无血清悬浮法培养结肠癌HT29细胞系及肿瘤干细胞样亚群筛选

目的:研究无血清培养基悬浮培养人结肠癌细胞系HT29,筛选并鉴定HT29结肠癌细胞系中肿瘤干细胞相关亚群。方法:通过无血清培养基筛选结肠癌细胞系肿瘤干细胞相关亚群。应用克隆形成实验、表面标志检测、双苯酰亚胺(Hoechst)33342染色检测来确定培养细胞中肿瘤干细胞比例及其培养后的肿瘤干细胞含量变化。结果:结肠癌细胞系HT29中约50%的肿瘤细胞在无血清培养基中能够存活、增殖,形成自由飘浮的细胞球。细胞球可连续传代,若重新接种于含血清培养基中可重新贴壁分化,分化后细胞与培养储存的HT29细胞形态无明显差异。流式细胞仪检测表面标志,HT29细胞系中CD44+细胞含量为(44.18±2.18)%,而细胞球中CD44+细胞含量为(83.41±11.21)%;双苯酰亚胺33342染色检测提示HT29中侧群(sidepopulation,SP)细胞含量为(3.82±0.08)%,而细胞球中含量明显增高。结论:结肠癌细胞系HT29可在含生长因子的无血清培养基中悬浮生长,并维持细胞系。该细胞系中含有一定比例的具有增殖和自我更新能力的结肠癌干细胞样细胞亚群。表面标志以及双苯酰亚胺33342检测差异提示细胞球中仅部分细胞具有干细胞的功能。     

结肠癌干细胞的肿瘤形成特征及其临床价值

目的:研究结肠癌干细胞的肿瘤形成价值及其可能的临床价值.方法:对结肠癌干细胞进行无血清培养,以流式细胞仪检测CD133和CD44表达,以5-氟尿嘧啶(5-flurouracil,5-Fu)处理结肠癌干细胞并观察其增殖抑制,对结肠癌干细胞进行动物实验以确定其成瘤特征.结果:Hoechest33342染色后,HT-29干细胞相对于HT-29细胞有明显的核拒染现象;流式细胞仪发现HT-29细胞CD133阳性表达比率为44.6%,C D44阳性表达比率为0.6%;而经过培养提纯后的HT-29干细胞CD133阳性表达比率为92.6%,CD44阳性表达比率为97.8%,C D 1 3 3与C D 4 4阳性表达比率显著提高;HT-29干细胞相对于HT-29细胞对5-Fu有明显的耐药作用,HT-29细胞IC50值为1.394μg/m L,HT-29干细胞的IC50值为13.087μg/m L,IC50值有明显的增高在恢复血清培养后,体外增殖活性明显增强;HT-29干细胞体内生长速度比H T-29细胞体内生长速度显著要缓慢,H T-29细胞的肿瘤体积是HT-29干细胞肿瘤体积的两倍多;HT-29干细胞在小鼠富血管区(腋下和腹股沟淋巴结)的生长速率比皮下生长速率有明显的提高,但与HT-29细胞的生长速率还有明显差距.结论:在结肠癌细胞株存在结肠癌干细胞,通过无血清培养能分离出具有干细胞特征的结肠癌干细胞株,结肠癌干细胞对5-Fu具有耐药性,在适当微环境下其能快速生长,转化为成熟的结肠癌细胞,提示了改变结肠癌干细胞微环境可能是防止肿瘤复发的一个途径.     

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。     

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.     

永生化骨髓间充质干细胞克隆形成能力和致瘤性的实验研究

目的探讨永生化人骨髓间充质干细胞（hBMSC）用于软骨组织工程的可能性及安全性。方法自健康志愿者骨髓中提取原代hBMSC,流式细胞仪检测表型后建立永生化hBMSC,将hBMSC及永生化hBMSC细胞悬液接种于双层软琼脂培养基中,2周后观察克隆形态并计算克隆形成率;将两种细胞接种于裸鼠背部皮下,术后3d及1、3个月肉眼观察成瘤情况。结果hMSC表面抗原CD34、CD45表达阴性,CD扮、CD71、CD106表达阳性;hBMSC未能在软琼脂中形成克隆,永生化hBMSC克隆形成率为0．3％;永生化hBMSC接种裸鼠后3d接种部位有细胞聚集的团块,1、3个月时均未见肿瘤形成。结论本研究通过转基因技术建立的永生化hBMSC增殖能力较强,但无致瘤性,可作为种子细胞用于软骨组织工程中。     

Cancer stem cell markers CD133 and CD24 correlate with invasiveness and differentiation in colorectal adenocarcinoma

AIM： To verify that CD markers are available for detecting cancer stem cell populations and to evaluate their clinical significance in colon cancer.METHODS： Immunohistochemistry for CD133, CD24 and CD44 was performed on the tissue microarray of 523 colorectal adenocarcinomas. Medical records were reviewed and clinicopathological analysis was performed.RESULTS： In colorectal adenocarcinoma, 128 of 523 cases （24.5%） were positive and 395 cases （75.5%） were negative for CD133 expression. Two hundred and sixty-four of 523 cases （50.5%） were positive and 259 cases （49.5%） were negative for CD24 expression. Five hundred and two of 523 cases （96%） were negative and 21 cases （4%） were positive for CD44 expression. Upon clinicopathological analysis, CD133 expression was present more in male patients （P = 0.002） and in advanced T stage cancer （P = 0.024）. Correlation between CD24 expression and clinicopathological factors was seen in the degree of differentiation （P= 0.006）. Correlation between CD44 expression and clinicopathological factors was seen in the tumor size （P = 0.001）. Survival was not significantly related to CD133, CD24 and CD44 expression.CONCLUSION： CD markers were related to invasiveness and differentiation of colorectal adenocarcinoma. However, CD expression was not closely related to survival.     

Molecular mechanisms of denbinobin-induced anti-tumorigenesis effect in colon cancer cells

AIM: To explore both the in vitro and in vivo effects of denbinobin against colon cancer cells and clarify its underlying signal pathways. METHODS: We used COLO 205 cancer cell lines and nude mice xenograft model to study the in vitro and in vivo anti-cancer effects of denbinobin. RESULTS: Denbinobin at concentration of 10-20 μmol/L dose-dependently suppressed COLO 205 cell proliferation by MTT test. Flow cytometry analysis and DNA fragmentation assay revealed that 10-20 μmol/L denbinobin treatment induced COLO 205 cells apoptosis. Western blot analysis showed that caspases 3,8,9 and Bid protein were activated by denbinobin treatment to COLO 205 cells accompanied with cytochrome c and apoptosis-inducing factor (AIF) translocation. Pretreatment of MEK 1 inhibitor (U10126), but not p38 inhibitor (SB203580) and JNK inhibitor (SP600125), reversed denbinobin-induced caspase 8, 9 and Bid activation in COLO 205 cells suggesting that extracellular signal-regulated kinase were involved in the denbinobin-induced apoptosis in COLO 205 cells. Significant regression of tumor up to 68% was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with denbinobin 50 mg/kg intraperitoneally. CONCLUSION: Our findings suggest that denbinobin could inhibit colon cancer growth both in vitro and in vivo. Activation of extrinsic and intrinsic apoptotic pathways and AIF were involved in the denbinobin-induced COLO 205 cell apoptosis.     

miR-383靶向Notch1对结肠癌HCT116细胞增殖和迁移的影响

目的 分析miR-383和Notch1在结肠癌中的作用及机制.方法 选择2017年4月至2020年3月在河北北方学院附属第一医院经病理检查确诊为结肠癌的74例患者,收集其经手术切除的肿瘤组织及癌旁正常组织(距肿瘤边缘>3 cm).采用TargetScan预测miR-383的潜在靶基因,采用双荧光素酶报告基因实验、逆转录...     

下调CD44基因表达对人胃癌SGC7901细胞裸鼠移植瘤的抑制作用及其机制

目的探讨靶向CD44的短发夹RNA(shRNA)对人胃癌裸鼠移植瘤生长抑制作用及其机制。方法利用CD44 shRNA细胞及对照细胞株建立裸鼠原位移植瘤及皮下种植瘤模型,监测肿瘤生长变化,采用RT-PCR及免疫组织化学方法检测瘤体内CD44表达的变化,并进一步检测上皮-间质转化(EMT)相关因子表达的变化。结果成功构建了CD44 shRNA转染荷瘤皮下及原位裸鼠模型。与对照shRNA组、空白对照组分别比较,CD44 shRNA组荷瘤裸鼠肿瘤生长速度减慢,皮下种植瘤及原位移植瘤质量减轻,差异均有统计学意义(P均0.01)。RT-PCR结果发现,CD44 shRNA组CD44mRNA表达在皮下种植瘤及原位移植瘤均显著下调,差异均有统计学意义(P均0.01)。CD44 shRNA组细胞EMT相关基因E-cadherin表达增加,N-cadherin、Vimentin表达降低。结论 CD44 shRNA可以有效抑制人胃癌SGC7901细胞裸鼠移植瘤生长,并下调CD44的表达水平,这可能与CD44基因参与EMT相关。     

Colon cancer stem cells:Controversies and perspectives

Tumors have long been viewed as a population in which all cells have the equal propensity to form new tumors,the so called conventional stochastic model.The cutting-edge theory on tumor origin and progression,tends to consider cancer as a stem cell disease.Stem cells are actively involved in the onset and maintenance of colon cancer.This review is intended to examine the state of the art on colon cancer stem cells(CSCs),with regard to the recent achievements of basic research and to the corresponding translational consequences.Specific prominence is given to the hypothesized origin of CSCs and to the methods for their identification.The growing understanding of CSC biology is driving the optimization of novel anti-cancer targeted drugs.     

Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines

BACKGROUNDColon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers. It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-expressing stem cells, these so-called “tumour-initiating” cells, reminiscent in their properties of the normal intestinal stem cells (ISCs), may explain the apparent heterogeneity of colon cancer cell lines. Also, colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5. Furthermore, in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIMTo investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODSA portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines (Caco-2 and LoVo). These cells were then characterized according to their proliferation capacity, gene expression signatures of ISC markers, and their tumorigenic properties in vivo and in vitro.RESULTSThe data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2, but not from the LoVo, cell lines, in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog (+4 crypt) stem cells. This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny. Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein (EYFP) from this cell line exhibit tumorigenic-like properties in vivo and in vitro. In contrast, cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties. Thus, cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells, but not LoVo cells.CONCLUSIONOur findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines. Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data. We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.     

Upregulated CD133 expression in tumorigenesis of colon cancer cells

AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging dro...     

MicroRNA在人结肠癌干细胞中的表达谱

目的:探讨微小RNA(microRNA,miRNA)在人结肠癌干细胞中的表达,为进一步研究miRNA调控结肠癌干细胞向结肠癌细胞分化的分子机制奠定基础.方法:应用miRNA表达谱芯片检测人结肠癌干细胞和已分化结肠癌细胞中miRNA的表达谱.利用实时定量PCR技术检测两种细胞中差异表达的miRNA,验证miRNA芯片结果的可靠性.应用软件对筛选出的显著差异表达miRNA的靶基因进行预测.结果:与已分化结肠癌细胞相比,人结肠癌干细胞中表达上调超过1.5倍的miRNA有35个.为:hsa-miR-192,hsa-miR-29b,hsa-miR-215,hsa-miR-194,hsa-miR-33a,hsa-miR-32等:表达下调超过1.5倍的miRNA有11个,为:hsa-miR-93,hsa-miR-1231,hsa-miR-524-3p,hsa-miR-886-3p等.PCR技术验证,与miRNA芯片结果相符合.表达显著上调miRNA的共同靶mRNA有:AFF2、MTF1、RUNDC2C和ZFHX4.表达显著下调miRNA的共同靶mRNA有:ONECUT2、SH3TC2、PTPRT、RNABP10、NR3C1、RGSL1、RNASEL和TANC2.结论:筛选出的差异表达miRNA可能参与结肠癌的发病.为该病诊治提供了新的思路.其共同靶基因可能具有重要的调控结肠癌干细胞生长和分化的作用.