态”(EMT)特性进行鉴定,并从粘附因素和细胞骨架蛋白角度分析其骨转移潜能获得的分子机制。方法:用W estern印迹法鉴定LNCaP及其亚细胞系C4、C4-2和ArCaP亚细胞系IF11、IA8,以及PC-3、Du145等细胞中上皮型钙粘素(E-cadherin)、神经型钙粘素(N-cadherin)和波形纤维蛋白(V im entin)的表达差异情况,并分析其在前列腺癌转移过程中的作用。结果:E-cad-herin在PC-3、LNCaP、C4、C4-2中表达较高,但在Du145、IF11、IA8中表达极低;而V im entin的表达情况恰恰与E-cadherin相反;N-cadherin在IF11、IA8细胞中呈现显著的高表达状态。结论:转移潜能不同的人前列腺癌细胞株之间存在EMT表型的表达差异,其中PC-3、LNCaP、C4、C4-2是未发生EMT改变的细胞,Du145、IF11、IA8却是EMT化的细胞。EMT表型差异蛋白在解释前列腺癌转移机制方面占据着重要地位。     

人胆管癌CD24+ CD44+ EpCAMhigh细胞亚群的分选及其肿瘤干细胞样特性的鉴定

目的 检测及分选人胆管癌中的CD24+ CD44+ EpCAMhigh细胞亚群,探讨其是否具有肿瘤干细胞样生物学特性.方法 流式细胞术检测6例人胆管癌中CD24、CD44、EpCAM的表达率;取2例人胆管癌新鲜标本种植到NOD/SCID鼠皮下,建立荷人胆管癌小鼠模型.流式细胞术分选CD24+ CD44+ EpCAMhigh亚群细胞,NOD/SCID鼠移植瘤试验鉴定其成瘤和分化能力.结果 6例人胆管癌组织标本和2例移植瘤中,CD24+ CD44+ EpCAMhigh在人胆管癌中表达率为0.58%～2.43%(平均值0.94%).运用NOD/SCID鼠移植瘤模型,分选得到CD24+ CD44+ EpCAMhigh亚群细胞.NOD/SCID鼠移植瘤试验,1×103个CD24+ CD44+ EpCAMhigh细胞能成瘤(3/8),而CD24- CD44- EpCAMlow/-细胞在5×104才能成瘤(1/8).CD24+ CD44+ EpCAMhigh胆管癌细胞NOD/SCID鼠移植瘤的组织类型及标记物的表达率和原代肿瘤相似.结论 人胆管癌中含有CD24+ CD44+ EpCAMhigh细胞亚群,具有强成瘤能力、自我更新和分化的能力,可能是胆管癌干细胞.     

环氧化酶2在不同前列腺癌细胞系中的表达

目的:研究环氧化酶2(COX-2)在不同前列腺癌细胞系中的表达,探讨COX-2在前列腺癌侵袭进展及转移潜能获得机制中的可能作用。方法:应用Western印迹及RT-PCR鉴定LNCaP及其亚细胞系C4-2和AR-CaP亚细胞系IF11、IA8,以及PC-3细胞中COX-2的表达情况,并初步分析其在不同特性前列腺癌细胞系转移侵袭过程中的作用。结果:Western印迹结果显示:COX-2蛋白在PC-3细胞中表达相对较高,在IF11、IA8、LNCaP和C4-2细胞中表达缺失,差异具有统计学意义(P<0.05)。COX-2mRNA表达结果同蛋白一致。结论:不同来源、不同转移潜能的前列腺癌细胞株中COX-2表达存在差异。高表达COX-2可能在PC-3细胞高侵袭转移潜能获得方面起着一定作用,而与其他细胞系转移作用无关。     

In vivo investigation of CD133 as a putative marker of cancer stem cells in Hep-2 cell line

Background

Mounting evidence suggests that most tumors consist of a heterogeneous population of cells with a subset population that has the exclusive tumorigenic ability. They are called cancer stem cells (CSCs). CSCs can self‐renew to generate additional CSCs and also differentiate to generate phenotypically diverse cancer cells with limited proliferative potential. They have been identified in a variety of tumors. In this study, we identify the marker of CSCs in the established human laryngeal tumor Hep‐2 cell line in vivo. Our in vitro experiment shown as CD133, a 5‐transmembrane glycoprotein expressed in Hep‐2 cell line. CD133 was supposed as a candidate of CSC in laryngeal carcinoma. In this study, the expression of CD133 was detected in a Hep‐2 cell line. Applying the magnetic cell sorting (MACS) technology, we reported the results of purifying CD133 positive cells from a Hep‐2 cell line. Three‐type cells' tumor‐forming ability was examined in vivo to identify the marker of CSCs in Hep‐2 cell line.

Methods

CD133 was selected as a putative marker of CSC in laryngeal carcinoma, Hep‐2 cell lines. Flow cytometry was used to detect the expression of CD133 in the Hep‐2 cell line. Immunomagnetic beads were applied to purify CD133‐positive cells. CD133(+), CD133(?) tumor cells, and unsorted Hep‐2 cells were injected into severe combined immune deficiency (SCID) mice individually to observe tumor‐forming ability.

Results

Only a small proportion (3.15% ± 0.83%) of cells in the Hep‐2 cell line express the CD133 marker. In comparison with CD133(?) tumor cells and unsorted cells, CD133(+) cells possess a marked capacity for tumor formation in vivo (p <.05).

Conclusion

CD133 is 1 of the markers for CSCs in human laryngeal tumors of the Hep‐2 cell line. Work on the characterization of these cells provides a powerful tool to investigate the tumorigenic process in the larynx and to develop therapies targeting the CSC. © 2008 Wiley Periodicals, Inc. Head Neck, 2009

     

Pancreatic Carcinoma Cell Lines Reflect Frequency and Variability of Cancer Stem Cell Markers in Clinical Tissue

Background: Pancreatic cancer is one of the most deadly malignancies with insufficient therapeutic options and poor outcome. Cancer stem cells (CSCs) are thought to be responsible for progression and therapy resistance. We investigated the potential of pancreatic cell lines for CSC research by analyzing to what extent they contain CSC populations and how representative these are compared to clinical tissue. Methods: Six pancreatic cancer cell lines were analyzed by flow cytometry for CD326, CD133, CD44, CD24, CXCR4 and ABCG2. Subsequently, 70 primary pancreatic tissues were evaluated for CD326, CD133 and CD44 by immunohistochemistry. Results: All the cell lines but one showed a stable expression pattern throughout biological replicates. Marker expression in clinical tissue of CD44 distinguished normal patients from pancreatic carcinoma patients with a sensitivity of 50% at 80% specificity and metastasized from nonmetastasized carcinomas with 69% sensitivity at 100% specificity. Conclusions: Our results indicate a link between elevated CD44 expression, malignancy and metastasis of pancreatic tissue. Furthermore, individual pancreatic cell lines show a substantial amount of cells with CSC properties which is comparable with interpatient variability detected in primary tissue. These pancreatic cancer cell lines could thus serve for urgently needed pharmacological CSC in vitro research.     

Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations

BACKGROUND: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. METHODS: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. RESULTS: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells. CONCLUSIONS: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.     

Prostate cancer stem cell biology

The cancer stem cell (CSC) model provides insights into pathophysiology of cancers and their therapeutic response. The CSC model has been both controversial, yet provides a foundation to explore cancer biology. In this review, we provide an overview of CSC concepts, biology and potential therapeutic avenues. We then focus on prostate CSC including 1) their purported origin as either basal-derived or luminal-derived cells; 2) markers used for prostate CSC identification; 3) alterations of signaling pathways in prostate CSCs; 4) involvement of prostate CSCs in metastasis of PCa; and 5) microRNA-mediated regulation of prostate CSCs. Although definitive evidence for the identification and characterization of prostate CSCs still remains unclear, future directions pursuing therapeutic targets of CSCs may provide novel insights for the treatment of PCa.     

人膀胱癌组织中肿瘤干细胞样细胞的分离培养及PIWIL2表达

目的从人膀胱移行细胞癌（BTCC）组织中分离培养肿瘤干细胞样细胞（CSLC）,鉴定其生物学特性,并研究PIWIL2在CSLC中的表达和意义。方法收集12例不同临床分期BTCC患者组织标本,通过酶消化和原代培养相结合等方法处理,采用无血清悬浮培养法分离培养获得含CSLC的悬浮细胞球,流式细胞仪检测细胞表面分子标志CD133和CD44的表达,免疫磁珠分选系统分离CD133＋CD44＋细胞;采用半定量逆转录聚合酶链反应检测PIWIL2mRNA在CSLC中的表达;裸鼠皮下接种CS-LC,观察其成瘤能力。结果成功地从人膀胱癌组织分离培养获得可悬浮生长、稳定传代的CSLC,CSLC高表达CD133和CD44,裸鼠皮下接种1×104、1×105个CSLC均可全部成瘤,PIWIL2mRNA在CSLC的表达显著高于正常膀胱组织细胞。结论采用无血清悬浮培养法成功从人膀胱癌组织中分离获得CSLC,具有肿瘤干细胞特性,能高度表达PIWIL2,为靶向肿瘤干细胞的治疗提供了实验依据。